Francis B. Ryan Peter D. Kube Scott A. Parkinson

Xiaoxu Li John A. Nell

September 2005

FRDC Project No.2005/227

ASIThoroughbred Oysters

Selection of genetic strategies

in Pacific oysters to maximise

commercial benefit

Selection of genetic strategies in Pacific oysters to maximise commercial benefit

Francis B. Ryan, Peter D. Kube, Scott A. Parkinson, Xiaoxu Li and John A. Nell

FRDC Project 2005/227

Selection of genetic strategies in Pacific oysters to maximise commercial benefit

Francis B. Ryan1, Peter D. Kube2, Scott A. Parkinson3, Xiaoxu Li4, and John A. Nell5

1 Australian Seafood Industries Pty Ltd (ASI). 51 Belar St, Howrah, Tasmania, 7018, Australia Tel: (03) 6244 6622 Fax: (03) 6244 1538 2 CSIRO Marine and Atmospheric Research. GPO Box 1538, Hobart, Tasmania, 7001, Australia Tel: (03) 6232 5222 Fax: (03) 6232 5000 3 Australian Seafood Industries Pty Ltd (ASI). 51 Belar St, Howrah, Tasmania, 7018, Australia Tel: (03) 6244 6622 Fax: (03) 6244 1538 4 South Australian Research and Development Institute (SARDI) 2 Hamra Ave, West Beach, SA 5024, Australia Tel: (08) 8207 5400 Fax: (08) 8207 5481 5 NSW Fisheries. Port Stephens Fisheries Centre, Private Bag 1, Nelson Bay, NSW, 2315, Australia Tel: (02) 4982 1232 Fax: (02) 49821107 Cover design: Louise Bell, CSIRO Marine and Atmospheric Research Copyright Fisheries Research and Development Corporation and Australian seafood industries Pty Ltd, Australia, 2006 This work is copyright. Except as permitted under the Copyright Act 1968 (Cth), no part of this publication may be reproduced by any process, electronic or otherwise, without the specific written permission of the copyright owners. Neither may information be stored electronically in any form whatsoever without such permission. The Fisheries Research and Development Corporation plans, invests in and manages fisheries research and development throughout Australia. It is a statutory authority within the portfolio of the federal Minister for Agriculture, Fisheries and Forestry, jointly funded by the Australian Government and the fishing industry ISBN 0-646-46031-5 Disclaimer: ASI and FRDC do not warrant that the information contained in this report is free from errors or omissions. ASI and FRDC shall not be in any way liable for any loss, damage or injury suffered by the user consequent upon or incidental to the existence of errors or omissions in the information.

Table of Contents

1 NON-TECHNICAL SUMMARY..................................................................................................... 1

2 ACKNOWLEDGEMENTS............................................................................................................... 4

3 INTRODUCTION ............................................................................................................................. 5

3.1 BACKGROUND ........................................................................................................................5 3.2 NEED..........................................................................................................................................5

3.3 OBJECTIVES.............................................................................................................................6

4 METHODS......................................................................................................................................... 7

4.1 TEAM STRUCTURE.................................................................................................................7 4.2 AIMS ..........................................................................................................................................8 4.3 ECONOMIC ANALYSIS ..........................................................................................................8

5 RESULTS AND DISCUSSION ...................................................................................................... 10

5.1 ENHANCED SELECTIVE BREEDING .................................................................................10 5.1.1 General Overview..........................................................................................................10 5.1.2 Discussion: Current status of breeding program............................................................10 5.1.3 Discussion: Enhancement of the selective breeding program .......................................13 5.1.4 IP Issues.........................................................................................................................14 5.1.5 Conclusions ...................................................................................................................14

5.2 TRIPLOIDY .............................................................................................................................14 5.2.1 Introduction ...................................................................................................................14 5.2.2 Patented technology.......................................................................................................14 5.2.3 Alternative Techniques for Triploid Production............................................................18 5.2.4 General considerations ..................................................................................................18 5.2.5 Conclusions ...................................................................................................................19

5.3 DOUBLE HAPLOIDY (DH) ...................................................................................................20 5.3.1 Double Haploidy in Plants (Dr Davies).........................................................................20 5.3.2 Technical Explanation of Double Haploidy (Mr Kent) .................................................21 5.3.3 The SARDI Oyster DH program (Dr Li).......................................................................22 5.3.4 General Discussion........................................................................................................26 5.3.5 IP issues.........................................................................................................................27 5.3.6 Conclusions ...................................................................................................................27

5.4 INTENSIVE INBREEDING ....................................................................................................28 5.4.1 General overview...........................................................................................................28 5.4.2 General considerations ..................................................................................................29 5.4.3 Conclusions ...................................................................................................................30

5.5 MARKER ASSISTED SELECTION.......................................................................................31 5.5.1 Technical explanation....................................................................................................31 5.5.2 General Discussion........................................................................................................31 5.5.3 Conclusions ...................................................................................................................32

6 COMPARISON OF TECHNOLOGIES........................................................................................ 33

6.1 ENHANCED SELECTIVE BREEDING .................................................................................33 6.2 TRIPLOIDY .............................................................................................................................35 6.3 DOUBLE HAPLOIDY.............................................................................................................36 6.4 INTENSIVE INBREEDING ....................................................................................................36

7 GENERAL CONCLUSIONS.......................................................................................................... 38

7.1 ENHANCED SELECTIVE BREEDING: ................................................................................38 7.2 TRIPLOIDY: ............................................................................................................................38

7.3 DOUBLE HAPLOIDY (DH):.................................................................................................. 38 7.4 INTENSIVE INBREEDING: .................................................................................................. 39 7.5 MARKER ASSISTED SELECTION (MAS): ......................................................................... 39 7.6 THE PROJECT TEAM CONSIDERATIONS: ....................................................................... 39

8 INDEPENDENT REVIEW ............................................................................................................. 40

8.1 INDEPENDENT REVIEWERS REPORT.............................................................................. 40

9 REFERENCES................................................................................................................................. 41

10 APPENDIX 1 REVIEW OF ASI PACIFIC OYSTER BREEDING PROGR AM .................... 44

10.1 INTRODUCTION ................................................................................................................... 44 10.2 CURRENT ASI SELECTIVE BREEDING STRATEGY....................................................... 44 10.3 IMPROVEMENTS TO CURRENT STRATEGY................................................................... 45 10.4 ECONOMIC WEIGHTS OF TRAITS .................................................................................... 47 10.5 BREEDING STRATEGY ISSUES ......................................................................................... 48 10.6 DEVELOPMENT OF SELECTIVE BREEDING PROGRAM SYSTEMS ........................... 50 10.7 DEVELOPMENT OF TECHNICAL PROTOCOLS .............................................................. 51 10.8 CONCLUSION........................................................................................................................ 51 10.9 REFERENCES ........................................................................................................................ 52

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1 NON-TECHNICAL SUMMARY

2005/227 Selection of genetic strategies in Pacific oysters to maximise commercial benefit PRINCIPAL INVESTIGATOR: Mr F B Ryan

ADDRESS: Australian Seafood Industries Pty Ltd 51 Belar Street Howrah, Tasmania, 7018 Australia

Telephone: 03 6244 6622

E-mail: barry_07@bigpond.net.au E-mail: scottasi@bigpond.net.au

REPORT AUTHORS: Francis B. Ryan, Peter D. Kube, Scott A. Parkinson,

Xiaoxu Li, and John A. Nell

OBJECTIVES: 1. Document the existing oyster genetic R&D technology and IP ownership.

2. Review and, where possible quantify the expected benefits and likely risks when using each of these technologies for Pacific oysters.

3. Document a pathway for the commercial deployment of Pacific oysters developed by triploidy, hybrid vigour, double haploidy and marker assisted selection with particular emphasis on the cost and time needed for both the R&D phase and full commercialisation.

4. Identify the way in which the use of triploidy, hybrid vigour, double haploidy and marker assisted selection fit in with a selective breeding strategy and identify aspects of a selective breeding program that are essential for the commercial use of these tools.

5. Identify any impediments to the commercialisation of these technologies.

6. Estimate the commercial production costs of triploid, hybrid and double haploid Pacific oysters and assess their economic value relative to stock produced from ASI’s conventional selective breeding program.

7. Identify IP ownership of each of the breeding tools, expected costs associated with commercial access to the IP and any limitations to commercial application that may occur due to IP ownership.

NON TECHNICAL SUMMARY OUTCOMES ACHIEVED The future priorities for Pacific oyster genetic research were clarified. Enhancement of the current ASI selective breeding program and development of direct triploidy methodologies were suggested as high priority. Existing, developing and proposed technologies in relation to oyster triploidy, double haploidy, intensive inbreeding and marker assisted selection were reviewed alongside the Australian Seafood Industries Pty Ltd (ASI) breeding program with a view to establishing which, if any, might be developed to replace or improve the selective breeding program currently operated by ASI. Enhanced selective breeding: It was accepted that with the adoption of improvements including the use of economic weights, variation of breeding strategies, development of

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improved breeding program systems and technical protocols, an enhanced selective breeding program could be expected to at least double the rate of genetic gain achieved by the current ASI program (e.g. the current program’s gain per generation is estimated at 3-4% for selection on both shape and growth. This could be increased to 6-8% in an enhanced multi-trait selection program). Such enhancement would require research input to identify the various parameters and the best strategies, systems and protocols to be adopted. However, the technology involved is already established and proven in other industries and therefore the risks are minimal and the benefits are assured and substantial. The R&D costs are estimated at $647 K for the necessary 3 year program. Although some of the beneficial outcomes would be available to begin modification of the breeding program within eighteen months. There are no impediments or IP issues. Moderate developmental cost, relatively short developmental time, assured successful application with quantifiable benefits across the entire Pacific oyster industry and the applicability of some of the outcomes to the wider aquaculture industries make this the highest priority recommendation for future ASI research. Triploidy: The use of triploid oysters is a proven and established concept for which the benefits are known and substantial in some environments, although variable in others - particularly where food supply is limited. The technology available in Australasia derives triploids via a tetraploid technology which is patented and exclusive to only one oyster hatchery. ASI should keep a watching brief on alternative methods for production of tetraploids that may become available in the future. The hatchery is not willing to incorporate improved ASI broodstock into its triploid program even though there is some evidence that the gains from selection and triploidy are additive. The technologies for direct triploid production are well developed. The main issues preventing their commercialisation are variations in egg quality, egg development and unacceptable proportions of diploids amongst the triploids. The Project Team considers this to be a worthwhile area for research in the immediate future. It is expected that further development of ‘direct’ triploid technologies to a commercial level would require an estimated three years and total research expenditure of perhaps $240K. If successful, the assimilation of broodstock from the ASI selective breeding program into a triploid program would be straightforward. This technology would be integrated with the ASI selective breeding strategy according to commercial demand. Double haploidy (DH): This technology remains at the “proof of concept” stage. It has not been established that double haploid larvae can be produced in reliable quantity, and that a satisfactory percentage of these can metamorphose, mature and breed. In some plant species only about 5% of DH individuals go on to produce seeds. This is of little significance in plants where it is possible to set up and test many individuals within each DH family, but similar (or even lower) levels of fertility in DH oysters could pose a serious obstacle to adoption of the technology. An estimated $1265 K expenditure and 6.5 years R&D work are needed before double haploid oysters could become available as part of the ASI breeding program. The technology would not replace the breeding program. In view of the potency of this technology in some areas of plant breeding, the Project Team assigned a moderate priority for a revised proposal taking the DH research to the stage of demonstrating production of adequate numbers of double haploid spats and their survival to 2-3 months of age. Research costs to this level were estimated at $92,000. IP issues are associated with DH technology. SARDI sees ASI as a vehicle for the commercialisation of double haploidy and has sought inter alia to negotiate for royalty payments but ASI does not consider the technology to be sufficiently developed at this stage.

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Intensive Inbreeding: As with double haploidy, this technology relies on development of highly homozygous lines which are eventually outcrossed in the expectation that hybrid vigour will result. However, development of the homozygous lines depends on the use of a series of “selfed” matings. The effectiveness of “selfing” as a technique has been demonstrated but the breeding capacity of successively selfed lines remains uncertain, and the existence of the hybrid vigour phenomenon in oysters has not been demonstrated. The Project Team therefore decided that “proof of concept” has not yet been established for this technology in oysters. The lead time to establishment of proof of concept and commercial production of hybrid spat by this route would be in the order of 8 to 13 years, the developmental cost is estimated to be well in excess of $1175 K. and there are uncertainties as to the likely benefits flowing from hybrid vigour. The Project Team therefore considered intensive inbreeding to be a low research priority. Marker assisted selection: The Project Team considered that this technology has not yet reached a developmental stage in oysters and is not ready for commercial application. OUTPUTS:

• Enhancement of the current selective breeding program is identified as the highest priority and most cost effective area for developmental research to supplement the ASI selective breeding program.

• Industry access to triploidy technology through the tetraploid route is limited by the existing IP ownership and patent protection. Developmental research to commercialise alternative technology for induction of triploidy is warranted.

• Intensive inbreeding, double haploidy and marker assisted selection cannot be considered as tools to replace or supplement the ASI selective breeding program at this time.

KEYWORDS: Pacific oyster, Crassostrea gigas, selective breeding, breeding strategies

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2 ACKNOWLEDGEMENTS

We would like to thank FRDC through project number 2005/227 for supporting this work. We would also like to thank CSIRO Marine and Atmospheric Research, Tasmanian Fishing Industry Council and South Australian Research and Development Institute (SARDI) for their support in the hosting meetings and facilitating the smooth operation of the project. The following people need thanks for their contribution to meetings and their expert opinion on specific topics. Mr Gary Zippel and Mr James Burke Australian Seafood Industries Pty Ltd Dr Nick Elliott, Dr Bob Ward and Dr Sharon Appleyard. CSIRO Marine and Atmospheric Research Dr Phil Davies SARDI Dr Greg Kirby Flinders University Mr Michael Cameron Cameron of Tasmania Pty Ltd Mr Richard Pugh and Mr Greg Kent Shellfish Culture Limited

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3 INTRODUCTION

3.1 BACKGROUND

This project was developed because the oyster industry needed a genetic R&D strategy to assess the economic benefit of the various developing breeding technologies for comparison with conventional selective breeding. The strategy was to be used as the basis for developing and prioritising genetic research for the aquaculture industry, particularly the oyster industry led by its R&D commercialisation company, Australian Seafood Industries Pty Ltd (ASI). The project focussed on evaluation of breeding technologies such as triploidy, double haploidy, hybrid vigour and marker-assisted selection and comparison of the benefits likely to be achieved by them with the benefits achievable through conventional selective breeding. These tools were being considered for ASI’s genetic improvement program. However, each one would require extra expenditure for R&D, and the means by which they might be utilized within ASI’s breeding program was unclear. Therefore ASI needed to assess the economic value of these breeding technologies and determine how each would fit within an overall genetic strategy. The project was also to include a review of Intellectual Property ownership, as it relates to the genetic technologies under consideration. As discussion developed it became clear that the term “hybrid vigour” applied in this case to a technique involving intra-species development of highly homozygous lines1 which are eventually outcrossed in the expectation that hybrid vigour will result. The method under consideration for development of the homozygous lines depended on the use of a series of “selfed” matings, whereby gametes from an oyster are stored and later fertilised by gametes from the same oyster after its sex has changed at the next spawning season(s). Within the project this technology came to be described as “intensive inbreeding”. Similarly it was decided that for appropriate comparison, the various technologies needed to be considered alongside the ASI breeding program as it could be with further research input, rather than as it currently presents. The term “Enhanced Selective Breeding” was therefore adopted.

