gene pyramiding for biotic stress tolerance in …...weekly science research journal primary article...

Weekly Science Research Journal

Primary Article

Gene Pyramiding For Biotic Stress Tolerance In Crop Plants

S. Suresh and D. Malathi

S. Suresh and D. Malathi

FromDepartment of Plant Breeding and

Genetics,Agricultural College and Research

Institute, Madurai, Tamil Nadu, INDIA.

Article Is Published On December 2013 Issue & Available At

www.weeklyscience.org

10.9780/ 2321-7871/1202013/53DOI :

ABSTRACT

Yield and quality are central to sustainable crop production. Due to biotic stress, yield loss upto 52 per cent. Chemical control of insect and disease is not feasible for environments and economical not viable. Host plant resistance is the only safe, economical, and environment friendly way for the absolute control of this devastating pest and disease. Historically, long-term cultivation of varieties carrying single resistance gene has resulted in a significant shift in pathogen or insect race frequency and consequent breakdown of resistance. One tangible solution to resistance breakdown is pyramiding of multiple resistance genes in the back-ground of modern high yielding varieties. Gene pyramiding aims to assemble multiple desirable genes into a single genotype. The main use of gene pyramiding is to improve an existing elite cultivar through introgression of a few genes of large effects from other sources, since the presence of the target genes has to be monitored by phenotyping, which is only effective for major genes. Gene pyramiding is difficult to achieve using conventional breeding alone because of linkage with some undesirable traits that is very difficult to break even after repeated backcrossing. .

Biotic Stress , environments , phenotyping.Keywords:

Biotic Stress

Host Plant Resistance

Biotic stress is that occurs as a result of damage done to plants by other living organisms, such as bacteria, viruses, fungi, parasites, harmful insects, and weeds. Due to biotic stress up to 35% of the total food production is lost. The estimated crop loss was of the value of Rs 90,000 Crores in 2004 and in 2009, Rs 1,40,000 Crores.

Is the collective heritable characteristics by which a plant species, race, clone or individual may reduce the possibility of successful utilization of that plant as a host by an insect species, race, biotype or individual.1. Identification of stable sources understanding components and inheritance

stress

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of resistance2. Utilization of wild relatives as gene sources to increase the levels and diversify the bases of resistance

3. Exploitation of novel genes and molecular marker approaches for pest resistance

4. Development of varieties with improved yields and better resistance to the target pests.

'Watson & Singh' (1953) first introduced the concept called gene pyramiding. Gene pyramiding is defined as a method aimed at assembling multiple desirable genes from multiple parents into a single genotype. The end product of a gene pyramiding program is a genotype with all of the target genes.

1) Enhancing trait performance by combining two or more complementary genes, 2) Remedying deficits by introgressing genes from other sources, 3) Increasing the durability.

Serial gene pyramiding: Genes are deployed in same plant one after other.* Pedigree breeding * Backcross breeding * Recurrent selection

Simultaneous gene pyramiding: Genes are deployed at a time in a single plant. * Marker assisted selection * Transgenic method

Backcross breeding

Gene Pyramiding Concept

Objectives of gene pyramiding include:

Types Of Gene Pyramiding

1. Conventional technique -

2. Molecular technique -

Conventional technique

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2321-7871 th Vol-1, Issue-23, 26 December 2013Gene Pyramiding For Biotic Stress Tolerance In Crop Plants

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Pedigree breeding

Recurrent selection

V. Disadvantages Of Conventional Methods

Molecular technique

Marker Assisted Selection

1. Gene pyramiding is mainly used to improve qualitative traits such as disease and insect resistance. This is associated with the fact that the presence of target trait genes must be confirmed by phenotyping mostly at the individual level and that individual phenotypic performance is a good indicator of the genotype only if genes have a major effect on phenotypic performance and the error of phenotyping is minimal. 2. In addition to the reliability of phenotyping at individual level other factors influencing the success of gene pyramiding are the inheritance model of the genes for the target traits, linkage and/or pleiotropism between the target trait and other traits. 3. For instance, allelic genes cannot be combined in the same genotype. The effect conferred by a recessive gene cannot be evaluated on heterozygous individuals and progeny testing is required. 4. If the target gene is tightly linked to genes with large negative effects on other traits, these undesirable genes may be transferred together with the target gene into the recipient line and result in reduced performance of other traits (linkage drag). Therefore, any improvement in the knowledge of the trait genetics (inheritance, genetic relationship, etc.) and techniques for inferring genotype-phenotype relationship will be useful.

