gene pyramiding in india

PresentationOnPYRAMIDING GENES PROCEDURE, IMPORTANCE AND

WORK DONE IN INDIA IN VARIOUS CROPS

Submitted ToDr. K.N.SinghProf. & HeadDeptt. of PMB & GE

Submitted byBrijesh YadavM.Sc. (Ag.) BiotechnologyId.No.-A-6936/12

Narendra Deva University of Agriculture & Technology

Kumarganj- Faizabad (U.P.)

Contents:

1.INTRODUCTION2.PROCESS OF DESIGNING A GENE PYRAMIDING STRATEGY3.MARKER-ASSISTED BACKCROSSING4.GUIDELINES FOR DESIGNING AN EFFICIENT GENE PYRAMIDING STARTEGY

5.EFFICIENCY OF GENE PYRAMIDING6.MAIN FACTORS AFFECTING GENE PYRAMIDING7.INTEGRATING GENE DISCOVERY, VALIDATION AND PYRAMIDING8.POLYGENIC TRAIT IMPROVEMENT BY GENE PYRAMIDING- A STEP FORWARD

9.WORK ON GENE PYREMIDING10.CONCLUSI ON

INTRODUCTIONGene 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. Generally speaking, the objectives of gene pyramiding include: 1) enhancing trait performance by combining two or more complementary genes, 2) remedying deficits by introgressing genes from other sources, 3) increasing the durability of disease and/or disease resistance, and 4) broadening the genetic basis of released cultivars .Traditionally, 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.

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It has also widened several aspects of the practical application of gene pyramiding:Firstly, for traits that are simply inherited, but that are difficult or expensive to measure phenotypically, and/or that do not have a consistent phenotypic expression under certain specific selection conditions, marker-based selection is more effective and /or economic than phenotypic selection. Secondly, traits which are traditionally regarded as quantitative and not targeted by gene pyramiding program can be improved using gene pyramiding if major genes affecting the trait are identified.Thirdly, genes with very similar phenotypic effects, which are impossible or difficult to combine in single genotype using phenotypic selection, can be pyramided through marker-assisted selection (MAS).Fourthly, markers provide a more effective option to control linkage drag and speed up the recovery of recurrent genome and make the use of genes contained in un adapted resources easier. Marker-assisted gene pyramiding is currently the method of choice for inbred line development targeted at improving traits controlled by major genes.

2. PROCESS OF DESIGNING A GENE PYRAMIDING STRATEGY

Bringing all the desirable alleles into a single genotype is the overall objective of a gene pyramiding program. When the number of parental lines containing the desirable genes (founding parents) is more than three, more than one crossing scheme can result in the generation of the target genotype. Therefore, the gene pyramiding scheme can be divided into two parts. The first part is aimed at cumulating one copy of all target genes in a single genotype (called root genotype). The second part is aimed at fixing the target genes into a homozygous state, that is, to derive the target genotype from the root genotype. Diagramatic representation of a gene-pyramiding scheme cumulating six target genes from six parental lines.

a. Designing the Fixation SchemeAssuming that a genotype with a copy of the desirable allele at each of the targeted loci (root genotype) is available, the design of an optimal strategy is aimed to find the minimum number of generations for genotyping and/or phenotyping required to fix all the loci for the desirable alleles within the limit of the largest possible population size applicable. The most commonly used methods for the production of homozygous individuals are the development of recombinant inbred lines (RIL), and doubled-haploid (DH) population. Therefore, it is advisable to investigate the feasibility of achieving the objective using RIL or DH.

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The objective of this step is to identify a selection scheme that leads to the production of the target genotype using the minimum number of generations and the practically allowable population sizes in each of the generations. The choice of the parent to use may be subject to particular considerations depending on the value of the founding parents, the position of the loci, etc.b. Designing the Cross SchemeA crossing scheme which leads to the production of the root genotype needs to be designed if the objective is achievable based on the above step.

3. MARKER-ASSISTED BACKCROSSINGBreeders 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.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 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.