3.2 NEED

The fundamental need was to clearly define the priorities for genetic R&D in the oyster industry for a five-year period. The potential economic benefits of the different technologies relative to their developmental costs were unclear, as were the logistics of applying the technologies to the ASI breeding program. IP issues also needed to be taken into account. The output required was a strategy for ongoing industry research which identified the best commercial returns from investment in R&D. The project aimed to evaluate genetic technologies such as enhanced selective breeding, triploidy, double haploidy, hybrid vigour (considered under intensive inbreeding), and marker-assisted selection. The immediate priorities were to:

• Document the existing oyster R&D technology; and IP ownership

1 As distinct from between species hybridization

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• Evaluate different R&D technologies and their respective outputs with regard to their potential contribution to ASI’s selective breeding program (or an alternative breeding program).

• Detail the steps required to commercialise these R&D technologies, including benefit cost analyses, and identification of any research gaps, adoption gaps, or bottlenecks.

Ultimately, it was recognised that the benefits of these technologies needed to be compared with those of enhanced selective breeding in its own right.

3.3 OBJECTIVES

The original objectives were to:

1. Document the existing oyster genetic R&D technology and IP ownership.

2. Review and, where possible, quantify the expected benefits and likely risks when using each of these technologies for Pacific oysters.

3. Document a pathway for the commercial deployment of Pacific oysters developed by triploidy, hybrid vigour, double haploidy and marker assisted selection with particular emphasis on the cost and time needed for both the R&D phase and full commercialisation.

4. Identify the way in which the use of triploidy, hybrid vigour, double haploidy and marker assisted selection fit in with a selective breeding strategy and identify any aspects of a selective breeding program that are essential for the commercial use of these tools.

5. Identify any impediments to the commercialisation of these technologies.

6. Estimate the commercial production costs of triploid, hybrid and double haploid Pacific oysters and assess their economic value relative to stock produced from ASI’s conventional selective breeding program.

7. Identify IP ownership of each of the breeding tools, expected costs associated with commercial access to the IP, and any limitations to commercial application that may occur due to IP ownership.

These objectives needed to be modified to include enhanced selective breeding as an additional technology requiring evaluation and to replace “hybrid vigour” and “hybrid” by the term “intensive inbreeding”. All of the objectives have been achieved with the exception of objective 6. While it was possible to estimate commercial production costs where applicable for the technologies examined, it was not possible to assess their economic value because in most cases there has been insufficient developmental research to provide the necessary data.

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4 METHODS

4.1 TEAM STRUCTURE

Good communication between ASI, industry and researchers was essential to the outcome of this project. A Project Team was set up to precisely define the scope of activities, perform a preliminary review of each of the technologies under scrutiny and assign initial responsibilities for preparation of discussion papers relating to each technology. Discussion then continued within the Team by correspondence, teleconferencing and face-to-face meetings as necessary to arrive at a collective view as to the attributes of each technology. ASI contracted two technical experts to assist with the project. These and two ASI representatives worked as a core Project Team to collaborate in covering all aspects of the project brief. Dr Xiaoxu Li (South Australian Research and Development Institute SARDI) and Dr Peter Kube (CSIRO Marine and Atmospheric Research, Hobart) were the two core technical experts. A third technical expert, Mr John Nell (NSW Fisheries), independent of both CSIRO and SARDI, functioned as an Independent Reviewer in case consensus could not be reached between the Project Team members. The ASI representatives on the core Project Team were Mr Scott Parkinson and Mr Barry Ryan. Mr Barry Ryan (Chairman of the ASI) was the project Principal Investigator and took responsibility for the coordination, management and operation of the team. While the technical experts contracted from CSIRO and SARDI took responsibility for the inputs from their respective agencies, ancillary personnel were involved to a varying degree in the discussions – these were Drs Nick Elliott, Sharon Appleyard and Bob Ward from CSIRO, Dr Phillip Davies from SARDI, Dr Greg Kirby from Flinders University, Mr Greg Kent from the University of Tasmania, Mr Gary Zippel from ASI, Mr Richard Pugh from Shellfish Culture Hatchery (Tasmania) and Mr Michael Cameron (Cameron of Tasmania). The special interest/expertise contributed by these participants was:

Mr Ryan: Australian Seafood Industries Pty Ltd Selective breeding techniques

Dr Kube: Quantitative Geneticist, CSIRO Marine and Atmospheric Research Selective breeding, inbreeding and hybrid vigour

Mr Parkinson: Australian Seafood Industries Pty Ltd Selective breeding

Dr Li: Senior Scientist, SARDI Double haploidy; triploidy, selective breeding

Dr Nell: Principal Research Scientist (Aquaculture), NSW Fisheries Triploidy in oysters

Dr Elliott: Principal Research Scientist, CSIRO Marine and Atmospheric Research Marker assisted selection, selective breeding, inbreeding and hybrid vigour

Dr Appleyard: Molecular Geneticist, CSIRO Marine and Atmospheric Research Marker assisted selection

Dr Ward: Principal Research Scientist, CSIRO Marine and Atmospheric Research Inbreeding and hybrid vigour

Dr Davies: Senior Scientist, SARDI Double haploidy in plant breeding

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Dr Kirby: Senior Lecturer, Plant Breeding and Biodiversity, Flinders University Double haploidy in plant breeding

Mr Kent: Research Fellow, University of Tasmania Triploidy, double haploidy in oysters

Mr Pugh: Hatchery Manager, Shellfish Culture Triploidy in oysters, hatchery techniques

Mr Cameron: Cameron of Tasmania Hatchery techniques, Tassie Gold oysters

The Independent Reviewer was to arbitrate if differences of opinion developed within the Review Team and has provided an alternative view of the outcomes by way of an independent report which is included later in this final report. Deliberations of the team involved two in-person meetings and two telephone conferences over a total period of 6 months. Work between the meetings to discuss and refine the meeting reports and discussion papers, was by telephone and email.

4.2 AIMS

Identify strategies by which enhanced selective breeding, triploidy, double haploidy, intensive inbreeding, and marker-assisted selection could be used to produce a commercial product by:

• Identifying the R&D that would be needed to get the technology to a fully operational stage, and the expected time required to complete this R&D.

• Listing the actions needed to produce a commercial product, beginning from selection of base material and through to the production of commercial quantities of seed.

• Constructing a timeline for these actions, showing the links between each action and the total time span needed to produce a commercial product.

• Listing new equipment, skills and licensed technology that would need to be sourced for an operational program.

• Listing existing IP ownership and IP ownership likely to arise with further R&D, and estimate all costs that would be associated with gaining access to this IP.

4.3 ECONOMIC ANALYSIS

Once the strategy was described, the intention was to calculate the economic value of each of the technologies in a cost-benefit study. The first step in this process was to estimate the potential benefits from each technology in terms of traits that would be improved, the magnitude of the improvement, and the expected value of the gains. However, it was found that data was not available to support such an analysis. Double haploidy and intensive inbreeding were found to be at the ‘proof of concept’ stage, with little evidence to suggest that the technologies would be workable at the commercial level and no evidence to indicate the extent of benefits if they were workable Similarly, the extent of benefits to be derived from marker assisted selection (MAS) was unknown at this stage, although the cost was likely to be high. The question of cost/benefit for triploidy by the direct route is complicated by the variability of its beneficial effects, and the likely success and penetration of the patented tetraploid technology. There is room for research, particularly in relation to which conditions will allow triploids to fully express (and which will not), but data to support comparative cost benefit analysis in any general sense is not presently available.

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Costs associated with the tetraploid induction of triploids are closely guarded as commercial-in-confidence by the operative company. In the case of enhanced selective breeding, double haploidy and intensive inbreeding, estimates of development and operational costs to advance the technology to the point of commercial application could be made to the satisfaction of all participants based on figures developed within FRDC project proposals. These analyses include development costs as well as operational costs.

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5 RESULTS AND DISCUSSION

5.1 ENHANCED SELECTIVE BREEDING

Dr Peter Kube led this discussion.

5.1.1 General Overview

Dr Kube summarised the basic purpose of a selective breeding program, which was the control of breeding to increase the frequency of desired traits in the next generation, and to concentrate desired genes into selected individuals. He instanced gains per year from selective breeding in poultry, salmon, radiata pine and other species which approximated to 10% per generation and pointed out that in a well run program gains could continue to be delivered over many generations. Due to improved knowledge, improved computing power and the need for greater economic focus there have been significant changes to the application of selective breeding over last 25 years or so, coupled with an increased flow of ideas across and between industries. Dr Kube described the features of modern selective breeding programs:

• Their goal is to increase profit through the use of objective economic analysis.

• They utilise well targeted selection based on the economic values of multiple traits including quality.

• They apply a high selection intensity utilising large and diverse populations.

• They are based on both within and between family selection.

• They take full account of underlying genetics including heritabilities, genetic correlations, dominance effects and inbreeding.

• They use objective selection methodologies such as the calculation of breeding values and the application of economic weights.

5.1.2 Discussion: Current status of breeding program

Participants noted that the current ASI breeding program falls far short of the modern selective breeding program described by Dr Kube. The ASI Circular Breeding Program provided by Dr Andrew Swan makes reference to the use of economic weights but in fact does not utilise any form of objective economic analysis. This is crucial to the success of ASI. In addition, the Program is based only on within family selection. Full realisation of the ASI breeding program’s potential is also limited by the number of families that can be raised in any one season. At present, the cost of facilities and operational limitations restrict the number to 24, whereas a greater number would afford a greater selection intensity and faster improvement. A larger number of families will also be important if more traits are to be included in the program. Clearly, a balance must be struck between the rate of improvement and the cost, but a number of questions arose:

• What can industry afford?

• What percentage of their value do other industries assign to selective breeding?

• Is there cheaper technology which would allow rearing more families for the same cost?

• What increased rate of improvement would follow if the number of families reared was increased i.e. what is the cost/benefit?

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It was suggested that the Cawthron Institute runs a small, simple and inexpensive flow through research system capable of producing 50 lines and that this system or similar might be an innovative low cost way of producing more families, but Mr Parkinson said the real costs lay in ongoing grow out, data collection, and grading. These costs would not be moderated by cheaper hatchery technology. Cash flow is the problem. Could ASI look towards exporting IP or technology? The option of breeding only once every two years but with larger family numbers was canvassed but rejected because it would create longer time lags between the initiation of breeding and subsequent release of thoroughbred spat to commercial oyster farms. External funding from FRDC, AusIndustry or some other source seems to be the most likely solution. The issue is to secure an adequate source of funding and for a sufficient time period to allow the breeding program to become established. It was pointed out that, in other industries, external funding occurred for many years to allow programs to become established, and new developments are frequently funded with external dollars. Aquaculture industries in Australia will eventually accept selective breeding as an integral cost of doing business and will then be prepared to pay the full cost of running a breeding program. However, that shift in mind-set takes many years and has not yet occurred in the Pacific oyster industry. The success of the original breeding plan is unquestioned in delivering genetic gains of reasonable proportion and at reasonable cost however, it was recognised that the ASI selective breeding program was due for major review. The urgency for this review has been accentuated because ASI has matured and the industry has changed. Previously the emphasis has been on growth, but now shape, uniformity and survival have emerged as very important traits. Meat/shell ratio and conditioning are also being considered important by industry. New information on the heritability of different oyster traits is emerging and new technologies are being examined during this review. There will be a need for assimilation of these developments into the breeding program. The Project Team agreed that this provided a strong case for taking ASI’s breeding program to the next step. They concluded that the original breeding plan is now outdated and there is need for development of a strategic breeding plan which describes revised breeding objectives, utilises economic weights for both old and new traits, defines appropriate population sizes and makes use of new analytical methods. Economically based breeding objectives should be the first priority. The skills needed for this development are those associated with genetic expertise – the development of a strategic breeding plan and advice about the collection, manipulation, assessment and analysis of data. Recognising that ASI funds currently do not allow for the development of an improved breeding program in the immediate future Mr Parkinson asked what were ASI’s options for the coming season. Modifications to the works program for the coming season are being discussed and considered, however this is a stop-gap measure and does not constitute a complete review. It was agreed that for the moment the only option was to modify the circular breeding plan and use it again. Enhancement proposal Following discussions about the current status of the ASI selective breeding program, the Project Team requested a discussion paper which would outline in broad terms the research required to bring the current ASI breeding program to its full potential. This paper was prepared by Peter Kube and Scott Parkinson and is attached as Appendix 1.

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This paper describes the current ASI breeding strategy in detail, indicates eleven problem areas and offers solutions to overcome them. The current program, which has operated successfully since 1998, is described as a simple and conservative strategy, appropriate for the resources and technical skills that were available. But Dr Kube said that the problems were fundamental and required major revision of the program if it was to be brought up to the standard of a modern breeding program. For the first five generations, selection had been based on fast growth. This produced an oyster which was bigger but tended to be undesirably elongated. For the current sixth generation, selection for fast growth is secondary to selection for shape, taking in to account both width and depth. The heritability of this trait is moderately high – approximately 0.3 (Ward et al. 2005). Condition/glycogen storage has been flagged as a major research priority but is not a trait considered for selection in the program yet because of its complexity. The present AusIndustry project being undertaken by James Burke (ASI) in association with TAFI is seen as a precursor project - examining the relation between speed of growth, shell density, meat/shell ratio and condition. Other aspects of this important trait which need to be studied are timing, trigger factors, glycogen content and non-destructive testing methods. Dr Davies stressed the importance of measuring performance under commercial conditions, because prevailing conditions may influence the genetic response. The discussion paper addressed four suggested initiatives to enhance the breeding program: Use of economic weights: The review paper states that of all the factors needing to be introduced into the breeding program, the use of economic weights to target selection had the first priority. As the number of traits under consideration increases (for instance by the addition of survival and condition to the present mix of growth and shape) it will become increasingly difficult to use intuitive selection. Already, adverse correlations are known to occur between improving growth rates and improving shape, and a system based on economic weights is the only way to achieve the best outcome in economic terms. Figure A3 in the appended paper (Appendix 1) taken from actual observations by Ward et al. (2005, Chapter 10) indicates how selection by economic weight can optimise the outcome over several traits. Adoption of different breeding strategies: The paper includes a theoretical demonstration of the effects of varying breeding strategies (see Figure A4 in Appendix 1). In that example, the variables are method of selection and numbers of families, but other variables such as best mate allocation (looking at economic weights, breeding values and inbreeding effects), population sizes, population structures, mating designs, number and nature of traits under selection, selection strategies and strategies to manage inbreeding – could all be subjected to similar evaluation using knowledge of theoretical genetics. Such a study could then be extended to consider the infrastructure required for different breeding strategies (e.g. hatchery requirements and farm grow-out requirements), evaluating the practicality and cost of each and finding the balance between genetic gain and a workable selective breeding program. Breeding program systems: An advanced selective breeding program involves assembling large amounts of data and regular, complex calculations of a variety of genetic parameters such as breeding values, economic weights, and predicted levels of inbreeding. This needs to be done on a regular basis (perhaps annually) and in a timely manner, and to do this, systems need to be developed that can be used operationally by ASI. Some of these systems would be generic systems with application across the whole of the aquaculture sector. It was acknowledged that the current ASI database was an important achievement, but it was noted that it required modification to incorporate automatic manipulation of the data in this way.