(1) Use of DNA markers that are tightly-linked to target loci as a substitute for / to assist phenotypic screening (2) A marker is a “genetic tag”


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(3) Use of molecular markers for indirect selection of different traits(4) Speeding up the process of conventional breeding (5) Facilitating the improvement of traits

Marker-assisted selection (MAS) is a method of rapidly incorporating valuable traits into new cultivars. Molecular markers, or DNA tags, that have been shown to be linked to traits of interest are particularly useful for incorporating genes that are highly affected by the environment, genes for resistance to diseases and pests, and to accumulate multiple genes for resistance to specific diseases and pests within the same cultivar – a process called gene pyramiding. One of the first wheat cultivars to be developed using MAS was the soft winter wheat cultivar “Madsen”, released in 1986 by the USDA-Agricultural Research Service (ARS) and Washington State University. “Madsen” was developed using the isozyme marker from the endopeptidase protein, EpD1b, to incorporate a gene for resistance to eyespot (Tapesia yallunde) (Allan et al. 1989). Since 1990, detailed molecular maps of wheat have been constructed that include more than 3,000 molecular markers and several important traits have been associated with DNA markers. Additional markers can be developed from the 8,000 expressed sequence tags (ESTs) that have been mapped in wheat. (Maps and references are available on-line (USDA-ARS 2005).)

In a gene pyramiding scheme, strategy is to cumulate into a single genotype, genes that have been identified in multiple parents. The use of DNA markers, which permits complete gene identification of the progeny at each generation, increases the speed of pyramiding process. In general, the gene pyramiding aims at the derivation of an ideal genotype that is homozygous for the favorable alleles at all the loci. The gene pyramiding scheme can be distinguished into two parts (Figure). The first part is called a pedigree, which aims at cumulating of all target genes in a single genotype called the root genotype. The second part is called the fixation step which aims at fixing the target genes into a homozygous state i.e. to derive the ideal genotype from the one single genotype. Each node of the tree is called an intermediate genotype and has two parents. Each of this intermediate genotype variety can resist. Moreover, pyramiding can also improve becomes a parent in the next cross. The intermediate genotypes are not just an arbitrary offspring of a given cross but it is a particular genotype selected from among the offspring in which all parental target genes are present.

Marker Assisted Gene Pyramiding

Distinct Gene Pyramiding Scheme


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Although the pedigree step may be common, several different procedures can be used to undergo fixation in gene pyramiding. Generation of a population of doubled haploids from the root genotype is a possible procedure for the fixation steps. Here, a population of gametes is obtained from the genotypes and their genetic material is doubled. This leads to a population of fully homozygous individuals, among which the ideotype can be found. Using this process, the ideal genotype can be obtained in just one additional generation after the root genotype is obtained. However, producing large population of doubled haploid is difficult and cumbersome in certain plant species. A possible alternative to this method is to self the root genotype directly to obtain the ideal genotype. However, selfing the root genotype will result in the breakage of linkage between the desired alleles and it will be difficult to derive this breaks as the linkage phase is rarely visible in selfed populations. As a result, it may span too many generations thereby stretching the gene pyramiding scheme. Another alternative to all this methods would be to obtain a genotype carrying all favorable alleles in coupling by crossing the root genotype with a parent containing none of the favorable alleles. This confirms that the linkage phase of the offspring is known and the genotype can be derived without any mixing. The ideal genotype will be reached within two generations after the root genotype. However, instead of crossing with a blank parent, a more simplified method would be to cross the root genotype with one of the founding parents. In such programs, the linkage will still be known, and the selection will be for genotypes that are homozygous for the target gene brought by the founding parent but heterozygous for other regions. The desired genes need not be fixed subsequently, thereby increasing the probability of getting the ideal genotype. This is called as marker assisted backcross gene pyramiding. By far this is the most accepted and efficient method to do the gene pyramiding.

