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12 Marker-Assisted Selection Lec

12 Marker-Assisted Selection Lec


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Published by: vijjumandula on Sep 17, 2010
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Marker Assisted Selection

Selection of a genotype carrying a desirable gene or gene combination via linked markers is called Marker assisted selection (MAS) Breeders practice MAS when an important trait that is difficult to assess, is tightly linked to another Mendelian trait which can be easily scored For e.g. gene for purple coleoptile color in some traditional rice varieties is closely linked to a gene that confers resistance to BPH in a segregating population like F2, about 95% of the plants showing purple coleoptile are found resistant to BPH

‡ In this case coleoptile color is a morphological marker which is used to assist selection for BPH resistance ‡ Marker-assisted selection is the most widely used application of DNA markers. ‡ MAS involves the scoring for the presence or absence of a desired plant phenotype indirectly based on DNA banding pattern of linked marker system 

The rationale is that the banding pattern revealing parental origin of the bands in segregants at a given marker locus indicate the presence or absence of a specific chromosomal segment which carries the desirable allele. This increases the efficiency of breeding in a number of ways. Once traits have been mapped and a closely linked marker has been found, it is possible to screen large numbers of samples for rapid identification of progeny that carry desirable characteristics. 

Marker-assisted selection (MAS) is a method of selecting desirable individuals in a breeding scheme based on DNA molecular marker patterns instead of, or in addition to, their trait values. When used in appropriate situations, it is a tool that can help plant breeders select more efficiently for desirable crop traits.  However, MAS is not always advantageous, so careful analysis of the costs and benefits relative to conventional breeding methods is necessary 

Most genes of economic importance behave in a dominant or recessive manner and require time consuming efforts to transfer. Sometimes the screening procedures are cumbersome and expensive and require large field space. If such genes can be tagged by tight linkage with DNA or isozyme markers, time and money can be saved in transferring these genes from one varietal background to another. A molecular marker very closely linked to the target gene can act as a "tag" which can be used for indirect selection of the gene(s) in breeding program.

A. Basic principles Wide Crosses 
Desirable traits such as disease resistance can often be found in wild relatives of domestic crops. However, when you cross a wild parent with an elite cultivar, the hybrid has a combination of genes from both parents. To breed the desirable gene back, many generations of backcrosses to the elite cultivar must be done.

Fig. 1. A. Conventional selection is based on direct measurement of important traits, such as yield, maturity, or disease resistance. B. In marker-assisted selection, plants are selected based on molecular marker patterns known to be associated with the traits of interest.

MAS can theoretically enhance selection efficiency because: I. It can be performed on seedling material II. MAS is not affected by environmental conditions III. When recessive alleles determine the trait of interest IV. Similarly, when multiple resistance genes are ³pyramided´ (combined) together in the same variety or breeding line V. Environmental variation in the field reduces a trait's heritability

vi. MAS may be cheaper and faster than conventional phenotypic assays, depending on the trait vii. A consideration that may affect cost effectiveness of MAS is that multiple markers can be evaluated using the same DNA sample Some limitations of the technique are as follows: i. MAS may be more expensive than conventional techniques ii. Recombination between the marker and the gene of interest may occur, leading to false positives iii. To avoid this last problem it may be necessary to use flanking markers iv. Sometimes markers that were used to detect a locus must be converted to "breeder-friendly" markers that are more reliable and easier to use.

‡ ‡

Imprecise estimates of QTL locations and effects may result in slower progress than expected Markers developed for MAS in one population may not be transferable to other populations 

The basic procedure for conducting MAS with DNA markers:

Extract DNA from tissue of each individual or family in a population. ‡ Screen DNA samples via PCR for the molecular marker (SSR, SNP, SCAR, etc.) linked to the trait of interest.

Separate and score PCR products, using an appropriate separation and detection technique. Identify individuals exhibiting the desired marker allele. Combine the marker results with other selection criteria (e.g., phenotypic data or other marker results), select the best fraction of the population, and advance those individuals in the breeding program. 
In cross between a wild plant variety and an elite cultivar, the wild parent (green) and the elite cultivar (magenta) each contribute one copy of each of three chromosome pairs to the hybrid (bottom left).