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Selective breeding programs in other industries have developed systems for this purpose and could be a source of information, but as part of the breeding strategy development ASI would first need to specify exactly what is required. Development of technical protocols: The discussion paper states that research is needed to develop a low cost, non-destructive procedure for measurement of oyster condition. Also, improved broodstock management systems are necessary to ensure the availability of elite animals to hatcheries and efficiency in their use. Strip spawning, for instance, is wasteful of a valuable asset.

5.1.3 Discussion: Enhancement of the selective breeding program

Demonstration of genetic gains achieved year by year was raised as an additional need for the program. Dr Li suggested a modern breeding program should spend about 10 to 20% of its breeding effort in demonstrating gains achieved against a control population. This comparison is required to further refine the breeding strategy in a timely manner, especially at the early development of the program. There are a few methods for establishing and maintaining control populations in other breeding programs with aquaculture species. Without this, it is difficult to get close comparison between successive generations because ‘controlled’ observation is not easy to arrange. Mr Parkinson said that he intended to establish a Technical Advisory Committee at the grower/hatchery level and that this group might be used to address this problem. It was also suggested that the development of economic weights might be difficult because the growers might not have good estimates or records of income and expenditure on the cost/values associated with some farming activities, or if they have they might not be prepared to divulge the information. The market signals for some traits such as meat quality and shell shape are not clear. In addition, standardisation of income and expenses across the industry might be very difficult due to the diversity of the farming techniques used in the industry. Dr Li said that his team had been working on establishment of economic weights in abalone, and that some of the methodology might be useful if economic weights were to be established for oysters. Consideration of these submissions led the participants to realise that for appropriate comparison, the various alternative genetic technologies needed to be considered alongside the ASI breeding program as it could be with further research input, rather than as it currently presents. The term “enhanced selective breeding” was therefore adopted. It was accepted that with the adoption of improvements including the use of economic weights, variation of breeding strategies and development of improved breeding program systems and technical protocols, an enhanced selective breeding program could be expected to at least double the rate of genetic gain achieved by the current ASI program. For example, the current program’s gain per generation is estimated at 3-4% for selection on shape and growth. This could be increased to 6-8% in an enhanced multi-trait selection program The Project Team agreed that enhancement of the type recommended by Dr Kube would require research input to identify the various parameters involved and the best strategies, systems and protocols to be adopted. However, the enhanced technology is already established, proven and quantified in terrestrial species and therefore the risks of adaptation to the oyster industry are minimal and the benefits are assured and substantial. The R&D costs are estimated at $647 K for a 3 year program. Some of the beneficial outcomes would be available to begin modification of the breeding program within eighteen months.

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5.1.4 IP Issues

The combined Management Agreement and License Agreement between the parties to FRDC 2000/206 (Ward et al. 2005) had originally raised some IP issues in relation to the ASI Breeding Program. However, it was reported that the issues had now been resolved so that none remained. The concepts identified for the enhanced selective breeding are all concepts that have been applied in other industries and there are no impediments or IP issues.

5.1.5 Conclusions

Moderate developmental cost, relatively short developmental time and assured successful application with quantifiable benefits across the entire Pacific oyster industry make enhanced selective breeding the technology of choice out of those reviewed within this project. ASI determined accordingly that this Final Report will recommend enhanced selective breeding as the highest priority for its genetic research. A successful project proposal entitled “Enhancement of the Pacific oyster selective breeding program” was lodged with FRDC in December 2005, with a project start date of July 2006.

5.2 TRIPLOIDY

Mr Pugh and Mr Kent led this discussion.

5.2.1 Introduction

A triploid oyster has three sets of chromosomes in every cell instead of the normal two sets in a diploid animal. When fertilisation takes place externally, as in oysters, there are opportunities to manipulate the numbers of chromosomes. In aquaculture, triploidy is used routinely to induce sterility and increase growth rates. Triploid oysters can be produced either by:

• Direct chemical/ pressure/ heat manipulation of diploid zygotes to produce triploid progeny (referred to as “direct technologies” hereafter) or

• Fertilisation of gametes from a tetraploid with the gametes from diploid broodstock. Currently the tetraploid broodstock are produced by manipulation of the eggs from triploid adults (which have been produced via one of the direct technologies).

The direct technologies produce commercial triploids immediately but the percentage recoveries of triploids has been variable at 90% or lower (e.g. Eudeline et al. 2000) whereas triploid progeny developed by the tetraploid route2 are said to be recoverable at levels close to 99%. Because the tetraploid route requires two maturation phases – first to grow out the triploids and then to mature the tetraploids – at least four years (and more likely 6 years) would elapse before the final tetraploid/diploid cross could be undertaken.

5.2.2 Patented technology

Triploid production via the tetraploid route is currently taking place at a commercial level under license at Shellfish Culture Limited (SC) in Tasmania using the method of Ximing Guo and Stan Allan which is patented in many countries including Australasia. It has been licensed through Rutgers University to a biotechnology company called 4Cs Breeding Technology Inc.

2 A tetraploid oyster has four sets of chromosomes, instead of the normal two sets.

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4Cs has the right to license to people or companies regarded as their partners and SC is the Australasian partner. The patent was filed in the US on 20 October 1998 (Patent Number 5,824,841). It cites a Foreign Patent Document 92106666X filed in China in August 1992, and lists 26 references relevant to ploidy manipulation. The patent document provides details of methods (used to produce tetraploids) which are to be protected’ and includes the following summary: “According to the present invention, the first polar body in the eggs of a triploid mollusc is manipulated so as to produce a viable tetraploid mollusc (shellfish). Typically, eggs are obtained from a triploid female by dissection and thereafter rinsed with filtered sea water. The eggs are fertilized with sperm obtained from a normal diploid male. At a suitable point in time after the fertilization of the eggs (5 minutes post-fertilization, for instance), a process for blocking the release of polar body 1 from the eggs is carried out for a suitable length of time. Subsequently, the eggs are incubated under standard hatchery conditions. In a preferred embodiment of this invention, said process for blocking the polar body I (PB1) is carried out by treating the fertilized eggs with cytochalasin B (CB) or other blocking agents dissolved in filtered sea water at a suitable concentration, but the process may also be carried out by administering a thermal or hydrostatic shock to the fertilized eggs. By virtue of this invention, viable tetraploid shellfish which can grow to maturity are produced.” Details of the above process are illustrated schematically in Figure 1. Interpretation of the patent document appears to be that any technique for tetraploid production which relies on suppression of the first polar body formation in the eggs produced by a triploid is protected whereas production of tetraploids by any other means (and therefore the subsequent production of triploids) would not be protected. However, legal interpretation would be needed if such a view were to be relied on.

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Figure 1: Schematic representation of meiosis with blocking of first polar body formation in the eggs produced by the triploid

The Project Team was aware of information that the French have different opinions on the patent, which suggests that it may not be watertight. In addition, a recent paper describes a ‘new’ method of producing tetraploid Pacific oysters by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males (McCombie et al. 2005). This offers a means of direct tetraploid production, avoiding the intermediate triploid multiplication step. It is fundamentally different to the Guo and Allan method, where the tetraploids are produced by crossing the eggs from triploid females with sperm from diploid males. However, the McCombie method still requires 2n sperm from tetraploid males that currently can only be produced by the Guo and Allan method.

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Figure 2: Production of triploid oysters from diploids via tetraploidy

Eggs from diploid females

Triploid spat

Chemical/physical Treatment

Adult triploid (few gametes)

Eggs from triploid females

Tetraploid spat

Adult tetraploid (many gametes)

Growout – 2 yrs

Triploid spat

Grow - out – 2 to 3yrs

Chemical/physical Treatment

Commercial triploids

Grow - out – 2 yrs

Tetraploid x Diploid

The production of triploid oysters using the Guo and Allan method involves two treatment steps which may be chemical, physical or a combination of the two (Figure 2). The first step is the treatment of diploid eggs to produce triploid oysters. These are grown out, a tissue sample is taken, and their ploidy status is verified using flow cytometry3. A small percentage of triploids develop advanced gonads and produce a few mature eggs. The eggs produced by the triploids are fertilised with sperm produced from diploid males and are then chemically treated to produce tetraploids by preventing the formation of the first polar body. Once the tetraploids are grown to maturity it is a simple process to produce commercial triploids using a tetraploid by diploid cross. SC considers that this technology for the production of triploid oysters is now proven, delivers 99% recovery of triploids and that there is no need for additional development or special skills. If ASI improved broodstock were to be introduced into the SC program a lead time to commercialisation of selectively bred triploids can be estimated at 8 years, or more if the 2.5 years required for the usual ASI pre-commercial testing is included. However, although there is evidence for Sydney rock oysters that a combination of triploidy and selective breeding would be additive (Hand et al. 2004; Nell. 2006) SC triploidy program remains independent of the ASI selective breeding program. At present and for the foreseeable future, the SC license is exclusive and the company does not intend to introduce ASI broodstock into its program. ASI

3 Flow cytometry is a fast and accurate method to measure the DNA content of a cell. Cell suspensions are stained with a fluorescent dye which binds to the DNA, and then the amount of fluorescent material is measured by passing the sample through a light beam.

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should therefore keep a watching brief on alternative methods for tetraploid induction which are outside of the patent protection and may become available for use in the future. Mr Kent warned that if ASI thoroughbred oysters were introduced into the SC triploidy program, the tetraploids developed might not have the same characteristics as the original thoroughbred.

5.2.3 Alternative Techniques for Triploid Production

In some species (such as salmon) triploids are routinely induced directly in populations intended for commercial grow-out and the two-stage process patented by 4Cs Breeding Technology Inc. is not necessary. This methodology usually involves applying temperature or pressure treatments to fertilised eggs and obtaining a very high proportion of triploids. If a ‘direct’ alternative to the patented technology such as this was available, the lead time to production of thoroughbred triploid oysters would be considerably reduced. There are a number of alternative fully developed technologies for producing triploids involving chemical, pressure and/or temperature treatment. The main issues preventing their commercialisation are variations in eggs characteristics which result in unacceptable proportions of diploids amongst the triploids. One US company (Hilton’s Coast Seafood Company) is known to be producing triploids at an unknown but commercially acceptable level using a process including temperature, but no details are known and the company is unwilling to divulge them. The Project Team considered that recovery of 90-95% triploids would be commercially acceptable and suggested that development of an alternative (or optimisation of an existing) ‘direct’ technology for triploid production would be a worthwhile research project. It is expected that the development of the direct triploid technologies would take an estimated three years and total research expenditure of perhaps $240K to achieve. If successful, the assimilation of broodstock from the ASI selective breeding program into a triploid program would be straightforward. This technology could be integrated with the ASI selective breeding strategy according to commercial demand. Theoretically, the treatments available for producing triploids could also be used to produce tetraploids directly (i.e. not via triploids) from eggs collected from diploid females and these techniques could possibly be adapted so as to differ from the technique patented by Guo and Allan. Gametes (sperm or eggs) from tetraploids could then be used to fertilise gametes (eggs or sperm) from diploids to produce triploid progeny outside of the patent protection.

5.2.4 General considerations

Positive attributes There is adequate NSW literature to substantiate that triploid Sydney rock oysters are on average 31% (range 6-99%) heavier when compared to diploids (Hand et al. 1998a). One wide scale field trial demonstrated mortality rates of only 12.2% for triploids compared with 35% for diploids when challenged by winter mortality Mikrocystis roughley (Hand et al. 1998b). The triploid performance in Pacific oysters is described by (Nell and Perkins 2005). This study also record better growth rates and lower mortalities than in diploids although unpublished observations by Kent (pers. comm. 2005) suggest that this better performance might vary with environment, and others have observed that triploid growth seems to be good in reasonably productive areas but less so in areas of poorer productivity. Mr. Kent said that in his experience, triploid Pacific oysters grow 15 – 20% faster than diploids. The advantages of triploid oysters centre on the suppression of spawning. This allows the sale of finished triploids year round, whereas diploid oysters go through a spawning period during

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which (and for some time afterwards) they are unsaleable. Production of triploids on the farm is therefore less seasonal, with better prices during the diploid spawning season and benefits for farm cash flows. The alteration in flavour and texture associated with pre-spawning diploids is also absent and there are opportunities for introduction of triploid Pacific oysters into NSW and other areas of environmental concern where diploid Pacific oysters are not accepted. Negative attributes and risks A major disadvantage of triploids is discoloration of the meat, or ‘brown staining’ (Nell and Perkins 2005). This has been widely observed in triploid oysters, particularly in the warmer months (Nell 2002, Maguire et al 1994 and Hand & Nell 1999). The discoloration manifests as distinct patches, pale brown to dark brown in colour, which can cover over 50% of the gonad surface. It can have a severely adverse effect on marketability. Discussion within the project group indicated that its cause was not known and might possibly be associated with environmental, seasonal and/or genetic factors. This was identified as an important area for research. Dr Nell said that triploid Pacific oysters have an enormous adductor muscle which might impart a different taste to the oyster and which might be objectionable to consumers. NSW does not attempt to evaluate taste factors, but the group felt that it would be worth consulting commercial food testing agencies to find out what might be involved and how practical this would be. Anecdotal evidence suggests that under some conditions triploid Pacific oysters are less robust than diploids and that they require good levels of nutrition to perform well.

5.2.5 Conclusions

No risks were identified in relation to the use of triploids by industry, but if research to develop an alternative to the tetraploid method was supported, there is a risk that such research might prove to be more costly than the project group anticipates. There was a consensus amongst the project group participants that the better performance of triploid oysters under some circumstances (compared with diploids) will result in increased demand for triploid stocks in Australia and that an alternative to the Guo and Allan method should be pursued to avoid monopolisation of triploid production. However, alternative technology has not yet been refined to a commercially acceptable level. ASI would lend support to a hatchery initiated research program to develop and refine an alternative triploid production technique incorporating improved broodstock. If such a commercially acceptable methodology was established ASI would cooperate with hatcheries to performance test the product, and would integrate triploid production with the selective breeding strategy according to commercial demand. High pressure and/or temperature induction of triploidy was considered by the group as the highest priority for research development after Enhanced Selective Breeding. The Review Team formed this opinion taking into account:

• The fact that the basic technology was already established.

• The costs of fine tuning and development of a technique was likely to be relatively modest (estimated as less than $100 000 plus $140 000 pre-commercial phase costs).

• Establishment of an industry monopoly on triploid production was undesirable.

• A view that demand for triploids was likely to increase in the future.

Brown staining and the possibility of increased sensitivity to stressors in triploid oysters were also identified as worthwhile areas for future research.

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5.3 DOUBLE HAPLOIDY (DH)

Dr Li led this discussion with contribution from Dr Davies and Mr Kent.