Breeders transfer a target allele from a donor variety to a popular cultivar by a repetitive process called backcrossing; which, unfortunately, is slow and uncertain. Breeding a plant that has the desired donor allele but otherwise looks just like the popular cultivar usually takes four years or longer. Worse, the augmented variety may look just like the popular cultivar, but it inevitably retains stray chromosome segments from the donor. Consequently, to a greater or lesser extent, it will fail to perform exactly like the popular cultivar, thus limiting its appeal to farmers. Marker-assisted breeding tackles both problems by allowing breeders to identify young plants with the desired trait and by facilitating the removal of stray donor genes from

Marker Assisted Backcrossing


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intermediate backcrosses. The result, in about two years, is an improved variety exactly like the popular cultivar except that it possesses the transferred advantageous gene. In principle, this technique can be applied to the breeding of any crop or farm animal. So far, however, breeders of trees and rice have dominated the field. Because markers allow breeders to select immature plants, the time saved in breeding slow-growing trees is immense. In the case of rice, the crop's relatively advanced state of genetic mapping has facilitated the application of molecular marker techniques. Markers are effective aids to selection in backcrossing in three ways. First, markers can aid selection on target alleles whose effects are difficult to observe phenotypically. Examples include recessive genes, multiple disease resistance gene pyramids combined in one genotype (where they can epistatically mask each other's effects), alleles that are not expressed in the selection environments (e.g., genes conferring resistance to a disease that is not regularly present in environments), etc. Second, markers can be used to select for rare progeny in which recombination near the target gene have produced chromosomes that contain the target allele and as little possible surrounding DNA from the donor parent. Third, markers can be used to select rare progeny that are the result of recombination near the target gene, thus minimizing the effects of linkage drag. In general, the marker assisted backcross based gene pyramiding can be performed in three strategies (Figure 2). In the first method, the recurrent parent (RP1) is crossed with donor parent (DP1) to produce the F1 hybrid and backcrossed up to third backcross generation (BC3) to produce the improved recurrent parent (IRP1). This improved recurrent parent is then crossed with other donor parent (DP2) to pyramid multiple genes. This strategy is less acceptable as it is time taking but pyramiding is very precise as it involve one gene at one time. In the second strategy, the recurrent parent (RP1) is crossed with donor parents (DP1, DP2, etc.) to get the F1 hybrids which are then intercrossed to produce improved F1 (IF1). This improved F1 is then backcrossed with the recurrent parent to get the improved recurrent parent (IRP). As such, the pyramiding is done in the pedigree step itself. However, when the donor parents are different, this method is less likely to be used because there is chance that the pyramided gene may be lost in the process. The third strategy is an amalgamation of the first two which involve simultaneous crossing of recurrent parent (RP1) with many donor parents and then backcrossing them up to the BC3 generation. The backcross populations with the individual gene are then intercrossed with each other to get the pyramided lines. This is the most acceptable way as in this method not only time is reduced but fixation of genes is fully assured. Marker assisted backcrossing to be effective, depends upon several factors, including the distance between the closest markers and the target gene, the number of target genes to be transferred, the genetic base of the trait, the number of individuals that can be analyzed and the genetic background in which the target gene has to be transferred, the type of molecular marker(s) used, and available technical facilities (Weeden et al., 1992; Francia et al., 2005). When these entire selection criterions are maintained properly, only then a well acceptable MAB based gene pyramiding scheme can lead to durable crop improvement.


