1 international consultation on pro-poor jatropha development, rome, apr 08hy genetic markers for...

1International Consultation on Pro-poor Jatropha Development, Rome, Apr 08 HY

Genetic Markers for Jatropha Biodiversity Evaluation and Breeding

• Introduction to jatropha• Genetic markers, various

techniques• Controlled pollination and genetic

mapping• Our findings with SSR and AFLP

on jatropha genetic diversity• Methylation sensitive diversity• Implication / discussion

HONG Yan, Group of Plant [email protected]


“It has not been really domesticated….will also require serious amounts of agronomic research”

Nature editorial


Breeding / domestication will increase productivity and improve other agronomic traits

Wild plants vs domesticated ones

Maize


Genotype and environment

• Morphology and agronomic traits are variable for jatropha– Between varieties, also within varieties– Variable performance of sibling plants under same growth conditions

proves the importance of genotype • A few provenance trials* conducted in Africa found:

– Genotype-environment interaction (GxE)– A great range of seed yield– Various crude fat content from 18.4-42.3%

• Trait specific relative contribution remains to be further investigated

*Heller 1996


Genetic Markers• A gene or DNA sequence having a known location on a

chromosome and associated with a particular gene or trait• Advantages of genetic markers:

– Profile of genetic markers will define a genotype – Uniform at all tissues at all ages of the organism– allows early detection– developmentally stable and not affected by the environment– highly specific pattern can be developed for each individual

• Utilities:– For biodiversity study– To choose right parents for cross pollination– Marker assisted breeding– To differentiate plant varieties– Quality control of plantation


Comparison of Different genetic marker techniques

Feature RFLP RAPD* AFLP SSR Sequencing Development cost medium low low high high Running cost high low medium low high Samples/day low high high high low Level of skill required low low medium low-

medium high

Automation difficult yes yes yes yes Radioactivity necessary

yes-no no yes-no yes-no yes-no

Reliability high low-medium high high high Dominant or codominant

codominant dominant dominant (codominant)

codominant codominant

Polymorphism medium medium medium high medium-low

Gustavo Caetano-Anolles and P.M.Gresshoff 1997Gustavo Caetano-Anolles and P.M.Gresshoff 1997

*ISSR as an variant


Genetic marker development and biodiversity studies of jatropha

• Few reports with RAPD and ISSR techniques– Potential problems of reliability and reproducibility

• Generally very low genetic diversity reported in local populations analyzed

• No report on global jatropha genetic diversity

Basha 2007; Xiang 2007


Controlled pollination for breeding in TLL

– Self pollination: for inbred lines and evaluating heredity of traits

– Cross pollination: to generate hybrid seeds between different varieties, also with other Jatropha species for hybrid vigor and new traits


Interspecies jatropha hybrids

• Objectives:– Genetic mapping

– Possible better traits

F1 hybrid plants


Genetic marker development in TLL

• >500 SSR loci were cloned • >50 primer combinations of AFLP

analysis• High throughput fluorescence marker

analysis with ABI 3730xl DNA analyzer

• Both techniques are used together for construction of genetic map of jatropha with BC populations

Examples of SSR markers

Example of SSR allele detection


SSR / AFLP evaluation of jatropha biodiversity

• Work in progress to analyze 192 samples from Asia, South America and Africa

• Preliminary findings:– SSR analysis showed very little diversity, most were homozygous

– EcoRI / MseI AFLP showed very little diversity, too (polymorphic loci <5%, mean heterozygosity < 1%)

Monomorphic band pattern shown for most SSR loci


Low genetic diversity as detected by general AFLP analysis


E2/H3-HpaII-methylation sensitive

PZ14A

SU6

MD42

MD44

E2/H3-MspI-methylation insensitive

Methylation sensitive AFLP

C C G GG G C C

m5m5

m5C C G GG G C C

m5

m5

Cut

X

No cut

Methylation sensitive AFLP analysis


Segregation of methylation sensitive alleles

E1H2-376 E1H9-590 E4H5-91 E4H3-253MD24 1 1 1 1Selfed progenies 18,7 25,0 18,6 20,5

E1H2-376 E1H9-590 E4H5-91 E4H3-253MD24 1 1 1 1TN02 0 0 0 0F1 hybrids 7,10 17,0 9,8 10,7Genotype Aa AA Aa Aa


Epigenetic diversity in jatropha

• Methylation sensitive AFLP

• Much higher diversity observed in methylation sensitive AFLP analysis (polymorphic loci >60%, mean heterozygosity >14%)

• Stable in various parts of a plant

• Heritable, follows Mendelian segregation

• Most diversity found within populations

• Biological significance not known yet


DNA methylation in plant

• Best characterized epigenetic modification

• Implicated in may important biological processes

Inheritance of methylation pattern

Interference of DNA methylationin Arabidopsisby down regulateMET1, a methylase gene


Discussion

• Reasons for the lack of biodiversity so far– Technical or inherent?

• If low genetic diversity confirmed, issues to address:– Genetic basis for phenotype?

– Breeding strategy?

– Any significance role of epigenetic in jatropha?

• Suggestions:– A global survey of genetic diversity is important and critical

– International collaboration and verification is necessary


Temasek Life Sciences Laboratory (TLL)

• A non-profit research institute owned by Temasek holding, NUS and NTU

• With 30 research groups, we work on both basic and applied science

• One center of excellence in the area of plant tissue culture, genetic modification, plant biology and development study

• Experience and mechanism to collaborate and work with industry

• A cluster with about 20 scientists working on jatropha:

– Biodiversity and breeding– Genetic markers and mapping– Platform technology development– Phytochemistry– Genomics– Molecular and cell biology studies

• Two experimental farms

1 Research Link, National University of Singapore, Singapore 117604

Tel: +65 6872 7000Fax: +65 6872 7007http://www.tll.org.sg


Jatropha International CongressJatropha International CongressDecember 17 -18, 2008, SingaporeDecember 17 -18, 2008, Singapore

First Circular First Circular

Organized by Temasek Life Sciences LaboratoryOrganized by Temasek Life Sciences Laboratory

A True Event for Biofuel Research and BusinessA True Event for Biofuel Research and Business

Themes of the congress

1. Genetic resources 2. Breeding technologies and strategies3. Genetics and Genomics4. Biosynthesis of fatty acids and toxins5. Tissue culture, propagation and transformation6. Plantation management and pest control 7. Oil processing 8. By-product utilization 9. Investment and business models10. Government policies and incentives

1 international consultation on pro-poor jatropha development, rome, apr 08hy genetic markers for...

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