5.3.1 Double Haploidy in Plants (Dr Davies)

Dr Davies said that DH use in the plant industries was at an advanced stage and widespread whereas in oyster breeding it remained at a very early stage. Plants present some similarities and a range of differences by comparison with oysters. In wheat crops, the male and female elements are present in the one flower, self pollination is the rule and crops are highly homozygous to begin with. On the other hand maize is more nearly analogous to the animal situation – it is naturally outcrossing so that the male and female DHs are genetically distinct and can be crossed with good effect to produce high performing hybrids. He said that in heterozygote populations the DHs exhibit some reduction in fertility, but he is not aware of any plant situation in which this is extreme. In a typical plant scenario it is possible to examine up to 20 000 or so plants each year and amongst these to detect perhaps 5000 DHs. In the case of barley for instance, using the male (pollen) as a starting point, about 5% of these DHs would go on to produce seeds. When DH is used in the plant industries the anticipated performance gains are roughly the same as for conventional selection, but the gains are achieved more quickly and efficiently. In any one plant species about 20-30 traits might be earmarked for improvement but these are not tackled simultaneously. Typically, improvement takes place over perhaps 20 years. Seed production companies vary in their attitude to the use of DHs for onward production of commercial seed. Some use 100% DHs, but others commit only a proportion of their production to DHs because some feel that there might be an associated risk. Dr Davies explained that in plants there were differences in applying DH techniques depending on whether the species was self pollinated (e.g. wheat, barley, rice), or cross pollinated (e.g. maize, rye). Within each of these basic groups a variety of strategies was available. In self pollinated crops DHs can be produced by manipulation of gametes from a donor plant. The donor plants (F1) are characteristically derived from a cross between two different varieties and the DHs resulting from manipulation of these F1s are each genetically different, but each is true breeding. The DHs are then multiplied and performance tested to identify those carrying a beneficial array of traits. A plant having 10 beneficial independently inherited traits would be expected at a frequency of 1/1,000 compared with 1/1,000,000 if DH were not used. If markers were available for each of the 10 traits and marker assisted selection (MAS) was applied to the donor parents, this frequency would be reduced to 1/50. Dr Davies said that MAS is routinely used in plant breeding. In cross pollinated crops an opportunity exists for producing “synthetic” varieties. A number of DHs are produced and multiplied by self pollination, resulting in a number of DH lines. Selected DH lines are then intercrossed and each cross produces a true breeding “variety”. These synthetic varieties would then need to be tested and the best performers identified for ongoing use. This is analogous to the situation which would apply to oysters where DHs would be crossed to produce lines (i.e. varieties) which are then performance tested to identify those suitable for commercial development. Dr Davies pointed out that plant “varieties” produced by DH are different to the varieties in some other species such as strawberries and peaches where the variety is actually a clone.

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In the plant field there is enormous investment in trialling hundreds of varieties each year, from which perhaps only one will come through every 4-5 years for commercial use. If a trait is controlled by a large number of genes (e.g. ~50) it takes a great many trials to identify the few with commercial potential. Dr Davies said that DHs selected on their phenotype may not produce the best crosses – sometimes the results are surprising. He also said that so far DH techniques have not been developed for legumes such as lucerne, because the industries are not big enough to warrant the research expenditure which would be needed.

5.3.2 Technical Explanation of Double Haploidy (Mr Kent)

Mr Greg Kent gave this presentation based on his University of Tasmania Masters research on DH in oysters. He said that homozygous oysters can be produced either by chromosome set manipulation or by breeding between relatives. With chromosome set manipulation the progenies produced by inhibiting meiotic division(s) are called meiogens while those produced by inhibiting mitotic division are referred to as mitogens. If the DNA in a progeny is derived from the female side only (DNA from the sperm having been artificially denatured) this progeny is called gynogen. If the DNA in a progeny is derived from the male side only (DNA from the egg having been artificially denatured) it is called androgen. According to the developmental stage (meiotic division or mitotic division) at which the chromosome set manipulation is applied, gynogens and androgens in molluscs can therefore be further defined as meiotic or mitotic gynogens and androgens, respectively. Meiogens are homozygous to some extent, but not double haploid. The true double haploid is a mitogen, with complete homozygosity. In the case of a mitotic gynogen, the male DNA is destroyed but fertilization proceeds and the mitogen is then derived by suppression of the first cleavage. The remaining DNA then replicates, thus restoring the oyster to a diploid state. The end result of these manipulations is an oyster that has two identical sets of chromosomes, as opposed to normal diploid oysters that have maternal and paternal derived chromosomes. Because DH oysters have two identical sets of chromosomes they will be homozygous at every locus (gene) and therefore may impart the following advantages to a selective breeding program: Traits can be fixed: Because any crossing-over during meiosis will exchange genetically identical material, traits can be fixed. By contrast, when normal diploid oysters undergo meiosis to produce gametes (eggs and sperm) crossing over of the maternal and paternal chromosome pair occurs and genetic information is exchanged between chromosomes so that variability is induced in the offspring. Clonal lines of good parental stock may be produced: The process of producing DHs results in the removal of deleterious genes which would otherwise lower survival of stock on-bred from the double haploid lines. In addition, activating (fertilizing without the addition of sperm DNA) eggs from animals which are already double haploids and then doubling their chromosomes, or fertilising eggs with the sperm from the same double haploid individual, would allow the production of clonal lines genetically identical to the parents. Highly outbred crosses can be made: Completely inbred (homozygous) double haploid individuals can be genetically typed (using micro satellites or allozymes). This information can then be used to select genetically dissimilar stock for crossing. The greater the genetic dissimilarity between broodstock the greater is the possibility of producing heterosis (hybrid vigour) in the offspring. Heterosis has been shown to impart growth and/or fitness advantages

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in some species (although the heterosis advantages over outcrosses in oysters need further investigation). Crossing two double haploid parents can potentially produce lines of identical outbred offspring: Not only would the offspring have a high degree of heterosis but individuals would be genetically the same as each other, thus reducing the potential for variation between siblings. Breeding lines can be protected: Offspring produced by mating two double haploids, although genetically the same as each other would be heterozygous. Any further production from these offspring cannot regenerate the homozygous parental stock. Mr Kent reported having explored various physical methods for the destruction of DNA in oyster sperm including radiation with ultraviolet light, X-rays and gamma rays. Diploid status can be restored by pressure, temperature, and chemical treatment (cytochalasin B (CB), 6-dimethylaminopurine (6-DMAP), and colchicine). When the first mitotic cleavage is targeted the resulting progeny will be DH. Summarising his results, Mr Kent said that:

• The optimum ultraviolet (UV) intensity and duration for denaturation of sperm was 3-5 minutes @1200uWcm-2.

• The optimum (but unreliable) UV intensity and duration for denaturation of eggs was 10-15 minutes @1200uWcm-2.

• CB had greater efficacy than 6-DMAP in suppressing both polar bodies and 1st cleavage.

Dr Li added that the use of DH could accelerate progress of a breeding program towards particular goals. While conventional selection would take 10–12 generations before offspring would breed true to a preselected type, the use of DH would achieve the same objective in about one generation although the process would have to proceed over 3-5 years to allow for performance testing and multiplication of the selected lines, before the lines were commercially ready. Purification of genes takes place at a much faster rate in the DH program by comparison with traditional selection. Also, he suggested that heterozygous clones would show better survival (and reproductive potential – although this is of little significance in the ASI context, where heterozygous clones are not used for breeding) due to masking of deleterious recessive genes that are expressed in homozygous clones. In addition, clonal lines allow more accurate selection for all traits, particularly those requiring destructive measurement.

5.3.3 The SARDI Oyster DH program (Dr Li)

Dr Li led this discussion based on his work with FRDC Project 2002/204: “Development of the Techniques to Produce Homozygous Pacific Oysters” (Li et al. (in preparation)). The first phase of this project had been to determine the genetic loads of Pacific oysters farmed in Australia to see whether they were appropriate subjects for DH manipulation. The effective number of lethal genes in each oyster was estimated from the relative survival of young oysters in selfed families relative to families from the same eggs but fertilised with mixed fresh sperm. The data for survival to days 10, 15 and 20 were used to test for significant variation between oysters, using the three time intervals as blocks in a randomised block ANOVA (see Table 1). Results from this work indicated that the genetic loading was high but also variable between individuals so that in some individuals the load was low enough for ongoing work to proceed.

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Two oysters (A013 and A089) consistently produced high genetic loads and two (A085 and A130) produced low genetic loads. The overall mean (based on the 4 means) is 12.6 lethal equivalents (standard deviation of 5.78). The 95% confidence limits on the true mean are 3.4 to 21.8 lethal equivalents. This estimate is consistent with published estimates (about 12). Dr Li said in the 2003/04 oyster spawning season, he then proceeded with the second phase of the project, which was to optimise the technologies for producing pure and/or inbred lines. Based on the best evidence available at the time Dr Li chose to use 7 markers to test the resulting ‘double haploid’ larvae for homozygosity. He provided an example of these tests (Table 2). The table details the results from a sample from a pool of about 5000 larvae, all of the same family. Dr Li says that this was the best result from a number of similar tests performed on different families produced by different technologies. About 1000 of the original 5000 metamorphosed initially but only 5 were left when they were eventually sent to the SA Oyster Hatchery. Table 1: Estimated number of lethal equivalents at 3 sample times

Oyster Day 10 Day 15 Day 20 Mean estimate A013 16.2 18.0 20.6 18.3 A085 3.1 9.4 8.0 6.8 A089 14.4 15.7 20.5 16.9 A130 7.3 9.3 9.0 8.5

Because the condition of the broodstock used in this study were highly variable, attempts to produce DHs resulted in very high variations in fertilisation rates and survival. There were also heavy losses due to bacterial contamination throughout the experiment but especially at metamorphosis. Of the 33 families produced only 5 were kept, with the number of spat per family ranging from 1~150. The spat remaining were kept at the South Australian Oyster Hatchery, and subsequently at the Zippel Enterprises farm at Smoky Bay S.A along with a number of other unrelated experimental groups from SARDI. There were about 250 of these “DH” spat in total originally. In December 2005 all the experimental oysters, perhaps including some DHs, were recovered from the Zippel Enterprises farm by Dr Li. However, most had lost their identification and only 2 or 3 could be tracked back to the “DH” family to which Table 2 refers. Had the group been identifiable they would have been mature enough to test and could have provided valuable information about DH survival and fertility. Mr Kent questioned Dr Li’s interpretation of the tests for homozygosity. In the best of the results from FRDC 2002/204 only 4 of the 9 larvae tested were judged to be homozygous and within these none could be shown to be completely homozygous because the test could not be completed on all 7 markers. The four presumed homozygotes could be assessed as 100% homozygous, but since the test could not be completed on all markers this assessment is based on only 4 to 5 loci. Others agreed with this summation and the Project Team concluded that if double haploidy was to be developed, testing of the preliminary technology would need to be repeated.

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Table 2: Homozygosity of the larvae (9 days old) produced by double haploid techniques in one family

Individual Marker and allele Cg49 CgTD01 CgTD02 Cg108 Cg44 L16 CmrCg02

A B A1 B1 A2 B2 A3 B3 A4 B4 A5 B5 A6 B6

Mother A B A1 B1 A2 B2 A3 B3 A4 B4 A5 B5 A6 B6 Larva 2* A A x x B3 B3 B4 B4 A5 A5 x Larva 3 x x x x A4 A4 A5 B5 A6 A6 Larva 7* B B x A2 A2 x A4 A4 A5 A5 B6 B6 Larva 8* A A B1 B1 x x B4 B4 A5 A5 A6 A6 Larva 9 A A A1 A1 x x A4 B4 A5 B5 x Larva 12 A A A1 A1 D E x A4 A4 A5 B5 A6 A6 Larva 13* A A A1 A1 x A3 A3 A4 A4 x x Larva 14 B B A1 A1 D E A3 A3 B4 B4 A5 A5 F B6 Larva 15 A B A1 B1 A2 B2 A3 B3 A4 B4 A5 B5 x * = Homozygous larvae, x = no result To establish absolutely that an organism is DH, it is necessary to demonstrate that each gene pair is homozygous. This is impossible in practice. Instead, geneticists rely on demonstration of homozygosity in a number of pairs of identifiable genetic sequences, or “markers”. A proportion of animals will by chance be homozygous for one marker, but if more than one marker is used and each is homozygous, the conclusion that the animal is DH becomes more reliable. The number of markers used in any series of tests such as those detailed in Table 2, is therefore critical to the interpretation of the test. While homozygosity across several markers may be adequate for identifying the number of “DH” larvae in a treated population, the identification of individual DHs from which DH lines are to be bred is more critical and would warrant the use of a greater number of markers. The Project Team considered the number of markers that should theoretically be used, and recommends the following. As a minimum, 10 markers should be used It is important that these markers be spread over all linkage groups (chromosomes) for a valid test. In addition, it is important that tests are done on different families and that the markers are independent. Data is publicly available to select markers that cover different linkage groups (for example, markers have been produced by Hedgecock’s group in the USA). The rationale for selecting this number of markers was based on the number of chromosomes, which is 10 pairs for Pacific oysters. The technique, timelines and adaptation of DH to selective breeding Dr Li indicated that he felt at this stage DH should be used as an occasional technique to fix a particular trait(s) and/or to produce uniform stocks, rather than as a continuing process alongside the selective breeding program. The procedure involved in applying DH techniques to oysters was not well understood. It required considerable effort from the group to clarify this. Dr Li indicated that the procedure could be considered as two phases: Phase 1: Production and selection of DH parents. This would involve the production of approximately 600 DH progeny from 15 DH families. Ten percent of these would be expected to survive to 2 years, and from them 10 males and 10 females would be selected. This step would take 2 years (from years 0 to 2).

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Phase 2: Performance testing of crosses between DH parents and replication of DH parents. This would involve making selected crosses between the 10 males and 10 females and performance testing the families to determine which combination of DH parents produces the best progeny. At the same time this is happening, the 20 DH parents would need to be replicated (effectively cloned) so there were sufficient numbers of DH parents for commercial spawning (say, more than 500 per parent). This step is expected to take 2 additional years (years 2 to 4). Commercial spawnings would then be done by crossing between the best parents identified as part of the performance testing. The detail of each phase is described in Figure 3, based on a possible (theoretical) scenario. Figure 3: Steps required for application of double haploid techniques to Pacific oysters – expected scenario

PHASE 1:Produce DHs

Step 1 - year 0

Step 2

Step 3

Step 4 - year 2

PHASE 2:Test

Step 5

Step 6

Step 7 - year 4

COMMERCIAL PRODUCTION

Select parents(30 diploid females)

Induce DHs

Growout DHs(approx 15 families by 40 individuals)

Select best DH's(10 male and 10 female)

Select best crosses and produce at commercial scale

Replicate all DH's(20 selections by >500 each)

Cross between selected DHs

Grow out families(between 40 - 90 families)

Grow out DH replicatesMeasure performance and

identify best crosses

If, in phase 1 at Step 3 there were insufficient families containing 100 individuals (including an estimated 40 DHs) per family, it would be a simple matter to select additional diploid females and treat them without loss of time until the required numbers at Step 3 were obtained. The attrition in numbers of double haploids between Steps 3 & 4 reflects an anticipated loss of DHs as they mature to 1 year old. At phase 2 performance testing proceeds to identify the best crosses of double haploid families, starting at year 2 and ending at year 4. At this stage problems could develop if the fertility

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levels in the double haploids are low. However, assuming satisfactory levels of fertility in the DH’s, taking 10 families as the starting point, and discarding families on the diagonal and reciprocal crosses, it would be possible to make 45 separate crosses. Using the formula [No. of crosses = n(n-1)/2 where n is the number of families] the crosses available would increase from 66 to 91 as the number of families rises from 12 to14. The number of families used would therefore be determined by the performance testing logistics, especially if multiple test sites were used. The Project Team noted that raising this number of families was well above the current capacity of ASI, particularly since these families would be in addition to those that need to be raised for the selective breeding program A number of uncertainties were expressed in relation to Dr Li’s models. The commercial breeding program used by ASI requires a pool of 500 broodstock from each line to ensure that adequate broodstock numbers from favoured lines can be retained for use over several years. The model outlined, as adapted to the needs of a commercial breeding program, therefore requires the production of large numbers of oysters (in the order of 5000 to 10 000) using the techniques of gynogenesis, androgenesis and/or selfing and there were doubts about how robust these techniques would be under commercial conditions. Some members of the Project Team felt that extra research and development would be necessary to bring these techniques to an operational scale. Dr Li thought the risk that these techniques would not work was minimal. He acknowledged that gynogenesis and androgenesis had never been attempted on an operational scale in oysters but drew attention to extensive experimental use of these techniques in Pacific oysters (Guo et al. 1993; Li and Kijima 2006) and various finfish species (Babiak et al 2002; Kirankumar et al. 2003; Bertotto et al 2005). In addition, cloned hirame breeds which show superior characteristics in disease resistance and/or high growth rates have been established and commercially applied in Japan (Yamamoto 1999). Dr Li expressed confidence that both techniques were feasible in oysters because the process was similar to, but easier than the process of producing double haploids which he now considered proven to the larval stage. The alternative, selfing, has been shown to be effective in experimental observations where animals were available which changed from male as 1-2 year olds to female in later years. However, in a double haploid program where the need may be to replicate “lines” consisting of only a few double haploid individuals this sex transition might not always be available, although Dr Li suggested that in the future techniques for artificial sex reversal might be developed in oysters. Further uncertainties were also expressed and these were as follows:

• What is the survival to maturity of DHs and what is their capacity to breed?

