12 Marker-Assisted Selection Lec

8/8/2019 12 Marker-Assisted Selection Lec

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Marker Assisted Selection


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MARKER ASSISTED BREEDING

Selection of a genotype carrying a desirable gene or genecombination via linked markers is called Marker assistedselection (MAS)

Breeders practice MAS when an important trait that isdifficult to assess, is tightly linked to another Mendelian traitwhich can be easily scored

For e.g. gene for purple coleoptile color in some traditionalrice varieties is closely linked to a gene that confers resistance toBPH in a segregating population like F2, about 95% of theplants showing purple coleoptile are found resistant to BPH


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I n this case coleoptile color is a morphological markerwhich is used to assist selection for BPH resistance

Marker-assisted selection is the most widely usedapplication of DNA markers.

MAS involves the scoring for the presence or absence of adesired plant phenotype indirectly based on DNA bandingpattern of linked marker system


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The rationale is that the banding pattern revealing parentalorigin of the bands in segregants at a given marker locus

indicate the presence or absence of a specific chromosomalsegment which carries the desirable allele.

T his increases the efficiency of breeding in a number of ways.

Once traits have been mapped and a closely linked marker hasbeen found, it is possible to screen large numbers of samples forrapid identification of progeny that carry desirablecharacteristics.


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Marker-assisted selection (MAS) is a method of selectingdesirable individuals in a breeding scheme based on DNAmolecular marker patterns instead of, or in addition to, their

trait values.

When used in appropriate situations, it is a tool that can helpplant breeders select more efficiently for desirable crop traits.

However, MAS is not always advantageous, so careful analysisof the costs and benefits relative to conventional breedingmethods is necessary


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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 andexpensive and require large field space.

I f such genes can be tagged by tight linkage with DNA orisozyme markers, time and money can be saved in transferringthese genes from one varietal background to another.

A molecular marker very closely linked to the target gene canact as a "tag" which can be used for indirect selection of thegene(s) in breeding program.


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MARKER ASSISTED BREEDING

A. Basic principles

Wide Crosses

Desirable traits such as disease resistance can often be foundin 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.


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


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MARKER ASS I ST ED BREED I NG

MAS can theoretically enhance selection efficiencybecause:

I. It can be performed on seedling material

II. MAS is not affected by environmental conditionsIII. When recessive alleles determine the trait of interest

IV. Similarly, when multiple resistance genes arepyramided (combined) together in the same varietyor breeding line

V. Environmental variation in the field reduces a trait'sheritability


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vi. MAS may be cheaper and faster than conventional phenotypicassays, depending on the trait

vii. A consideration that may affect cost effectiveness of MAS isthat multiple markers can be evaluated using the same DNAsample

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 interestmay occur, leading to false positives

iii.T

o avoid this last problem it may be necessary to use flankingmarkers

iv. Sometimes markers that were used to detect a locus must beconverted to "breeder-friendly" markers that are morereliable and easier to use.


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I mprecise estimates of Q TL locations and effects may result

in slower progress than expectedMarkers developed for MAS in one population may not betransferable to other populations

T he basic procedure for conducting MAS with DNAmarkers:

Extract DNA from tissue of each individual or family in apopulation.

Screen DNA samples via PCR for the molecular marker(SSR, SNP , SCAR, etc.) linked to the trait of interest.


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Separate and score PCR products, using an appropriateseparation and detection technique.

I dentify individuals exhibiting the desired marker allele.

Combine the marker results with other selection criteria (e.g.,phenotypic data or other marker results), select the bestfraction of the population, and advance those individuals inthe breeding program.

I n cross between a wild plant variety and an elite cultivar, thewild parent (green) and the elite cultivar (magenta) eachcontribute one copy of each of three chromosome pairs to thehybrid (bottom left).


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A disease resistance gene in the wild parent is indicated by ared tick mark. I n the next generation (right), the hybrid isbackcrossed to the original elite cultivar.