A disease resistance gene in the wild parent is indicated by a red tick mark. In the next generation (right), the hybrid is backcrossed to the original elite cultivar. 

Due to genetic recombination (ie. crossing over) in the hybrid, genetic material is exchanged between wild and elite chromosomes. In the resultant offspring, one copy of each chromosome is solely derived from the elite parent, while the second copy of each chromosome may have genes from both parents.

In each generation of back crossing, more of the wild parent genes are lost. In effect, repeated back crossing reconstitutes the elite parent genome. If the resultant offspring have the same genes as the original elite cultivar, they will also have the same agronomic traits. In this case, however, we wish to retain the small piece of the wild chromosome bearing the disease resistance gene

selection of plants in a given generation of backcross generation is done by scoring dozens of markers using DNA from individual plants. Each band on a gel can be scored as comming from one parent (presence of a band) or the other parent (absence of a band). In the figure at right, map locations of markers from the elite culitvar are shown in magenta, and those from the wild parent are shown in green. If we screen enough plants, we can find those plants that maximize the number of markers derived from the elite parent, while retaining the important gene of interest. In this map, a marker located very close to that gene is indicated in red. The majority of the time, if we have the marker, we have the gene.

In each backcross generation, a hybrid is crossed with the elite parent. The amount of the wild parent genome passed on to the next generation will vary among offspring. The middle panel shows several of the possible chromosome complements that could be donated by the hybrid parent, each containing varying amounts of wild genes.  In some offspring, even the disease resistance gene will not be passed on. 

What we'd like to do, in each generation, is to select those individuals that received the largest amount of the recurrent parent genome, and also carry the desired gene (bottom panel).  For this purpose, molecular markers can be used to keep track of both the gene of interest, as well as to distinguish between DNA from the donor parent, and DNA from the recurrent parent.  The goal of backcrossing is to move a single trait of interest (e.g. disease resistance from a wild relative or a transgene from a donor line) into the genome of a commercially valuable variety without losing any part of the commercial variety's existing genome.

The plant with the gene of interest is the donor parent, while the commercially viable variety is the recurrent parent. Using DNA markers can accelerate a backcrossing program significantly. For example, most conventional corn backcrossing programs require 4-6 backcross generations before sufficient recurrent parent genome is recovered to release the line commercially. Using markers allows a breeder to reach the same goal in 2 backcross generations thereby cutting 1-2 years of the product development cycle.

Hillel, J. et al. (1990) DNA fingerprints applied to gene introgression in breeding programs. Genetics 124:783-789. 
Introgression is the repeated crossing of a hybrid derived from a donor and recipient, back to the recipient, which is referred to as the recurrent parent. Typically, in each generation, the individuals with the desired trait are selected, and crossed back to the recurrent parent. To approximate 100% return to the recurrent parent genetic background, approximately 6 to 8 backcross generations are required. 

Using markers for trait selection has numerous advantages when compared to conventional plant breeding: Speed ± DNA can be extracted from tissue from the first leaves or the cotyledons of a plant. Trait information can be discovered with markers prior to pollination allowing more informed crosses to be made. Consistency ± Markers remove the impact of environmental variation that often complicates phenotypic evaluation. Biosafety ± Using markers in screening for disease resistance means not having to introduce the pathogen into breeding populations. Particularly for livestock breeding this delivers a very important level of biosafety. 

Efficiency ± Screening progeny early in the process allows a breeder to drop ³also-rans´ from the program more quickly. Most breeding programs that use markers still evaluate the same number of plants in the field however the level of genetic quality is vastly increased because of the early-stage screening that has been carried. Complex traits ± Most multigenic traits are very difficult to manage through conventional plant breeding. The statistical chance of getting the required allele at each of a number of loci is very low. Markers allow you to skew the odds in your favour.