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Different schemes of backcrossing for gene pyramiding. RP- Recurrent parent; DP- Donor parent; BC- Backcross; IRP- Improved recurrent parent. A. Stepwise

transfer; B. Simultaneous transfer; C. Simultaneous and stepwise transfer.

(1) Agrobacterium mediated method (2) Biolistics (particle bombardment) (3) Protoplasts (4) Chemical method (5) Whole -Tissue Electroporation (6) Silicon Carbide Whiskers (7) Viral Vectors (8) Laser Micropuncture (9) Nano?ber Arrays (10) DNA uptake in Pollen (11) Pollen Tube Pathway (12) Electrotransformation of Germinating Pollen Grain (13) Floral Tiller Injection (14) Lipofection (15) Microinjection (16) Direct DNA uptake by mature zygotic embryos.

Agrobacterium is a soil borne bacterium that has been rightfully called the “natural plant genetic engineer.” Over its evolutionary journey, this bacterium has developed the unique ability to transfer part of its DNA into plant cells. The DNA that is transferred is called the T-DNA (for transferred DNA) and this DNA is carried on an extra chromosomal plasmid called the Ti (tumor-inducing) plasmid. Through intervention of scientists, the Ti plasmid no longer causes tumor formation in infected plant cells, but the T-DNA region is still transferred. As opposed to DNA transfer methods that utilize direct uptake of DNA into plant cells, the use of Agrobacterium may appear to be more complex because two different biological systems (bacteria and the target plant cells) are involved. This might have been true in the early years of plant transformation, but today, Agrobacterium provides the method of choice for most plant transformation efforts. With methods utilizing introduction of DNA without a biological vector (direct DNA uptake), it appears to be necessary to deliver the DNA to the nucleus of the target cell, but with Agrobacterium, the T-DNA itself possesses the necessary signals for delivery there. Most direct DNA introduction systems require expensive instrumentation, but Agrobacterium is simply prepared by growth on an appropriate medium and inoculated on the plant tissue. Additional claims of simpler foreign DNA insertions and more consistent transgene function in plants transformed with Agrobacterium may or may not be valid, and this appears to depend more on how the DNA is delivered with direct DNA introduction systems than on any inherent problem with the method. Considering primarily overall transformation efficiency, advances since the mid-1980s in our understanding of the Agrobacterium-mediated DNA transfer process have led

TRANSGENIC METHOD

Gene transfer methods

(i). Agrobacterium mediated transformation


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to tremendous increases in efficiency and use of this transformation vector.

The transition from forming tumors on tobacco stems to routinely transforming wheat and corn with speci?c genes of interest resulted from multiple advances in the understanding of both the T-DNA process and the interaction of bacteria with plant cells. Since there are many thorough reviews on the mechanism of Agrobacterium T-DNA transfer (Binns and Thomashow 1988; Zamb ryski 1992; Tz?ra and Citovsky 2006), only the basic features as relating to transformation are presented here (Figure 1).

To start, the plasmid that is used as a vector for Agrobacterium-mediated transformation has been whittled down to contain only the essential components. Agrobacterium vectors are called “binary vectors” because they are the second of two plasmids that are involved in the overall process. Many of the transfer functions are retained on a modified Ti plasmid with the T-DNA removed. The second binary vector contains the T-DNA, but the hormone and opines biosynthesis genes have all been deleted. What is left on the binary vector, aside from the components that allow the plasmid to be retained in the bacterium, are the le ft and right “borders” of the T-DNA region. Genes of interest are cloned between the borders, which are recognition sequences for the T-DNA processing machinery. The genes for the T-DNA processing machinery are still located primarily on the modi?ed Ti plasmid where they direct T-DNA processing on the binary plasmid. After Agrobacterium is inoculated on the appropriate plant tissue, the bacteria may recognize the target tissue as a suitable host; remember that this bacterium is a pathogen that infects plant tissue. Chemical signals are put out by both the plant tissue and the bacteria. Wounded plant tissues from appropriate plant tissues produce acetosyringone, which activates the bacterial virulence (vir) genes, which initiates the T-DNA transfer machinery. Not all wounded plant tissues produce acetosyringone, and the lack or poor production of acetosyringone by monocot cells originally made it difficult to impossible to produce transgenic monocots using Agrobacterium. Addition of synthetic acetosyringone to the inoculated plant tissues allows Agrobacterium-mediated transformation of monocots to proceed and tremendously enhances transformation of other moderately susceptible target plants. Once the bacteria infect plant tissue, most plants will respond by trying to fight off the invasion by either producing antipathogenic compounds or sacrificing cells adjacent to the infected region to prevent spread of the invasion. Pathogens, in turn, have developed methods to introduce regulatory compounds into plant cells, in an attempt to shut down the defensive machinery of the target cell.