• From how many selected diploid animals should these DHs be derived?

• Might they all be of one sex?

• Is it possible that all performance tested DHs might be inferior to the best conventionally selected lines – or alternatively, is it safe to assume they will be better?

• How will the improvement in the DH commercial product compare with the improvement in the conventionally selected stock over the same period?

5.3.4 General Discussion

It was generally agreed that improvement through the use of DH technology was potentially quicker but riskier than the use of intensive inbreeding which provides a progressive and more gentle build-up of homozygosity and allows the purging of deleterious genes in a gradual way (rather than suddenly as in DH). The potential for loss of useful genetic variation is therefore greater than with intensive inbreeding. There was discussion as to what constituted ‘proof of concept’ in relation to the oyster work undertaken by Dr Li. He felt that this work had already established proof of concept in so far as testing of several groups of larvae had shown that some individuals were homozygous for the

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markers used. However, the Project Team was of the view that this original work was insufficient to establish proof of concept except tentatively and in relation to demonstration of a research technique only. To support a case for developmental research of the DH technique it was at least necessary to demonstrate capacity to produce a substantial number of DH larvae which survived until 2-3 months of age. From ASI’s point of view the proof of concept needs to extend beyond this to demonstrate DH survival to maturity with a capacity to produce viable gametes. It is clear that the DH technology is still at the early research stage in oysters. The concept has been proven, if at all, only at a very basic research level. Work to establish that DH larvae can be produced in reliable quantity, and that these can mature and breed has not yet been done. Participants expressed concern that efforts were not made to move toward this point by more careful management of the experimental oysters which were residual from FRDC Project 2002/204. At least four years of further research is necessary before a commercialisation program could be considered. A further 2.5 years would elapse before double haploid oysters were then available for commercial use, but substantial benefits could be available from the technology at that point.

5.3.5 IP issues

Intellectual property issues are associated with this technology. SARDI sees ASI as a vehicle for the commercialisation of DH technology and has sought to establish a contractual agreement with ASI (which included royalty payments) for its use. SARDI have said that income from royalties would be recycled back to fund further industry research. However, ASI is not ready to consider such an agreement until proof of concept to a commercial level can be demonstrated.

5.3.6 Conclusions

It has not been established that DH larvae can be produced in reliable quantity, and that a satisfactory percentage of these can metamorphose, mature and breed. As Dr Davies indicated, in some plant species only about 5% of DH individuals go on to produce seeds. This is of little significance in plants where it is possible to test many individuals within each DH family, but similar (or even lower) levels of fertility in DH oysters could pose a serious obstacle to adoption of the technology. An estimated $1265 K expenditure and at least 6.5 years R&D work are needed before DH oysters could become available as part of the ASI breeding program. While ultimate benefits from DH might be substantial in the long term, the attainment of these must be regarded as uncertain. However, in view of the potency of this technology in some areas of plant breeding, the project group was prepared to assign a moderate priority for further DH research at least to the stage of demonstrating production of adequate numbers of double haploid spats and their survival to 2-3 months of age. Research costs to this level were estimated at $92 000 using 6-10 markers.

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5.4 INTENSIVE INBREEDING

Dr Ward led this discussion, based on his 2003 FRDC project application “Hybrid vigour and inbreeding in Pacific oysters”.

5.4.1 General overview

It was decided that the technology previously referred to as “hybrid vigour” in the application for this project would be better described as “intensive inbreeding” with the consequence that hybrid vigour might occur.4 It involves the production of highly homozygous lines and their eventual outcrossing in the expectation that hybrid vigour will result. Dr Ward proposed (Ward 2003) the production of homozygous lines by ‘selfing’ which involves the cryopreservation of sperm from a particular male which is then used to fertilise eggs of the same animal after it has changed to female in the following years. Not all oysters change sex in this way, but the proportion which do so is high enough that the ‘selfing’ of lines (as distinct from individuals) is a reliable technique for increasing homozygosity. This technique accelerates the normal inbreeding process, which is crossing between close relatives. Dr Ward proposed that homozygous lines produced in this way could be outcrossed in an effort to demonstrate and quantify hybrid vigour effects. Hybrid vigour is a long recognised phenomenon in animal and plant breeding. The opposite to ‘inbreeding depression’, it comes about from crossing genetically differentiated inbred or purebred lines with the consequence that performance of the outcrosses is improved. Dr Davies said that clear species differences in the expression of hybrid vigour are recognised in plants. For instance, hybrid vigour is not seen at all in wheat whereas there are strong hybrid vigour effects in maize. He therefore warned that the occurrence of hybrid vigour in oysters should not be assumed. There is uncertainty as to whether the phenomenon occurs in oysters, but research interest in this area is intensifying, particularly in the United States where published work (Hedgecock et al. 2005) claims to have demonstrated hybrid vigour. These studies have focussed exclusively on growth rate. However, in the view of the review panel, there is uncertainty as to whether the phenomenon occurs in oysters. The technique, timelines and adaptation to selective breeding Figure 5 indicates the way in which intensive inbreeding through selfing might be achieved and how it might be integrated within a selective breeding program. Contact with Hedgecock (pers. comm. 2005) in the US and examination of some of his papers (Hedgecock et al. 2005) reveals that the hybrid vigour US workers claim to have demonstrated is a comparison between the growth of outcrossed animals and that of the inbred lines from which they were bred. Therefore the hybrid vigour measured in these trials could be an expression of escape from inbreeding depression rather than true hybrid vigour. The level of inbreeding in their work was not high; however, inbreeding depression in Pacific oysters is known to be significant even at relatively low levels (Evans et al. 2004). In addition, the reported hybrid vigour appears to be occurring at very low frequencies. Out of 100 outcrosses in the US program only one line has demonstrated substantially better performance in the field than comparable commercial lines (Hedgecock. pers. comm. 2005). The Americans are investing heavily in this area of research and it deserves to be explored more thoroughly, but at this stage it appears hybrid vigour in oysters is a concept yet to be proven.

4 This was to avoid confusion with hybrid vigour associated with interspecies crosses.

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Figure 4: Intensive inbreeding compared with conventional selective breeding

Selective breeding program Inbreeding and Hybridisation

Select lines

Self inbreed lines

Commercial lines

Repeat cycle

Year 12

Year 1

Year 4

Year N

Year 2

Year 3 Self inbreed lines

Performance test lines (2.5 yrs)

Produce array of crosses

*Assuming 3 years for each “selfing” cycle and 3 cycles

5.4.2 General considerations

It was accepted that if it were adopted, intensive inbreeding would need to be associated with an ongoing selective breeding program. Contact with Hedgecock (pers. comm. 2005) in the US and examination of some of his papers (Hedgecock et al. 2005) reveals that the hybrid vigour US workers claim to have demonstrated is a comparison between the growth of outcrossed animals and that of the inbred lines from which they were bred. Therefore the hybrid vigour measured in these trials could be an expression of escape from inbreeding depression rather than true hybrid vigour. The level of inbreeding in their work was not high; however, inbreeding depression in Pacific oysters is known to be significant even at relatively low levels (Evans et al. 2004). In addition, the reported hybrid vigour appears to be occurring at very low frequencies. Out of 100 outcrosses in the US program only one line has demonstrated substantially better performance in the field than comparable commercial lines (Hedgecock. pers. comm. 2005). The Americans are investing heavily in this area of research and it deserves to be explored more thoroughly, but at this stage it appears that hybrid vigour in oysters is a concept yet to be proven. Each “selfing” cycle would take 2-4 years to complete because the oysters first need to attain maturity (1-2 yrs) and then wait a further 1-2 yrs for the sex change to occur before breeding can be finalised. If 3 cycles are used, the total time could extend to perhaps 9 years or more before the first commercial stock were ready for ASI performance testing. In crop breeding using selfing, where each cycle takes two years it takes 4-6 years before the stock is commercially ready.

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Dr Li said that the selfing technique had been used experimentally 10 times by him and always worked with an average 30% of progeny surviving. Discussion ensued about the complexity of sex determination in oysters, the reliability of sex changes with age and the consequent uncertainty of reliance on selfing. Referring to diploids, Dr Li said that in one of his observations of 130 diploid 2 year old males, 30% had become female by 3 year old. Conversely, of 60 2 year old females, 11 had become male by 3 years old. The Project Team noted however, that while the effectiveness of “selfing” as a technique has been demonstrated on a research basis, the capacity to successfully and successively self fertilise lines in an operational situation and for the oysters so derived to survive and breed remains uncertain. The loss of flavour and palatability in some hybrid strains such as chicken and strawberries was mentioned. It was suggested that development of hybrid oyster lines should not proceed without an eye to these traits and that attention should be given to these attributes throughout the breeding program generally. Positive attributes Intensive inbreeding would achieve a progressive and slower build-up of homozygosity than the sudden change which occurs with double haploidy. The result would be the purging of deleterious genes in a gradual way so that the chance of loss of potentially useful genetic variation might be reduced. The technology would provide a high level of intellectual property protection because homozygous lines could remain under the control of the breeding program and the commercially released crossbreds could not be used to re-create them. No IP difficulties are foreseeable. Negative attributes and risks Slow introduction of beneficial traits into the commercial breeding program (12 years or more) and high cost because of the need to generate and maintain a large number of families over several generations, additional to those generated for the selective breeding program, mitigate against support for this technology. In addition, the cost estimated for a developmental program is at least $1145 (possibly substantially more) and there are a number of risks which are:

• Increased gains through hybrid vigour in oysters can only be assumed and there is a risk that they may not be realised.

• Sex change in inbred oysters may not occur reliably.

• Selfed lines may be lost after 3-4 generations because inbred oysters have a lower genetic fitness.

• The risk that selection for oysters which are suitable for selfing (i.e. those which change sex) might eventually impact adversely on sex ratios.

5.4.3 Conclusions

Because of the uncertainty that benefits would be achieved, the long lead time, high cost and logistical difficulty of this technology the Project Team rated intensive inbreeding as a low priority for developmental research.

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5.5 MARKER ASSISTED SELECTION

Dr Sharon Appleyard led this discussion.

5.5.1 Technical explanation

Marker assisted selection (MAS) is selection of potential broodstock based on laboratory measures for the presence of a particular part of the DNA sequence (called a genetic marker) rather than on physical characteristics of the animal (such as shape, growth rate or disease resistance). For MAS to work, associations need to be established between a particular DNA sequence (the marker) and the commercially important trait (say, disease resistance). To establish these associations, studies are required to ‘map’ the presence of markers on the DNA sequence, take physical measurements of large numbers of individuals for the commercial trait in question, and then test the DNA of that individual for the presence of the marker. Once an association has been established and shown to hold-up over different families, it can then be used commercially by DNA typing of potential broodstock instead of taking physical measurements. MAS studies require highly specialised laboratories, specialised skills, and measurements on large numbers of individuals. The ultimate form of MAS is gene assisted selection (GAS) where the DNA marker is actually within the specific gene. Selection on the basis of an animal’s own performance is never 100% accurate. To overcome this limitation, selective breeders use progeny testing, the rationale being that the performance of the progeny is an indicator of the genes of the parent. In some situations MAS is an alternative technique that allows selection at a young age, sometimes before the desired traits can be observed, leading to shorter generation times and faster genetic improvement. MAS does not have application for traits that can be easily and cheaply improved traditionally. For example, in most selective breeding programs growth rate is relatively easy and cheap to measure and therefore is not a candidate for MAS. MAS is useful when a trait is difficult and expensive to measure, when a trait cannot be measured until a later age, or when traditional measurement methods give a low heritability. In terrestrial livestock breeding quality traits are often difficult and expensive to measure and therefore are the focus of MAS programs. For example, in beef cattle MAS is being used to select for beef marbling and for beef tenderness (Burrow et al. 2001).

5.5.2 General Discussion

An MAS R&D strategy should concentrate on traits that are difficult or expensive to improve by conventional selective breeding, for example (in the oyster industry) quality traits or perhaps disease resistance. The cost to develop MAS would be very high because of the steps necessary before the technology became commercially viable. These steps would include:

• The production of linkage maps.

• The need to understand the number of markers affecting the trait.

• The need to establish the mode of inheritance of markers.

• The need to determine linkage interactions.

Consideration would also have to be given to how to use the genetic marker information in the genetic analyses. This would depend on the size of the associated effects for a particular genetic marker. If the effect of a marker was small then physical measurements of the progeny in

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addition to assessment for the presence of a marker may be required. The selection program would also need to take account of the frequency of the alleles (if a particular allele is fixed, MAS will not add much genetic improvement) and the effects of recombination (marker alleles do not always reflect the inheritance of a genotype). If MAS was to be implemented it would be as a tool within a selective breeding program rather than a breeding technology in its own right. It would require the resources of the selective breeding program to be effective. Identification of traits that would benefit from MAS (those that are difficult to measure/identify, low heritable traits) would also be necessary. Dr Appleyard explained that the preferred genetic markers for MAS are microsatellites and, although hundreds of oyster microsatellites have been documented the associations with particular commercial traits have not been established. She said that DNA sequences of approximately 370 microsatellites containing clones from C. gigas are deposited in GenBank (http://www.ncbi.nlm.nih.gov/). Of these, 123 have been shown to be useful markers which have developed into amplifiable markers with confirmed inheritance (Li et al. 2003; Hubert et. al. 2004). MAS is not a viable technology for ASI to consider in the foreseeable future, although ultimately it could enhance traditional breeding methods. Linkage maps in fish and shellfish are not advanced and currently no aquaculture species is selected based on marker information. Currently, this genetic strategy is not even at the research stage although Dr Appleyard said that Dennis Hedgecock in the US is investigating it. It would be a major undertaking for the oyster industry to develop this technology. She added however, that apart from MAS, genetic markers could already be used for pedigree affirmation and validation of parentage. No IP issues were identified with the concept of MAS. However, the associations between genetic markers and a particular commercial trait, once identified, have IP value and a fee is usually associated with the use of that particular marker. There is a worldwide consortium (but mainly associated with US and French scientists) called the Oyster Genome Consortium, which shares information about genetic linkage maps and markers in oysters. To develop MAS technology it would be necessary to be a member of this international group and to contribute to MAS research. CSIRO, through Dr Robert Ward, is already a member of this consortium and is maintaining a watching brief on developments but has no intention at present of going further than this. It would take a major project (from ASI) to persuade CSIRO to do otherwise.