Application MAS in crops: Some examples of rice genes of agronomic importance mapped with molecular markers
Gene Pi1 Pi2 Xa21 RTSV Bph1 Gm1 Fgr Trait Chromosome Link marker Blast resistance 11 Npb181 Blast resistance 6 RG64 BLB resistance ` 11 RG103 Rice tungro spherical virus 4 RZ 262 resistance Brown Plant hopper 12 XNpb248 resistance Gall midge resistance OPK-7 Fragrance 8 RG28

Essential Requirements - marker(s) should cosegregate or be closely linked ( 1 cM or less) with the desired trait - Efficient screening of mol marker for large population ( easy analysis, cost, etc.) - Highly reproducible scoring technique - Choice of Markers - Ease of marker assay technique - Extent of polymorphism - Reproducibility

Marker ±Assisted Selection
Advantages of MAS: -Segregants can be scored at the seedling stage for traits expressed late in the development -Difficult ,expensive and time-consuming traits( e.g. drought tolerance) can be scored -Several selection for many traits can be carried out simultaneously -Heterozygotes are easily identified -Hasten the BC breeding programme -MAS is well-suited for introgressing exotic germplasm

Marker ±Assisted Selection
Use of MAS: 1. For traits difficult to phenotype 2. Gene Introgression 3. Gene Pyramiding 4. Accelerated BC breeding 5. Follow a recessive gene 6. Development of heterotic hybrids

Marker ±Assisted Selection Use of MAS: 1. Gene Introgression: 
markers are used to hasten the recovery of the recipient genome during an introgression breeding program  Gene flow from cultivated to landraces, from transgenics to cultivated and wild species  Wild relatives of crops constitute a source of genetic variation which can be efficiently utilized for improvement of both qualitative and quantitative traits

Marker ±Assisted Selection 

It is often observed that the desirable genes such as those for disease resistance remain linked with undesirable weedy characteristics of the alien species  During gene introgression by backcrossing the linked undesirable gene also gets transferred to the recipient parent. This is referred to as linkage drag  Molecular markers can reduce linkage drag at least 10-fold in a fraction of the time needed by the traditional breeding

Marker ±Assisted Selection

1.Gene Introgression: 
Introgression is the repeated crossing of a hybrid derived from a donor and recipient, back to the recipient, which is referred to as the recurrent parent.  Typically, in each generation, the individuals with the desired trait are selected, and crossed back to the recurrent parent.  To approximate 100% return to the recurrent parent genetic background, approximately 6 to 8 backcross generations are required.

1.Gene Introgression: 
The key observation is to make is that when you say you want to breed a gene into some genetic background that has all the important quality traits you want, you are really asking to maximize the number of chromosomal segments from the recurrent parent, while at the same time bringing along the gene of interest.  If you get most of the recurrent parent genome back, you automatically expect to get back all of its qualities.  This strategy is sometimes referred to as genomic selection. (GS)

‡ The pyramided lines having three or four genes in combination showed an increased and wider spectrum of resistance to bacterial blight than those having a single resistance gene. ‡ The pyramided lines showed a wider spectrum and a higher level of resistance than lines with only a single gene ‡ Sequence-tagged site (STS) markers were used to pyramid three genes for BB resistance in an elite breeding line of rice.

2.Gene Pyramiding 
Pyramiding of genes has been suggested as an effective way to provide durable form of disease and insect resistance in crop plants  MAS was applied for pyramiding four genes for BB resistance e.g. Xa4, xa5, xa13, and Xa21.  Breeding lines with two or three genes were also developed.

3.Development of Heterotic hybrids: 
Molecular markers based on genetic analysis can be used to identify specific genomic regions containing QTLs for yield showing dominance or overdominance gene action in the F2 of a cross between two inbred lines  This will enable analysis of existing cross in forms of the no. of QTL involved and magnitude of their effect on the measured trait  Subsequently additional inbred lines can be tested against standard lines for the presence of additional dominant or overdominant QTLs 

This could be then incorporated into standard line s by marker aided introgression  In this way the existing inbreds can be improved first and then used to develop hybrids for realizing higher heterosis than in the existing ones

e.g. maize and rice 
Genetic markers can be used for the prediction of hybrid performance

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