Use of the T-DNA Transfer Process for Transformation


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Although some of these mechanisms are known, some are still being investigated, and a more thorough understanding of the infection process will allow further increases in the ef?ciency of Agrobacterium-mediated transformation. Once the vir genes are activated, the T-DNA on the bi nary vector is processed for transport to the target plant cell. Some of the vir gene products excise the T-DNA from the bi nary plasmid as a single-stranded DNA molecule, while other vir gene products coat the T-DNA to prevent degradation. Yet additional vir gene products bind to the T-DNA to act as navigators or signals to direct the DNA out of the bacterium, through the plant cytoplasm, and to the nucleus. Through the action of other vir genes, the bacterium produces a pillus, which is the conduit for transfer of the T-strand (the single- stranded, coated, signal containing T-DNA is the “T-strand”) from the bacterium to the target plant cell. The pillus is essentially a protein tube, which extends from the bacterium through the cell wall and into the cytoplasm of the target cell. After the T-strand is delivered to the nucleus, the last role of the signal protein on the T-strand is to find and nick the host DNA as an insertion point for the T-DNA. The T-DNA appears to insert primarily into gene-rich and transcriptionally active regions of DNA that are more exposed and accessible.

Fig. 1 Schematic of Agrobacterium-mediated transformation of a plant cell, showing production of the T strand from the binary vector, transport through the

bacterial pillus, and integration into plant chromosomal DNA.

For DNA introduction using particle bombardment, DNA is first precipitated onto the particles using either calcium chloride or ethanol, which are commonly used for DNA precipitation. When the DNA precipitates, it sticks to whatever is at hand. It is unclear how “tightly” the DNA is bound to the particles, but it must be able to withstand the incredible force of acceleration and cell wall /cytoplasm penetration and also come off the particles after delivery. During bombardment, the majority of the metal particles do not find their target. Most of the particles embed in the cell wall, enter the vacuole, or end up somewhere else in the cytoplasm; only a few reach the nucleus. After all, thousands of particles are delivered using literally a “shotgun” approach. Evaluation of those cells that express the introduced DNA shows that the overwhelming majority of cells (>90%) have particles either adjacent to or in the nucleus (Figure) (Yamashita et al., 1991; Hunold et al., 1994). Unlike Agrobacterium, where integration of the introduced DNA into the plant chromosomal DNA is orchestrated by bacterial proteins that are bound to the T-strand, particle bombardment results in the introduction of naked DNA. Clues to the fate of the introduced DNA can be taken from studying the final arrangement of the integrate d DNA within plant chromosomal DNA. In general, the patterns of DNA integration in the plant chromosome are very complex. To be more speci?c, it can be a real mess. Usually, the introduced DNA