5.5.3 Conclusions

It was established that this technology was still at an early experimental/developmental stage and not ready for commercial application in the foreseeable future. Its complexity and high developmental cost would be impediments if an application for its use developed in the future. Development of MAS would be expensive, and because other selection techniques are available and effective, it was Dr Appleyard’s opinion that ASI should not promote MAS research at this stage. However, the Project Team agreed to recommend that Australia remains on the Consortium, perhaps in a more active role, and that FRDC might fund this. Other than maintaining a watching brief, the Project Team considered that the priority for MAS research is low.

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6 COMPARISON OF TECHNOLOGIES

Table 3 details the essential findings of the Project Team. These details are amplified as follows.

6.1 ENHANCED SELECTIVE BREEDING

Expected increased genetic gain: Gains up to 10% in some initial selectively bred oyster lines (Ward et al. 2005) indicate that the capacity for genetic improvement of oyster performance through selection is comparable with that of poultry, salmon, radiata pine and other terrestrial species. In those species, adoption of best practice selective breeding techniques has doubled the rate of gain achieved by older style conventional breeding programs (for example, compare rates of gain in Table 1 of Powell et al. 2004) and it is reasonable to expect that the same would occur in oyster breeding. The current ASI program’s gain per generation is estimated at 3-4% for selection on both shape and growth and it is anticipated that this could be increased to 6-8% in an enhanced multi-trait selection program. Proof of concept: The concept of enhancing the current selective breeding program in the manner proposed has been demonstrated and applied in terrestrial species. Examples of the use of economic weights in other industries are widespread.5 A good description of the methodology is given in Ponzoni (1988), and some examples of their use are given in Dekkers and Gibson (1998), Greaves et al. (2004) and Wu et al. (2005). The procedure to estimate gains with different selection strategies has a long history (e.g. Hazel 1943, Cunningham 1975) and these are now standard procedures described in text books (e.g. Falconer 1993) and an example of an application of these procedures is given in Cotterill (1986). The need for breeding program systems is well understood in other Australian industries and systems have been developed. For example, the Australian beef industry has developed BREEDPLAN (see http://breedplan.une.edu.au), the Australian sheep industries have developed LAMBPLAN (see http://www.sheepgenetics.org.au/lambplan) and the Australian forest industries have developed TREEPLAN (see http://www.stba.com.au/treeplan.html). Steps necessary: The establishment of economic weights, systems, strategies and technical protocols for the oyster industry would need to be developed within a research project. Outcomes from the project would be realised and implemented within the selective breeding program at various stages as the research project develops. Because the enhancements would be modifications to the selective breeding program currently operating rather than completely new technology, ASI would not require commercial phase testing, which adds 2.5 years to the developmental time for each of the other technologies. Commercial testing is necessary after the research and development phase for each of the new technologies, to demonstrate that the technology is practicable on a commercial scale. ASI also makes use of this phase to performance test the initial progeny. Time involved: The research project would require three years to complete, but some enhancements would be available for implementation during the course of the project. Costs: The cost of $647K is derived from a fully costed 2005 project application to FRDC entitled “Enhancement of the Pacific oyster selective breeding program”, scheduled to commence, if approved, in July 2006.

5 The web pages of many selective breeding organisations will make reference to breeding for profit and the use of economic weights

Table 3: Comparison of genetic technologies

TECHNOLOGY EXPECTED INCREASED GENETIC GAIN

PROOF OF CONCEPT

STEPS NECESSARY TIME INVOLVED

EST. COST TO FRDC

EST. COST OUTSIDE OF FRDC (CASH + IN-KIND)

EST. TOTAL COST

Development 3 yrs $300K $347K Enhanced selective breeding program *1

Double present yes In other aquaculture, and terrestrial plant and animal industries

Commercial Phase *1 Immediate nil nil $647K

Successive selfing to produce inbred lines. Outcross to produce F1 hybrids & compare with controls to demonstrate hybrid vigour

6-11 yrs $405K $600K Intensive inbreeding *3 Unknown No (Selfing works on a restricted, once only basis, but not demonstrated on a successive basis as proposed.)

Commercial Phase *2 2.5 yrs nil $140K

>$1145K

Validate DH production and quantity. Demonstrate viability, fertility. Multiply to produce DH larvae in quantity

2 yrs

Mature, breed DH larvae to produce F1 hybrids. Grow out F1 hybrids & compare with controls or commercial products to demonstrate hybrid vigour. Demonstrate multiplication technique efficiency.

2 yrs

>$475K

>$650K

Double haploidy *4

Unknown (Known to be highly beneficial in some plant species)

No (In plant industries and some marine spp. the technology is well established. In oysters, proof only to a preliminary research level)

Commercial Phase *2 2.5 yrs nil $140K >$1265K

Triploidy*5 - tetraploid method

Yes (Patented & not available to ASI)

Establish commercial thoroughbred triploid lines (if ever technique became available.) Commercial Phase *2

6 yrs 2.5 yrs

? nil

? $140K

6-DMAP 6-dimethylaminopurine

Yes (proven but techniques not fully developed)

Heat & or pressure

Better growth rate (up to 40%) than diploids, but dependent on conditions. Non spawners enabling sales during spawning season.

Yes (commercial technology not available to ASI – Hilton’s Coast Seafood Co., USA)

Refine techniques to achieve satisfactory egg quality, egg development and percentage recovery of triploids. Commercial Phase *2

0.5 yrs 2.5 yrs

$40K nil

$60K $140K

$240K

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Table 3 comments *1 Costs taken from FRDC Application Nov 2005 “Enhancement of the Pacific oyster

selective breeding program”. Enhanced selective breeding would be applied commercially immediately the technology was established and would not involve costs outside of normal ASI operating costs. Some enhancements would become available within the three year development period.

*2 These are the costs of commercial testing that ASI would need to undertake, additional to

its existing breeding program. They are derived as follows: Hatchery costs $15 000 Labour (FTE 0.5) $62 500 Stock management $62 500 *3 Costs taken from FRDC application Nov 2003 “Hybrid vigour and inbreeding in Pacific

oysters.” and are minimal. Simulated demonstration of gains could be a precursor, and if no gains were demonstrated the rest of the project could be aborted, in which case the total cost would be confined to $30K.

*4 Costs taken from FRDC application Nov 2003 “Production of F1 hybrids from

homozygous Pacific oysters” with the added cost of the Commercial Phase. The project could be staged (eg the later steps would only be undertaken if the initial DH larval production was successful with survival to 2-3 months of age). If the project failed at that early stage the total cost would be confined to about $91 500.

*5 “The technologies for direct triploid production are fully developed. The main issues

preventing their commercialisation are the low percentages of triploidy achieved associated with variations in egg quality and egg development.

6.2 TRIPLOIDY

Expected increased genetic gain: In virtually all comparisons between diploid and triploid oysters, the triploids have grown faster and bigger than diploids, although this difference is best expressed in areas which support high production levels. There is adequate NSW literature to substantiate that triploid Sydney rock oysters are on average 31% (range 6-99%) heavier when compared to diploids (Hand et al. 1998a). There are fewer studies for Pacific oysters; however available data indicates triploid Pacific oysters also appear to have greater growth rates than diploids. In NSW growth rates of Pacific oyster triploids were more than double that of diploids (Nell and Perkins 2005) and in Tasmania Pacific oyster triploids have grown 15 – 20% faster than diploids depending on the prevailing conditions (Kent, pers. comm. 2005). Importantly, data from rock oysters suggests that the gains from selective breeding and triploidy are additive. Triploids may also have greater disease resistance. One wide scale field trial demonstrated mortality rates of only 12.2% for triploid rock oysters compared with 35% for diploids when challenged by winter mortality Mikrocystis roughley (Hand et al. 1998b). Proof of concept: Proof of concept is fully established for triploid production by the tetraploid route, but this patented technology is not available to ASI. The concept for ‘direct’ technologies (that is, technologies to produce triploids directly from diploid broodstock and not via the tetraploid route) is also fully established but there are issues preventing their commercialisation. These are variations in egg quality and egg development, resulting in an unacceptable proportion of diploids amongst the triploids. Steps necessary: If direct induction techniques were successful the assimilation of broodstock from the ASI selective breeding program into a triploid program would be straightforward. The

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project group regards this as a high priority project. The technology would be integrated with the ASI selective breeding strategy according to commercial demand. Time involved and cost: It is expected that the development of the ‘direct’ triploid technology(ies) would take an estimated three years and total research expenditure of perhaps $240K (including $140K for a commercial phase).

6.3 DOUBLE HAPLOIDY

Expected increased genetic gain: There is currently no information to allow potential genetic gain through this technology to be quantified. Proof of concept: The Project Team considered that ‘proof of concept’ has not been established for DH technology in oysters except perhaps, at the most basic level. Importantly, it has not been established at a level which would lend support to commercial development of the technology. DH technology is well established and highly beneficial in some plant species, although in others the cost relative to the size of the industry is inhibiting its development. Steps necessary: To develop ‘proof of concept’ to a commercial level a research project would be necessary to demonstrate:

a. That true homozygous DH larvae can be produced and survive to maturity.

b. That mature DH oysters are sufficiently fecund to produce suitable families for performance testing, and are capable of producing commercial quantities of eggs and sperm.

c. That the techniques of gynogenesis, selfing and/or androgenesis are effective in producing the numbers of homozygous broodstock required for commercial production.

d. That the F1 hybrid lines produced from DH oysters perform better than selectively bred lines, and the magnitude of this improved performance is sufficient to warrant the expected higher production costs for DH stock.

Time involved: 6.5 years in total, including 2 years for steps a) and b) above, 2 years for steps c) and d) and 2.5 years for the ASI commercial phase. Costs: The above project is virtually a repeat of FRDC project 2002/204: “Development of the Techniques to Produce Homozygous Pacific Oysters” but with modified objectives and the addition of the ASI commercial phase. It is therefore reasonable to estimate costs on the basis of the original SARDI projections plus the cost of ASI commercial testing i.e. $1265K. The project could be staged (e.g. the later steps would only be undertaken if the initial DH larval production was successful with survival to 2-3 months of age). If the project failed at that early stage the total cost would be confined to about $91 500, allowing for use of 6-10 markers to evaluate the DH status of the larvae.

6.4 INTENSIVE INBREEDING

Expected increased genetic gain: There is currently no information to allow potential genetic gain through this technology to be quantified. Doubts exist as to whether hybrid vigour, on which genetic gains would rely, does occur in oysters. Proof of concept: The concept has not been proven on two counts. Firstly, there is no clear proof that hybrid vigour exists in oysters and that genetic gains will occur. And secondly,

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although “selfing” has been used successfully as a research technique, the capacity to successfully and successively self fertilise lines in an operational situation and for the oysters so derived to survive and breed remains uncertain. Steps necessary and timing: A major study is underway in the United States6 as to the presence and extent of hybrid vigour in oysters and the project team considered a watching-brief of this project was the most appropriate action at this stage. If gains are demonstrated in the US study and if an Australian intensive inbreeding study were to be done, then at least two self fertilisation cycles would need to be undertaken, each requiring 2-3 years or more depending on the speed of maturation of the oysters, and the reliability of the sex reversal. The inbred lines would then be outcrossed and the progeny assessed for hybrid vigour (2 years) – say 6 to 11 years in all, depending on the number of self fertilisation cycles undertaken and the time required for each cycle. The final commercial testing of heterozygote lines by ASI would take an additional 2.5 years. Time involved: The entire program would take at least 8.5 years, but possibly up to 13.5 years. Costs: The costs are derived from estimates contained in Dr Ward’s FRDC project application (Ward 2003). This application (and the costs detailed in Table 3) was for a program of five years duration. It depended on only one “selfing” cycle (estimated 2.5 years) before the outcross to demonstrate hybrid vigour (2.5 years to grow out and performance test). During the grow out period, two further successive generations of “ selfed” lines were to be produced on the expectation that positive hybrid vigour effects would be demonstrated and the latter “selfed” lines would be needed for onward breeding. The Project Team considered that at least two and possibly three successive “selfed” generations would be needed if this technology were to be tested commercially, and that the time for each cycle could vary up to three years. The cost could therefore be substantially greater (in the order of double) than the $1145K suggested in the Table 3.

6http://hmsc.oregonstate.edu/projects/wrac

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7 GENERAL CONCLUSIONS

Existing, developing and proposed technologies in relation to triploidy, intensive inbreeding, double haploidy and marker assisted selection have been reviewed and compared with an enhanced breeding program with a view to establishing which, if any, might be developed to replace or improve the selective breeding program currently operated by ASI. None of the technologies (other than enhanced selective breeding) could replace the current selective breeding program although access to triploidy could improve it.

7.1 ENHANCED SELECTIVE BREEDING:

It was accepted that with the adoption of improvements including the use of economic weights, variation of breeding strategies and development of improved breeding program systems and technical protocols, an enhanced selective breeding program could be expected to at least double the rate of genetic gain achieved by the current ASI program (e.g. the current program’s gain per generation is estimated at 3-4% for selection on both shape and growth. This could be increased to 6-8% in an enhanced multi-trait selection program). The technology involved is already established and proven in other industries. To apply the technology to the Pacific oyster industry research would be required to collect appropriate data, develop protocols, identify the best strategies and develop specifications for systems. The risks are therefore minimal and the benefits are assured and substantial. There are no impediments or IP issues. Moderate developmental cost, relatively short developmental time and assured successful application with quantifiable benefits across the entire Pacific oyster industry, and the applicability of some of the outcomes to the wider aquaculture industries make this the highest priority recommendation for future ASI research.

7.2 TRIPLOIDY:

The use of triploid oysters is a proven and established concept for which the benefits are known and substantial in some environments. The technology available in Australasia derives triploids via a tetraploid technology which is patented and exclusive to only one oyster hatchery. ASI should keep a watching brief on alternative methods for tetraploidy induction of triploids that may become available in the future. The hatchery is not willing to incorporate improved ASI broodstock into its triploid program even though there is some evidence that the gains from selection and triploidy are additive. The technologies for direct triploid production are fully developed but protocols need refinement if these techniques are to be acceptable on a commercial scale. The main issues preventing their commercialisation are variations in egg characteristics which result in unacceptable proportions of diploids amongst the triploids. The project group considers that the development of direct triploid production to a commercially acceptable standard is a worthwhile area for research in the immediate future.

7.3 DOUBLE HAPLOIDY (DH):

This technology remains at the “proof of concept” stage. It has not been established that double haploid larvae can be produced in reliable quantity, that a satisfactory percentage of these can metamorphose, mature and breed, that DH individuals can produce commercial quantities of gametes, and that there are sufficient gains from DH production to warrant the extra production costs.

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An estimated $1265 K expenditure and 6.5 years R&D work are needed before double haploid oysters could become available as part of the ASI breeding program. The technology would not replace the breeding program. In view of the potency of this technology in some areas of plant breeding, the review team assigned a moderate priority for a revised proposal taking the DH research to the stage of demonstrating production of adequate numbers of viable DH spats and their survival. IP issues are associated with DH technology. SARDI sees ASI as a vehicle for the commercialisation of double haploidy and has sought inter alia to negotiate for royalty payments but ASI does not consider the technology to be sufficiently developed at this stage.