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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.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.

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In general, the marker assisted backcross based gene pyramiding can be performed in three strategies

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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.

4. GUIDELINES FOR DESIGNING AN EFFICIENT GENE PYRAMIDING STARTEGY

a. Guidelines for designing a gene pyramiding crossing schemei. Founding parents with fewer target markers enter the schedule at earlier stagesThis guideline is based on the following facts:1)Once a target gene has been incorporated into an intermediate genotype, genotyping must be done in all later stages to ensure its presence. Therefore, founding parents with more target genes should be used in later stage.2) Target genes containing in a founding parent are in desired linkage phase, which may be broken down due to recombination. The more the meiosis involved the lower the probability of maintaining the desired linkage.

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ii. A cross that invokes a strong repulsion linkage should be performed as early as possibleWhen the target genes are linked, genes linked in repulsion at some stages of the pyramiding is unavoidable and selection for recombinants is required. As the frequency of recombinant type is always lower than that of the parental types, larger population sizes are required to recover the desired recombinant. iii. More crosses should be conducted at each generation if genotyping cost is low and the practically applicable population size is largeWhen the maximum number of crosses is performed at each generation, the number of generations required to generate the root genotype is reduced and thus the total duration of the pyramiding program is reduced.

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iv. One cross per generation is required if the practically applicable population size is small or genotyping cost is highIn this type of crossing design, from the second generation the desirable genotype is formed by a recombinant gamete produced by the selected genotype in the last generation and a gamete of the newly introduced parent.The drawback to this crossing design is that the number of generations is large and the production of the new line is delayed.v. Using backcrossing before assembling more genesWhen the required population size at any stage is too large to be practicable, the use of backcrossing before assembling more genes is advisable.

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b. Methods for enhancing the efficiency of the fixation stepi. Crossing between selected individualsAs aforementioned, the success of a gene pyramiding strategy depends on obtaining the target genotype within the time frame and cost defined by breeders irrespective of inter-mating between selected individuals at each generation desirable alleles in all target loci is missing at any generation, crossing two plants with the best complementary genotypes can be used to secure the program. Crossing between complementary genotypes may also speed up the breeding process even if a satisfactory genotype is present.

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ii. Crossing the root genotype to a genotype with several desirable genesCrossing the root genotype to an elite line without the desired genes can also be used to convert the undesirable phase to the desirable one. This will effectively reduce the required population size in subsequent generations. This idea can be extended to generations before the formation of root genotype.iii. Advancing all satisfactory genotypesIn some generations more than one (partially heterozygous) genotype has the potential to produce the genotype desired for the next generation. These satisfactory genotypes may have different frequencies and different progeny sizes are needed to best realise their potential. The most efficient strategy will be the one that promotes the most satisfactory genotype (fewer loci are segregating, and the segregating loci are in the desirable linkage phase) at each generation.

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5. EFFICIENCY OF GENE PYRAMIDINGComputer 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 schemeA 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.

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6. MAIN FACTORS AFFECTING GENE PYRAMIDINGa. Characteristics of the target traits/genesWhen the genes to be pyramided are functionally well characterised 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.When the target genes are QTL with moderate or small effects, pyramiding may be less successful due to the following reasons. Firstly, the identified QTL may be more likely to be a false positive. Secondly, inaccurate QTL localizations result in the need to select for more marker locicovering large genomic segments to be certain that target QTL alleles are retained in selected progeny (Hospital and Charcosset 1997). Thirdly, QTL effects may be specific to a particular genetic background.

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b. Reproductive characteristicsThe propagation capability of a crop is determined by the number of seeds produced by a single plant. c. Operating capitalAll 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 Increasing the number of generations (duration) will it. Increasing 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.