(ii). Particle Bombardment


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integrates into a single site (locus) on the chromosomal DNA. However, the introduced DNA can also integrate at multiple sites, which makes analysis more difficult. To complicate the situation further, it is common to obtain multiple copies of the transgene in each integration site. And (it gets worse) the copies can be partial copies, with varying orientations. In addition (last thing), the introduced DNA appears to be mixed or interspersed with plant genomic DNA (Pawlowski and Somers 1998). Imagine the replication and repair machinery of the nucleus as an army of overworked, frantic, multiarmed, DNA tailors. The DNA tailors are supposed to make exact copies of chromosomes and fix any small mistakes, while they are sewing huge amounts of new DNA strands. They are working fine until the whoosh of this huge boulder (1- mm particle) overhead that is carrying DNA. It looks like plant DNA, so they take what they can and use it in their sewing operation. It is not a perfect fit, but they are frantic and under the time constraints to get the entire chromosomal DNA replicated before the cell divides. For particle bombardment, it is unclear whether the particles actually physic ally break the chromosomal DNA or merely deposit DNA in the proximity of the replicating parts of chromosomes. It is clear that the introduced DNA can integrate into chromosomal DNA, with very complex patterns. However, complex integration patterns can be largely controlled by manipulating the configuration of the introduced DNA.

Two different particle bombardment devices: (a) the commercially available PDS-1000/He (Bio-Rad) and (b) the noncommercial particle inflow gun (right).

Computer simulations and theoretical calculations have provided powerful tools for analyzing the efficiency of gene pyramiding programmes. Three different gene pyramiding schemes, one based on a cascading pedigree, and two based on the order of crosses of the founding parents were evaluated to check the transmission probabilities of the target genes and the cumulated population size needed in each scheme (Ribaut and Hiosington, 1998; Ribaut et al., 2001; Hospital et al., 2004). The simulation was based on identical recombination fractions between adjacent loci and spaced about 20 cM.

A MAS based gene pyramiding scheme based on a cascading pedigree is less expensive as it spans five generations in general and requires the smallest cumulated population size of all the schemes. The average transmission probability is 0.9975. Gene pyramiding scheme based on the crosses of founding parents spans four generations but the population size is somewhat higher. The average transmission probability is 0.9967. When gene pyramiding is carried out involving a larger number of target genes, each trait starts as a founding parent resulting in intermediate genotypes by subsequent crossing. It is based on a cascading pedigree and span one or two less generation in general.

1. Widely used for combining multiple disease resistance genes for specific races of a

Efficiency Of Gene Pyramiding

Advantages Gene Pyramiding


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pathogen2. Pyramiding is extremely difficult to achieve using conventional methods3. Consider - phenotyping a single plant for multiple forms of seedling resistance – almost impossible4. Important to develop 'durable' disease resistance against different races5. Main used to improve existing elite cultivar6. Eliminates extensive phenotyping 7. Control linkage drag8. Reduces breeding duration

1. Characteristics of the target traits/genes2. Reproductive characteristics3. A breeder's capability to identify the 'desired' genotypes4. Operating capital

When the genes to be pyramided are functionally well characterized and markers used for selection equal to the gene itself (perfect markers), gene pyramiding will be more successful. For qualitative traits controlled by one or a few genes, the identification of the genes and tightly linked markers is easier provided phenotyping is carefully conducted. One or two markers per gene can be used for tracing the presence/absence of the target genes. Bulk segregant analysis (BSA) is the preferred method for the identification of markers tightly linked to a major gene (Michelmore et al., 1991). For BSA plants from a segregating population are grouped according to phenotypic expression of the trait into two bulks. The bulks are screened with a large number of markers to identify those that distinguish the bulks and, by inference, must be genetically linked to the trait locus. When the target genes are QTL with moderate or small effects, pyramiding may be less successful due to the following reasons.

(1) The identified QTL may be more likely to be a false positive. (2) Inaccurate QTL localizations result in the need to select for more marker loci covering large genomic segments to be certain that target QTL alleles are retained in selected progeny (Hospital and Charcosset 1997). (3) QTL effects may be specific to a particular genetic background. Moreover, markers identified for a QTL can be ineffective in monitoring the QTL since the marker-QTL association might be different from population to population. (5) More QTL need to be pyramided to achieve a significant improvement.