7.4 INTENSIVE INBREEDING:

As with double haploidy, this technology relies on development of highly homozygous lines which are eventually outcrossed in the expectation that hybrid vigour will result. However, development of the homozygous lines depends on the use of a series of “selfed” matings. The effectiveness of “selfing” as a technique has been demonstrated but the breeding capacity of successively selfed lines remains uncertain, and the existence of the hybrid vigour phenomenon in oysters has not been demonstrated. Participants therefore decided that “proof of concept” has not yet been established for this technology. The lead time to establishment of proof of concept and commercial production of hybrid spat by this route would be in the order of 8 to 13 years and the developmental cost is estimated at well in excess of $1175 K. Moreover, there are uncertainties as to the likely benefits flowing from hybrid vigour. A major intensive inbreeding project is underway in the US and the review team considered that a watching-brief of this project was the most appropriate action at this stage. The review team therefore considered intensive inbreeding to be a low research priority.

7.5 MARKER ASSISTED SELECTION (MAS):

It was established that this technology is still at an early experimental/developmental stage and not yet ready for commercial application. Research in this area is expensive and highly specialised and the review team considered this to be beyond the scope of ASI’s current resources. The review team recommended that ASI should keep a watching brief on MAS (and other genomics work).

7.6 THE PROJECT TEAM CONSIDERATIONS:

• Enhancement of the current selective breeding program is identified as the highest priority and most cost effective area for developmental research to supplement the ASI selective breeding program.

• Industry access to triploidy technology through the tetraploid route is limited by the existing IP ownership and patent protection. Developmental research to commercialise direct high pressure/temperature induction of triploidy is warranted.

• Double haploidy, Intensive inbreeding, and Marker assisted selection cannot be considered as tools to replace or supplement the ASI selective breeding program at this time.

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8 INDEPENDENT REVIEW

The Independent Reviewer was included within the Review Team to assist with resolution at the technical level in the event that unresolvable differences of opinion arose between team members. In the event, full and free discussion allowed all issues to be resolved without argument, and the Independent Reviewer was not called on to arbitrate at any stage. However, his additional views in relation to the technologies under examination were valuable, and he has lent strength to this report by attesting to the fact that the review proceeded without bias.

8.1 INDEPENDENT REVIEWERS REPORT

Five genetic improvement techniques/strategies, triploidy, marker assisted selection, double haploidy, intensive inbreeding and enhanced selective breeding were considered on their suitability for ASI. All views of the participants in the project team meetings were considered without bias. The project team assessed the techniques/strategies on their ability to improve desirable characteristics quickly at an affordable cost. I agree that enhancement of the current breeding program is the highest priority and most cost effective area for R& D to support the ASI breeding program. However, research providers and research funding organisations should be encouraged to fund research into intensive breeding, double haploidy and marker assisted selection on the merit of R&D proposals. I recommend that:

1. ASI not support R&D into alternative tetraploidy induction techniques (for triploid production), as I expect that other research providers are well advanced in this work. However, if alternative tetraploidy induction techniques are developed it is likely that they will be patented like the currently available technique. ASI should keep a watching brief on the development of new tetraploid induction techniques and assess them on their merit as they come available.

2. ASI supports R&D into triploidy induction in oysters using hydrostatic pressure and or heat in an attempt to develop an effective non-chemical triploidy induction technique. As far as I am aware this research is not subject to patents and I expect it to have a high chance of success (>80% triploidy).

3. ASI supports research into the development of biochemical or other tools to allow for selection of meat yield in oysters. It should be noted that both peak meat condition and the length of time that oysters are in a minimum marketable meat condition are important. The development of tools to allow for the selection of meat yield in oysters should get the highest research priority.

Dr J A Nell (2006)

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9 REFERENCES

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Bertotto, D., Cepollaro, F., Libertini, A., Barbaro, A., Francescon, A., Belvedere, P., Barbaro, J., and Colombo, L. (2005). Production of clonal founders in the European sea bass, Dicentrarchus labrax L., by mitotic gynogenesis. Aquaculture 246:115;124.

Borralho, N.M.G.. Cotterill, P.P. & Kanowski, P.L. (1993). Breeding objectives for pulp production of Eucalyptus globulus under different industrial cost structures. Canadian Journal of Forest Research 23: 648-656.

Burrow, H.M., Moore, S.S., Johnston, D.J., Barendse, W., and Bindon. B.M. (2001). Quantitative and molecular generic influences on properties of beef: a review. Aust J. Exp. Agric. 41: 893-920.

Cotterill, P.P. (1986). Genetic gains from alternative breeding strategies. Silvae Genetica 35: 212-223.

Cunningham, E.P. (1975). Multi-Stage Index Selection. Theoretical and Applied Genetics 46: 55-61.

Dekkers J.C. and Gibson, J.P. (1998). Applying breeding objectives to dairy cattle improvement. Journal of Dairy Science 81 Suppl 2:19-35.

Eudeline, B., Allen Jr, S.K. and Guo, X. (2000). Optimization of tetraploid induction in Pacific oysters (Crassostrea gigas) using the first polar body as a natural indicator. Aquaculture: 187, 73-84.

Evans, F., Matson, S., Brake, J. and Langdon, C. (2004). The effects of inbreeding on performance traits of adult Pacific oysters (Crassostrea gigas). Aquaculture 230: 89-98.

Falconer, D. S. (1993). Introduction to Quantitative Genetics. Third edition. Longman Scientific and Technical. p 230-240.

Greaves, B., McGranahan, M., and Harding, K. (2004). Breeding objectives and selection criteria to maximise the economic value of sawn timber. Summary report. Forest and Wood Products Research and Development Corporation. Project no. PN001.96. 9 pp.

Gjedrem, T. (2000). Genetic improvement of cold-water fish species. Aquaculture Research 31: 25-33.

Gjerde, B. (2005). Design of breeding programs. In: T. Gjedrem (ed.), Selection and breeding programs in aquaculture. Springer. Pp 173-195.

Guo, X. and Gaffney, P.M. (1993). Artificial gynogenesis in the Pacific oyster, Crassostrea gigas: II. Allozyme inheritance and early growth. Journal of Heredity, 84: 331-315.

Guo, X., Hershberger, W.K., Cooper, K. and Chew, K.K. (1993). Artificial gynogenesis with ultraviolet light-irradiated sperm in the Pacific oyster, Crassostrea gigas, I: induction and survival. Aquaculture: 113: 201-214.

Hand, R.E., Nell, J.A., Maguire, G.B. (1998a). Studies on triploid oysters in Australia. X. Growth and mortality of diploid and triploid Sydney rock oysters, Saccostrea commercialis (Iredale and Roughley). Journal of Shellfish Research 17: 1115-1127.

Hand, R.E., Nell, J.A., Smith, I.R., Maguire, G.B., (1998b). Studies on triploid oysters in Australia. XI. Survival of diploid and triploid Sydney rock oysters, Saccostrea commercialis

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(Iredale and Roughley) through outbreaks of winter mortality caused by Mikrocytos roughleyi infestation. Journal of Shellfish Research 17: 1129-1135.

Hand, R E ., Nell, J A., Reid, D D., Smith, I R. and Maguire, G B. (1999). Studies on triploid oysters in Australia: effect of initial size on growth of diploid and triploid Sydney rock oysters, Saccostrea commercialis (Iredale and Roughley) Aquaculture Research 30: 35-42.

Hand, R E. and Nell, J A. (1999). Studies on triploid oysters in Australia: XII: Gonadal discolouration and meat condition of diploid and triploid Sydney rock oysters Saccostrea commercialis (Iredale and Roughley) in five estuaries in New South Wales, Australia. Aquaculture 171: 181-194.

Hand, R.E.; Nell, J.A. and Thompson, P.A. (2004). Studies on triploid oysters in Australia: XIII: Performance of diploid and triploid Sydney rock oysters Saccostrea commercialis (Gould, 1850) progeny from a third generation breeding line. Aquaculture 233: 93-107.

Hazel, L. N. (1943) Genetic Basis for selection indices. Genetics 28: 476-490

Hedgecock, D., Manahan, D.T., Langdon, C.J., and Davis, J.P. (2005). Crossbreeding Pacific Oyster for High Yield http://hmsc.oregonstate..edu/projects/wrac Project Termination Report.

Hubert, S. and Hedgecock, D. (2004). Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas. Genetics 168: 351-362.

Kirankumar, S., and Pandian, T.J., (2003). Production of androgenetic tiger barb, Puntius tetrazona Aquaculture 228:37-51.

Li, X. et al (in preparation) Development of Techniques to Produce Homozygous Pacific Oysters. Final Report FRDC Project 2002/204. South Australian Research and Development Institute and Fisheries Research and Development Corporation.

Li, G., Hubert. S., Bucklin, K., Ribes, V. and Hedgecock, D. (2003). Characterisation of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas. Molecular Ecology Notes 3: 228-232.

Li, Q. and Kijima, A. (2006). Microsatellite analysis of gynogenetic families in the Pacific oyster, Crassostrea gigas. Journal of Experimental Marine Biology and Ecology 331: 1-8.

Maguire, G.B., Gardner, N.C., Nell, J.A., Kent, G.N., Kent, A.S., 1994. Studies on triploid oysters in Australia: II. growth, condition index, glycogen content and gonad area of triploid and diploid Pacific oysters, Crassostrea gigas (Thumberg), in Tasmania. In Nell, J.A., and Maguire, G.B. (Eds), Evaluation of Triploid Sydney rock oysters (Saccostrea commercialis) and Pacific oysters (Crassostrea gigas) on commercial leases in New South Wales and Tasmania, Final Report to FRDC, September, 1994 NSW Fisheries Port Stephens Research Centre, Taylor’s Beach, NSW and University of Tasmania, Launceston, Tas., pp 37-71

McCombie, H., Ledu, C., Phelipot, P., Lapègue, S., Boudry, P., and Gérard, A. (2005). A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments. Marine Biotechnology 7:318-330.

McRae, T.A., Dutkowski, G.W., Pilbeam, D.J., Powell, M.B., and Tier, B. (2004). Genetic evaluation using the TREEPLAN system. In: “Forest Genetics and Tree Breeding in the Age of Genomics: Progress and Future”. 2004 IUFRO Joint Conference of Division 2. Charleston, South Carolina, USA, 1-5 November 2004. (available at http://www.stba.com.au/articles.html).

Nell, J.A. (2002). Farming triploid oysters. Aquacultuer 210: 69-88.

Nell, J A (2006). Manual for mass selection of Sydney rock oysters for fast growth and disease resistance. NSW Department of Primary Industries – Fisheries Research Report Series, No.13.

Nell, J.A. and Perkins, B. (2005). Studies on triploid oysters in Australia: farming potential of all-triploid Pacific oysters, Crassostrea gigas (Thunberg), in Port Stephens, New South Wales, Australia. Aquaculture Research 36: 530-536.

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Parkinson, S.A (2005). Enhancement of the Pacific oyster selective breeding program

FRDC Application Nov 2005.

Ponzoni, R.W. (1988). The derivation of economic values for combining income and expenses in different ways: An example with Australian merino sheep. Journal of Animal Breeding Genetics 105: 143-153.

Powell, M.B., McRae, T.A., Wu, H.X., Dutkowski, G.W. and Pilbeam, D.J. (2004). Breeding strategy for Pinus radiata in Australia. In: “Forest Genetics and Tree Breeding in the Age of Genomics: Progress and Future”. 2004 IUFRO Joint Conference of Division 2. Charleston, South Carolina, USA, 1-5 November 2004. (available at http://www.stba.com.au/articles.html).

Swan, A.A., Thompson, P.A. and Ward, R.D. (2004). Breeding plans for Australian Seafood Industries Pacific oysters. Confidential Report to Australian Seafood Industries by CSIRO Livestock Industries and CSIRO Marine Research. 42 pp.

Ward, R.D., Thompson, P.A., Appleyard, S.A., Swan, A.A. and Kube, P.D. (2005). Sustainable genetic improvement of Pacific oysters in Tasmania and South Australia. Final Report FRDC Project 2000/206. CSIRO Marine and Atmospheric Research and Fisheries Research and Development Corporation.193 pp.

Ward, R.D. (2003). Hybrid vigour and inbreeding depression in Pacific oysters. FRDC application Nov 2003.

Wu, H.X., Ivkovic, M., McRae, T.A. and Powell, M.B. (2005). Breeding radiata pine to maximise profit from solid wood production: Summary report. Forest and Wood Products Research and Development Corporation. Project no. PN01.1904 11 pp.

Yamamoto, E. (1999). Studies on sex-manipulation and production of cloned populations in hirame, Paralichthys olivaceus (Temminck et Schlegel). Aquaculture 173: 235-246.

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10 APPENDIX 1: Review of ASI Pacific oyster selective breeding program

Prepared as part of FRDC Project number 2005/227 By Peter Kube and Scott Parkinson, July 2005

10.1 INTRODUCTION

This document is part of a review of the genetics R&D strategy of ASI. A number of breeding technologies are being reviewed including triploidy, double haploids, and hybrid vigour. This document reviews the R&D needs for the selective breeding program. The current breeding strategy is briefly described and areas where improvements are needed are identified. The problems are briefly explained and the R&D needed to address them is outlined.

10.2 CURRENT ASI SELECTIVE BREEDING STRATEGY

The current breeding population and strategy has been developed as part of the FRDC funded projects FRDC 97/321 and 2000/206. This was a joint project between CSIRO and TAFI. An outcome of these projects was the formation of ASI, a company formed to run a commercial selective breeding program. A detailed account of the work done up to and including the 2002/03 spawning year (termed the F5 generation) is given in Ward et al. (2005). The F6 generation, which is the current generation undergoing improvement, was produced by ASI in 2003/04 and the strategy for this spawning was described in Swan et al. (2004). Following the F6, an F7 generation was produced by ASI in 2003/04. A simplified flowchart of the current breeding strategy for the F6 generation is shown in Figure A1. The key features are as follows:

• The recommended breeding population size is 30 families. For the F6 generation (2003/04 spawning), there are 19 families and most parents originate from the F1, F2, F3, and F4 breeding populations, with some new introductions and some from the mass selection lines. There are an additional 10 families in the F7 generation (2004/05 spawning).

• The mating design has been a circular mating design, which uses a set ‘roster’ of crossing designed to minimise inbreeding. Under this design, no EBV information is used for mate allocation.

• Families are grown out on four sites. At each site, 3 replicates (or bags) of 100 animals are used giving 1,200 animals per family. If the recommended 30 families were used, this would involve 9,000 animals per site and 36,000 animals in total.

• Assessments for selection are made at 24 months. Growth is assessed using total bag weight with 12 measurements (bags) per family. Survival is assessed on numbers of animals per bag. Shape is based on measurements of length, width and depth and is expressed as a width ratio (= width/length) and a depth ratio (= depth/length). Measurements are taken from 10 animals per family on 2 sites (20 per family). Condition, which is a measure of the amount of meat in the shell, is measured from 10 animals per family on a single site.

• Selection for the breeding population is based upon within family selection, with the best 2 individuals being selected from all families. The best individuals are selected

45

based on phenotypes after inspecting about 1,000 animals. No genetic evaluation is required for this breeding strategy.

• The genetic evaluation and selection for commercial crosses is based upon family means of assessed traits and a visual inspection of families by ASI staff and growers. After a family is selected, about 100 siblings of the original parents are sourced and commercial quantities of spat produced. The relationships between the tested families and the commercially deployed families are therefore double first cousins7 (coefficient of relationship is r =0.25).

• The generation time is 2 years. This results in a 2 year work cycle for the breeding program, with spawning being done every second year. (However, to date spawnings have occurred every year for a variety of reasons.)