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7. INTEGRATING GENE DISCOVERY, VALIDATION AND PYRAMIDING

When the expression of a trait is controlled by multiple genes with relatively small effects, it is a quantitatively trait. Most of the important agronomic traits such as yield, stress resistance and quality are quantitativeGenes for quantitative traits are more difficult to traits. identify. Quantitative traits loci (QTL) mapping using purposely generated mapping populations such as F2 plants, backcross plants, Recombinant Inbred Lines (RIL), Backcross Inbred Lines (BIL) or Doubled Haploid Lines (DHL), as well as a linkage map constructed using molecular markers are currently the standard approach for identifying QTL controlling quantitative trait. A large population size is required to provide sufficient detection power.

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a. Advanced backcross QTL analysis (AB-QTL)The advanced backcross QTL analysis (AB-QTL) was proposed by Tanksley and Nelson (1996) to simultaneously identify and introgress favourable alleles from unadapted donors into elite background. The general AB-QTL analysisis comprised of the following experimental phases:1) Generating an elite by donor hybrid,2) Backcrossing to the elite parent to produce BC population which is subjected to marker/or phenotypic selection against undesirable donor alleles,3) Genotyping BC2 or BC3 population with polymorphic molecular markers,4) Evaluating the segregating BC F22 or BC F23 population for traits of interest and QTL analysis,5) Selecting target genomic regions containing useful donor alleles for the production of NILs in the elite genetic background and6) Evaluation of the agronomic traits of the NILs and elite parent controls in replicated environments.The AB-QTL approach has been evaluated in many crop plant species to determine whether genomic regions derived from wild or unadapted germplasm have the poten- tial to improve yield.

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b. Introgression lines (ILs)ILs are produced by systematic backcrossing and introgression of marker-defined exotic segments in the background of elite varieties.identifying QTL genes using ILs does not require linkage map construction or sophisticated statistical analysis for QTL, this is a more user-friendly method for practical breeding programs and also for biological science.

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c. Functional markersMarkers linked to the QTL identified by linkage mapping using one or a few populations may or may not be useful in gene pyramiding because different subsets of QTL will be polymorphic in each population, and the linkage phases between the marker and QTL alleles can differ even between closely related genotypes. The linkage phase also tends to be more consistent if the source of QTL is from a gene pool which is very distinct from the one used by the breeders. Thus, markers linked to novel alleles from exotic germplasm or wild relatives are more likely to be successfully implemented

8. POLYGENIC TRAIT IMPROVEMENT BY GENE PYRAMIDING- A STEP FORWARD

Many economically important traits such as yield, quality and tolerance to abiotic stresses are of a quantitative nature. Genetic variations affecting such traits are controlled by a relatively large number of loci each of which can make a small positive or negative contribution to the final phenotypic value of the traits. These loci are termed QTLs. Molecular markers provide the opportunity to manipulate QTLs as Mendelian entities. Several QTLs for traits of economic importance like rice blast resistance (Wang et al., 1994) black mold resistance in tomato (Robert et al., 2001), flour colour in wheat (Parker et al., 2000), have been tagged with molecular markers. There have been some successful uses of MAS for polygenic traits in plantsGenetic enhancement, through AB-QTL strategy have been undergone by pyramiding various traits of agronomic importance, including fruit quality and black mould resistance in tomato were accomplished using wild relatives (Robert et. al 2001). A broad spectrum project is under progress at CIMMYT to pyramid major QTLs for durable physiological expression in maize.

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9. WORK ON GENE PYREMIDING

Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106S. Singh, J. S. Sidhu, N. Huang, Y. Vikal, Z. Li, D. S. Brar, H. S. Dhaliwal, G. S. KhushTheoretical and Applied Genetics, May 2001, Volume 102, Issue 6-7, pp 1011-1015

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in several countries. Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106, which is widely grown in Punjab, India, using marker-assisted selection. Lines of PR106 with pyramided genes were evaluated after inoculation with 17 isolates of the pathogen from the Punjab and six races of Xoo from the Philippines. Genes in combinations were found to provide high levels of resistance to the predominant Xoo isolates from the Punjab and six races from the Philippines. Lines of PR106 with two and three BB resistance genes were also evaluated under natural conditions at 31 sites in commercial fields. The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo. Only 1 of the BB isolates, PX04, was virulent on the line carrying Xa21 but avirulent on the lines having xa5 and xa13 genes in combination with Xa21.