The propagation capability of a crop is determined by the number of seeds produced by a single plant. This capacity determines the population size applicable if seed has to be collected from only a single plant. In a gene pyramiding program, in most generations this is the case, since the chance of selecting two or more individuals of exactly the same genotype in previous generation is very low. For example, although a fairly large F2 population can be obtained by collecting seed from many F1 plants of the cross between two homozygous parents, from the F3 gene-ration seed can only be collected from a single plant. The fact that F1 plants of the cross between two homozygous parents are genetically the same can also be used to increase the size of a progeny population of the F1 plants of two crosses (double cross) or of the F1 plants of one cross and an inbred line (Three-way cross or testcross). The efficiency of hybridization may be an important constraint for some crop species. When wild relatives are used as the donor of desirable genes, many more reproduction related constraints may exist including cross incompatibility between the wild species and cultivated crop. F1-hybrid sterility, infertility of the segregating generations, reduced recombination between the chromosomes of the two species. Appropriate techniques that may include chemical treatment and immature embryo culture for overcoming these problems must be established.

Main Factors Affecting Gene Pyramiding

1. Characteristics of the target traits/genes

2. Reproductive characteristics


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3. A breeder's capability to identify the 'desired' genotypes

4. Operating capital

It is obvious that the desirable genes must be present in all generations leading to the target genotype. To ensure the presence of the target genes individuals of desired genotype (which may change with generation advance) must be identified among all individuals in each generation. Breeder's capability to identify the desired genotypes has been greatly enhanced by the use of tightly linked or diagnostic markers. It might be appropriate to consider the importance of marker and trait gene linkage here. The efficiency of marker-based gene pyramiding will decrease substantially if the markers are not perfectly or tightly linked with useful trait genes. Association of a marker with a trait allele and consequently the reliability of the marker-based selection decreases with increasing cycles of meiosis. With a recombination value of r between a marker and trait allele, the

mprobability of this linkage being maintained across m cycles of meiosis is equal to (1 - r) . To keep this probability higher than a certain critical value, say P, m must not exceed lnP/ ln (1 - r), suggesting that a phenotypic test should be performed every m generations of selection to confirm the persistence of the initial linkage. Put in another way, the

tprobability of losing the target allele by recombination is 1 - (1- r) . For example, if the marker locus exhibits 10% recombination with the target gene, there is a 10% chance of losing the target allele each gene-ration, and a 27% chance of losing the target allele after three generations of meiosis. However, if the recombination frequency is 1%, there is only a 3% chance of losing the target allele after three generations of meiosis. When tightly linked markers are not available, selection on a pair of markers flanking the target locus can be very effective. If two marker loci M1 and M2 flank the target locus, one would select progeny that have both M1 and M2 alleles. The probability of losing the target allele with flanking marker selection is equal to the probability of selecting a double recombinant progeny from among the doubly heterozygous backcross progeny. If the flanking loci have recombination frequencies r1 and r2, respectively, with the target locus, the probability of losing the target allele due to double crossovers within the selected region is:

This probability can be much lower than the probability of losing the target allele based on selection for a single marker. For example, if the flanking markers each have 10% recombination frequency with the target locus, there is only a 1.2% chance of losing the target allele after a single generation. In any case, with tighter linkage, the chance of losing the target allele is reduced. However, this requires more plants to be tested and higher cost per plant. It is imperative to use markers that are tightly linked with trait genes. Multiple marker loci closely linked to the target gene, permits discrimination on the basis of the haplotype of several markers rather than just the genotype at one marker. For example, Cregan et al. (1999) developed two SSR markers tightly linked to the rhg1 gene. Neither marker alone could distinguish all resistant from all susceptible genotypes, because of identity in state alleles shared by some resistant and susceptible lines, but the two markers together could discriminate almost all resistant and susceptible lines. One resistant cultivar carried the susceptible allele at both loci, presumably due to recombination between marker and resistance loci during line development. Thus, recombination can change the linkage phase between markers, but if MAS is used first to select putatively resistant lines, followed by phenotypic evaluation of resistance, the linkage phase will remain intact in all selected progeny. Therefore, MAS can be self-reinforcing, ensuring that the same set of markers will be effective in future crosses.