Figure A1: Flowchart of the ASI Pacific oyster selective breeding program.

BREEDING POPULATION COMMERCIAL POPULATION

BREED POP. SELECTIONF6 within-family selectionplus additional families~ 2 per family = 60 total

ASSESSMENTweight, survival: 90 bags/site

shape: 10/family x 2 sitescondition: 10/family x 1 site

COMMERCIAL SELECTIONSelect best ~ 5 families

100 per family from F6 parental families

EXTRA SELECTIONSF5, F7 & new

families

COMMERCIAL SPAT

~ 20 M per year

Identify families with desired traits

GENETIC EVALUATIONdata

MATING

circular mating30 families

19 familiesFrom F1-F4, plus new

4 sites300/family/site = 9000 total

GROWOUT

BREEDING POPULATIONbroodstock(siblings of

original parents)

NEXT GENERATION

2-year breeding cycle

10.3 IMPROVEMENTS TO CURRENT STRATEGY

The current ASI selective breeding strategy has operated successfully since 1998 (for 6 generations). It is a simple and conservative strategy but has been appropriate for the resources and technical skills that were available. However, it is now apparent that there are some shortcomings to this strategy and improvements are needed. These shortcomings have been 7 Double first cousins have all grandparents in common, and therefore have a higher coefficient of relationship than normal first cousins.

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identified through knowledge of and comparison to selective breeding strategies in other industries (e.g. Gjedrem 2000, Powell et al. 2004), through ASI’s operational experience over the last 4 years, and from feedback from ASI’s customers with regard to the traits required in commercial families. Figure A2: Flowchart of the ASI Pacific oyster selective breeding program with eleven priority areas for improvement indicated.

NEXT GENERATION

9. Optimum breeding cycle

COMMERCIAL POPULATIONBREEDING POPULATION

EXTRA SELECTIONS

BREED POP. SELECTION COMMERCIAL SELECTIONCOMMERCIAL

SPAT 7. Optimum selection method 8. Economic weights for selection

10. Economic weights for selection 11. Broodstock management

4. Optimum no. of assessments 5. Assessment of condition

GENETIC EVALUATIONdata

6. Genetic evaluation system

MATING

2. Best mate allocation 3. Apply economic weights

GROWOUT

ASSESSMENT

BREEDING POPULATIONbroodstock

1. Optimum population size (siblings oforiginal parents)

In total, eleven issues have been flagged as priority areas for improvement with regard to the selective breeding program, and these are shown in the flowchart in Figure A2. These can be grouped into four broader themes as follows:

• Economic weights for selection: This includes determining the economic weights for selection in the breeding population (8), for mate allocation in the crossing program (3) and for selection in the commercial population (10).

• Breeding strategy issues: This includes determining the optimum population size (1), optimum number of assessments (4), optimum selection method (7), and optimum breeding cycle (9).

• Development of selective breeding program systems: This includes development of best mate allocation systems (2), and development of a genetic evaluation system (6).

• Development of technical protocols: This includes development of methods to assess condition (5), and refinement of protocols for broodstock management (11).

Each of these themes is now discussed with the problem being defined and solutions proposed.

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10.4 ECONOMIC WEIGHTS OF TRAITS

The problem The goal of a commercial selective breeding program is to maximise profit. To achieve this goal there is a need to clearly identify the economic value of changes in traits. This is particularly important in the ASI program because:

• The true economic values of traits known to be important to the Australian Pacific oyster industry (such as growth rate, shape, uniformity and survival) and how these vary in different environments are not clearly understood.

• Adverse correlations are known to occur between improving growth rate and improving shape (width index and depth index) and therefore trade-offs are required.

• Applying different relative weightings to growth and shape can result in very different outcomes. These affects can be seen in Figure A3 where the gains from 5 different selection indices are shown. Good gains in growth rate can be obtained, but these involve sacrifices in shape. Conversely, maximal gains in shape are made with a decline in growth rate. Finding the correct balance should be done using the economic values of traits.

• Decisions of ‘what type of oyster to breed’ will become more complex as additional traits are added to the breeding objective, such as condition and survival. It will, therefore become increasingly less likely that the intuitive decision about the mix of traits is the best economic decision.

Figure A3: Genetic gains in growth rate and shape (as determined by width index and shape index) for 5 different selection strategies.8

-10

0

10

20

30

40

Gro

wth

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8 The selection traits used in these indices were weight, width index and shape index. The relative economic weights were, respectively, 3:0:0 for the growth index, 3:1:1 for the growth emphasis index, 1:1:1 for the equal emphasis index, 1:3:3 for the shape emphasis index, and 0:1:1 for the shape only index. See Ward et al. (2005, Chapter 10) for further details.

48

The solution There is a need for an R&D project to develop and quantify breeding objectives for Pacific oysters in all growing regions in Australia. Methodologies have been developed in other industries and are now routinely applied in commercial breeding programs (e.g. Ponzoni 1988; Borralho et al. 1993). This is, essentially, an economic issue rather than a genetic issue. Specifically, the tasks of such a project would be as follows:

• Define the production system and identify all factors that influence costs and returns (i.e. profitability). The production system needs to be defined as a mathematical model that predicts enterprise profitability for the grower (and possibly for other parts of the value chain).

• Determine the genetic traits that influence profitability and calculate the economic weights of each trait. The economic weights measure how changes in a trait (say, through selective breeding) influence overall profitability of an enterprise.

• Construct an Excel based model that can calculate economic weights after inputting data on costs and prices (e.g. Greaves et al. 2004). This model will allow economic weights to be continually updated, and tailored for specific sites and regions.

10.5 BREEDING STRATEGY ISSUES

The problem Different selective breeding strategies will give different genetic gains (Falconer 1993; Gjerde 2005). For a given population, some of the key variables influencing the amount of genetic gain that will be obtained are:

• Selection methodology: This may include within-family selection, where the best individuals are taken from every family and no families are culled; family/within-family selection where the best individuals are taken from the best families; and combined selection, where information from relatives is used to improve the efficiency of genetic selections.9

• Population sizes: This includes the number of parents and the number of times they are crossed (which dictates the number of families in a population), and the number of individuals in each family.

The selection strategy currently used by ASI is conservative. It is simple and avoids the risks of inbreeding, but does not maximise potential gains. Specifically, some important issues are:

• The selection methodology used is within-family selection, and this gives considerably less gain than other methodologies. The amount of gain that is theoretical possible with different selection methodologies is shown in Figure A4. Genetic gains of at least twice the magnitude of those currently obtained should be possible.

• Gains will be maximised by generating more families and selecting from the best families. This is shown in Figure A5, where gains are predicted with different numbers of families. The ASI strategy is currently only generating about 25 families per generation.

• Increasing the numbers of individuals per family over current practise (which is about 1,200 per family) will have little impact on gains. This is shown in Figure A4, where gains are predicted with different numbers of individuals per family.

9 BLUP (Best Linear Unbiased prediction) is a statistical methodology that is now the method of choice for genetic evaluation. It can be considered a more sophisticated form of combined selection, and should deliver better gains, especially for traits of low heritability.

49

• The genetic gains shown in these examples have been predicted using the equations of Falconer (1993, Table 13.4) and assume a heritability of h2 = 0.25, a coefficient of variation of 20% and an intraclass correlation of t = ½ h2 (Falconer 1993, p 148).

Figure A4: Predicted genetic gains using 3 different selection strategies and different numbers of individuals per family.10

0

5

10

15

20

25

30

0 500 1000 1500 2000 2500 3000

Numbers per family

Ge

netic

ga

in (

% o

f me

an)

Combined

Family/within-family

Within family

Figure A5: Predicted genetic gains using different numbers of families.11

0

5

10

15

20

25

0 20 40 60 80 100 120 140 160 180 200

Numbers of families

Ge

netic

ga

in (

% o

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Family/within-family

Family

10 Gains for family/within-family selection assume 30 families, of which the best 15 are selected. 11 Gains assume selections from the best 15 families, and 1,200 individuals per family.

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The solution There is a need for a major revision of the ASI selective breeding strategy. This is a complex activity, and a well planned approach is needed that encompasses the following tasks:

• Objectively evaluate the potential genetic gains from different options using knowledge of theoretical genetics. There are a large number of factors that affect gains, and these include population size (numbers of parents, progeny, and families), population structure (single or multiple breeding populations), mating designs, number and nature of traits under selection, selection strategies, and management of inbreeding. The examples shown above illustrate the importance of some of these factors.

• Determine the infrastructure that is required for different breeding options (such as hatchery requirements, farm grow-out requirements, and broodstock holding requirements), evaluate the practicalities of accessing or developing suitable infrastructure, and find the appropriate balance between obtaining good genetic gains and a workable selective breeding program.

• Estimate the costs of different options (labour and operating costs) and determine a suitable work cycle. For example, should all the families for a single generation be spawned in a single year, or should they be done over two years?

10.6 DEVELOPMENT OF SELECTIVE BREEDING PROGRAM SYSTEMS

The problem Implementing an advanced selective breeding program involves a large amount of data analysis and the use of data to make a variety of technical procedures. Most of these procedures need to be done on a regular basis and in a timely manner. ASI has recognised the importance of efficient data management by developing a suitable database, but there is a need to either develop, or negotiate access to, systems that allow these other procedures to occur routinely. Specifically, the priorities are as follows:

• Develop suitable systems allowing breeding values to be calculated or provided. A breeding value is a calculated value that measures the genetic value of traits for either an individual or family.12 Breeding values use information from all relatives to increase the accuracy of predicted genetic values. For example, families/individuals in the ASI breeding program would have breeding values for economically important traits such as growth rate, width index, depth index, survival and condition. Breeding values allow objective decisions to be made and become increasingly important for advanced generation breeding programs, as more traits are included in the breeding objective, and when traits have lower heritabilities.

• Develop a best mate allocation system. Currently ASI does not have a system that determines which animals are mated with which. A mate allocation system would use economic weights, breeding values, and predicted levels of inbreeding to predict the mate allocation that maximises economic value. This would result in higher gains and greater economic value.

• Modify the database to accommodate these systems. The introduction of new systems will require new data outputs on inputs from the database. Therefore it is expected that some degree of database redevelopment will be required.

The solution The development of suitable systems is not a simple task, and is an issue that many selective breeding programs struggle with. This is a frequent cause of bottlenecks and a frequent limitation to genetic progress (e.g. McRae et al. 2004).

12 A breeding value is different from the observed or measured value. This is the phenotypic value.

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A possible approach is as follows:

• Evaluate the approach taken by selective breeding programs in other industries. This would allow the pros and cons of different approaches to be examined. It is recommended this be done by visiting and talking to program managers – this is more likely to yield a frank assessment of approaches that haven’t worked so well.

• Develop a specification of exactly what is required. (This needs to be as part of the breeding strategy development.)

• Discuss the possibilities of obtaining access to these systems from potential providers. This would include deciding what is done by ASI, what is outsourced, and what level of skills ASI staff need to develop to implement the systems.

10.7 DEVELOPMENT OF TECHNICAL PROTOCOLS

The problem Two areas were identified as needing improved technical protocols, and these are as follows:

• Methodologies to assess condition: Condition, which is a measure of the amount of meat in the shell, has been identified as an important trait. However, this appears to be a complex trait to measure and use in a selective breeding program. The expression of condition may vary at different times and on different sites. Research is needed to determine if the same genes control this trait at all stages and on all sites. Furthermore, condition measurements are expensive and destructive and this limits gains. Selective breeding programs always require a large number of measurements and therefore simple and low cost measurement techniques are required is a trait is to be included. Destructive measurements limit the potential for genetic gains because breeding can only be done by applying information from relatives.

• Broodstock management protocols: An important factor affecting the genetic gains that are realised by industry is the availability of genetically elite broodstock for producing commercial quantities of seed. The best gains are achieved when the best animals from the best families are available for broodstock. There is a large variation within families and simply selecting families will not deliver the best gains – especially when multi-trait selection is being practiced.

The solution The R&D needed to develop these protocols is as follows:

• Find novel ways of measuring condition which are, ideally, low-cost, simple and non-destructive.

• Develop systems to ensure sufficient quantities of elite animals are available as broodstock for commercial hatcheries, and to ensure these animals are managed and used in the most efficient way. This will require working closely with hatcheries to identify issues with broodstock management and the development of suitable solutions.

10.8 CONCLUSION

The ASI selective breeding program is functional and is delivering genetic change to the Australian Pacific oyster industry. However, it has not adopted some of the important features of a modern selective breeding program – features that have been shown to increase gains when applied to the selective breeding programs of other industries. A revised Pacific oyster program could deliver greater gains (approximately double the rate of genetic gain) and greater economic value to the Australian Pacific oyster industry. In addition, the ASI program needs to develop, or, gain access to data management systems. Such systems are essential for good and timely decision making and for the efficient operation of a modern selective breeding program.

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10.9 REFERENCES

Borralho, N. M. G., Cotterill, P. P. & Kanowski, P. L. (1993). Breeding objectives for pulp production of Eucalyptus globulus under different industrial cost structures. Canadian Journal of Forest Research 23: 648-656.

Falconer, D. S. (1993). Introduction to quantitative genetics. Third edition. Longman Scientific and Technical. p 230-240.

Greaves, B., McGranahan, M. and Harding, K. (2004). Breeding objectives and selection criteria to maximise the economic value of sawn timber: Summary report. Forest and Wood Products Research and Development Corporation. Project no. PN001.96. 9 pp.

Gjedrem, T. (2000). Genetic improvement of cold-water fish species. Aquaculture Research 31, 25-33.

Gjerde, B. (2005). Design of breeding programs. In: T. Gjedrem (ed.), Selection and breeding programs in aquaculture. Springer. pp 173-195.

Hedgecock, D; Gaffney, P.M; Goulletquer, P; Guo,X; Reece, K; and Warr, G.W. (2005) The case for sequencing the Pacific oyster genome J. Shellfish Res 24: 2; 429 – 441

McRae, T. A., Dutkowski, G. W., Pilbeam, D. J., Powell, M. B., and Tier, B. (2004). Genetic evaluation using the TREEPLAN system. In: Forest Genetics and Tree Breeding in the age of genomics: Progress and Future. 2004 IUFRO Joint Conference of Division 2. Charleston, South Carolina, USA, 1-5 November 2004. (available at http://www.stba.com.au/articles.html)

Ponzoni, R. W. (1988). The derivation of economic values for combining income and expenses in different ways: An example with Australian merino sheep. Journal of Animal Breeding Genetics, 105: 143-153.

Powell, M. B., McRae, T. A., Wu, H. X., Dutkowski, G. W. and Pilbeam, D. J. (2004). Breeding strategy for Pinus radiata in Australia. In: Forest Genetics and Tree Breeding in the age of genomics: Progress and Future. 2004 IUFRO Joint Conference of Division 2. Charleston, South Carolina, USA, 1-5 November 2004. (available at http://www.stba.com.au/articles.html)

Swan, A. A., Thompson, P. A. and Ward, R. D. (2004). Breeding plans for Australian Seafood Industries Pacific oysters. Confidential Report to Australian Seafood Industries by CSIRO Livestock Industries and CSIRO Marine Research. 42 pp.

Ward, R. D., Thompson, P. A., Appleyard, S. A., Swan, A. A. and Kube, P. D. (2005). Sustainable genetic improvement of Pacific oysters in Tasmania and South Australia. Final Report FRDC Project 2000/206. CSIRO Marine and Atmospheric Research and Fisheries Research and Development Corporation. 193 pp.