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Tagging and mapping of a rice gall midge resistance gene, Gm8, and development of SCARs for use in marker-aided selection and gene pyramidingA. Jain, R. Ariyadasa,  A. Kumar, M. N. Srivastava,  M. Mohan, S. NairTheoretical and Applied Genetics, November 2004, Volume 109, Issue 7, pp 1377-1384

Using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNAs (RAPDs), tagged and mapped Gm8, a gene conferring resistance to the rice gall midge (Orseolia oryzae), a major insect pest of rice, onto rice chromosome 8. Using AFLPs, two fragments, AR257 and AS168, were identified that were linked to the resistant and susceptible phenotypes, respectively. Another resistant phenotype-specific marker, AP19587, was also identified using RAPDs. SCAR primers based on the sequence of the fragments AR257 and AS168 failed to reveal polymorphism between the resistant and the susceptible parents. However, PCR using primers based on the regions flanking AR257 revealed polymorphism that was phenotype-specific. In contrast, PCR carried out using primers flanking the susceptible phenotype-associated fragment AS168 produced a monomorphic fragment. Restriction digestion of these monomorphic fragments revealed polymorphism between the susceptible and resistant parents. Nucleotide BLAST searches revealed that the three fragments show strong homology to rice PAC and BAC clones that formed a contig representing the short arm of chromosome 8. PCR amplification using the above-mentioned primers on a larger population, derived from a cross between two indica rice varieties, Jhitpiti (resistant parent) and TN1 (susceptible parent), showed that there is a tight linkage between the markers and the Gm8 locus. These markers, therefore, have potential for use in marker-aided selection and pyramiding of Gm8 along with other previously tagged gall midge resistance genes [Gm2, Gm4(t), and Gm7].

Genetic analysis and pyramiding of two gall midge resistance genes (Gm-2 and Gm-6t) in rice ( Oryza sativa L.)Sanjay Katiyar, Satish Verulkar, Girish Chandel, Yang Zhang, Bingchao Huang, John BennettEuphytica, 11-2001, Volume 122, Issue 2, pp 327-334

Asian rice gall midge (Orseolia oryzae) is a major pest across much of south and south-east Asia. The two genes, Gm-2 andGm-6(t), are known to confer resistance against a number of biotypes in India and China, respectively. An F3 population derived from a cross between Duokang #1 (donor of Gm-6(t)) and Phalguna (donor of Gm-2) was screened against Chinese gall midge biotype 4 at Guangdong, China, and Indian gall midge biotype 1 at Raipur, India. At each location, separately,a single gene governed resistance. The parallel segregation of 417 F3 progenies for both biotypes at two locations revealed that recombination had occurred between the two genes, establishing that the two genes are not allelic. However, the two genes Gm-2 and Gm-6(t), were found to be linked with a distance of ∼16.3 cM. A number of lines homozygous at one locus and segregating for the other locus were identified and selected. These lines were selfed to obtain lines homozygous for the favourable alleles at both loci (two locus pyramids). This is the first report on use of conventional host-pest interaction method for pyramiding two closely located Gm-resistance loci of dissimilar effects. The implications of deployment of these pyramids within and across country borders, with reference to the prevailing gall midge populations are discussed.

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10. CONCLUSI ON

1.Gene pyramiding is an important strategy for germplasm improvement. 2. Pyramiding requires that breeders consider the minimum population size that must be evaluated to have a reasonable chance of obtaining the desired genotype. 3.Gene pyramiding with marker technology can integrate into existing plant breeding programmes all over the world to allow researchers to access, transfer and combine genes at a rate and with a precision. 4. MAS based gene pyramiding has the potential To increase the rate of genetic gain when used in conjunction with traditional breeding . 

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