All breeding programmes are operated within the limits of available operating capital. Therefore, reducing the overall cost is always an important consideration when choosing a strategy. In addition to the use of the most economic mating and testing approaches, other factors affecting the cost also need to be considered. In the context of gene pyramiding, cost affects both what can be achieved and how to achieve it. Increasing


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the number of generations (duration) will reduce the pressure on population size required in each generation and may result in the reduction of the total cost. However, increasing the duration delays the release of the new cultivar and consequently reduced market share. The well-known trade-off between duration and cost in breeding has no exception in gene pyramiding. To find an optimum balance between duration and cost is desired but very difficult to achieve. In practice, the best strategy may be defined as the one that enabled the breeder to achieve the objectives with the shortest duration and within a fixed expected investment.

Gene pyramiding is an important strategy for crop improvement. Pyramiding requires that breeders consider the minimum population size that must be evaluated to have a reasonable chance of obtaining the desired genotype. Molecular marker genotyping can facilitate the gene pyramiding process by reducing the number of generations that breeders must evaluate to ensure they have the desired gene combination.

Barloy D., Lemoine J., Paulette A., Tanguy M., Roger R., Joseph J. (2006). Marker-assisted pyramiding of two cereal cyst nematode resistance genes from Aegilops variabilis in wheat. Mol. Breed. 20: 31-40. Bates S.L., Zhao J., Roush R.T., Shelton A.M. (2005). Insect resistance management in GM crops: past, present and future. Nat. Biotechnol. 23: 57-62.Bertrand Servin, Olivier C. Martin, Marc Mezard and Frederic Hospital. (2004). Toward a Theory of Marker-Assisted Gene Pyramiding. Genetics Society of America.Francia E, Tacconi G, Crosatti C, Barabaschi D, Bulgarelli D, Dall'Aglio E, Vale G (2005). Marker assisted selection in crop plants. Plant. Cell. Tissue. Organ. Cult. 82: 317-342.

Success Stories

CONCLUSION

References

Crops Traits Pyramided Genes References

Rice

Blight resistance Xa4, xa5, xa13, Xa21 Xa 5, Xa13 and X a21

S ingh et al., 2001, Narayanan et al., 2002 Joseph et al., 2004

Blast resistance Pi(2)t, Piz5, Pi(t)a H ittalmani et al., 2000

Gall-midge resistance Gm2,Gm6 Gm1, Gm4

Katiyar et al., 2001 Kumaravad ive l et al., 2006

BPH resistance Bph1 and Bph2 Sharma et al., 2004

Multip le res istance

Bacterial blight (Xa21 ) Sheath b light (RC7) Yellow stem borer Bt fusion gene (cry1AB /cry1Ac)

Datta et al., 2002

Wheat

Leaf rust resistance Lr41, Lr42, Lr43 Cox et al., 1994

Powdery mildew resistance Pm2 + Pm4a; Pm2 +Pm21; Pm4a+ Pm21

L iu et a l., 2000

Aphid res istance Gn2 and Gn4

Cereal cyst nematode CreX and CreY Domin iques et al., 2007

Cotton Insect pest resistance Cry 1Ac, Cry 2Ac Gahan et al., 2005

Bacterial blight and Sheath blight chi11, tlp and Xa21 Maru thasa lam et al., 2007

Pea Nodulation abil ity Sym9, Sym10 Schneider et al., 2002

Barley Yellow mosaic virus resistance rym4, rym5, rym9,rym11 Werner et al., 2005

Stripe rust res istance 3 QTL Castro et al., 2003

Broccoli Diamond back moths resistance cry1 Ac +cry1c Cao et al., 2002

Soybean Soybean mosaic virus resistance Rsv1, Rsv3, Rsv4 Z hu et al., 2006

Lepidopteran resistance cry1Ac +corn ear worm QTL Walker et al., 2002

Chickpea Lepidopteran resistance cry1Ac + cry1Ab Meenakshi et al., 2011


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