Due to genetic recombination (ie. crossing over) in thehybrid, genetic material is exchanged between wild and elitechromosomes. I n the resultant offspring, one copy of eachchromosome is solely derived from the elite parent, while thesecond copy of each chromosome may have genes from bothparents.


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I n each generation of back crossing, more of the wild parentgenes are lost.

I n effect, repeated back crossing reconstitutes the elite parentgenome. I f the resultant offspring have the same genes as theoriginal elite cultivar, they will also have the same agronomictraits. I n this case, however, we wish to retain the small piece of the wild chromosome bearing the disease resistance gene


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selection of plants in a given generation of backcross generation is done by scoringdozens of markers using DNA from individualplants. Each band on a gel can be scored ascomming from one parent (presence of a band)or the other parent (absence of a band). In thefigure at right, map locations of markers fromthe elite culitvar are shown in magenta, andthose from the wild parent are shown in green.

If we screen enough plants, we can find thoseplants that maximize the number of markersderived from the elite parent, while retaining theimportant gene of interest. In this map, amarker located very close to that gene isindicated in red. The majority of the time, if we

have the marker, we have the gene.


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Backcrosses

I n each backcross generation, a hybrid is crossed with the elite

parent.T

he amount of the wild parent genome passed on to thenext generation will vary among offspring.

T he middle panel shows several of the possible chromosomecomplements that could be donated by the hybrid parent, eachcontaining varying amounts of wild genes.

I n some offspring, even the disease resistance gene will not bepassed on.


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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 distinguishbetween DNA from the donor parent, and DNA from therecurrent parent.

T he goal of backcrossing is to move a single trait of interest(e.g. disease resistance from a wild relative or a transgenefrom a donor line) into the genome of a commerciallyvaluable variety without losing any part of the commercialvariety's existing genome.


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Backcrosses

T he plant with the gene of interest is the donor parent, whilethe 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 recurrentparent genome is recovered to release the line commercially.

Using markers allows a breeder to reach the same goal in 2backcross generations thereby cutting 1-2 years of the productdevelopment cycle.


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Hillel, J. et al. (1990) DNA fingerprints applied to geneintrogression in breeding programs. Genetics 124:783-789.

I ntrogression is the repeated crossing of a hybrid derived froma donor and recipient, back to the recipient, which is referred toas the recurrent parent.

T ypically, in each generation, the individuals with the desiredtrait are selected, and crossed back to the recurrent parent.

T o approximate 100% return to the recurrent parent geneticbackground, approximately 6 to 8 backcross generations arerequired.


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Using markers for trait selection has numerous advantageswhen compared to conventional plant breeding:

Speed DNA can be extracted from tissue from the first leavesor the cotyledons of a plant. T rait information can be discoveredwith markers prior to pollination allowing more informed crossesto be made.

Consistency Markers remove the impact of environmentalvariation that often complicates phenotypic evaluation.

Biosafety Using markers in screening for disease resistancemeans not having to introduce the pathogen into breedingpopulations. Particularly for livestock breeding this delivers avery important level of biosafety .


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Efficiency Screening progeny early in the process allows abreeder to drop also-rans from the program more quickly.Most breeding programs that use markers still evaluate the samenumber of plants in the field however the level of genetic qualityis vastly increased because of the early-stage screening that has

been carried.

Complex traits Most multigenic traits are very difficult tomanage through conventional plant breeding. T he statisticalchance of getting the required allele at each of a number of loci isvery low. Markers allow you to skew the odds in your favour.


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Application MAS in crops:

Some examples of rice genes of agronomic importancemapped with molecular markers

Gene Trait Chromosome Link marker Pi1 Blast resistance 11 Npb181Pi2 Blast resistance 6 RG64Xa21 BLB resistance ` 11 RG103RTSV Rice tungro spherical virus 4 RZ 262

resistanceBph1 Brown Plant hopper 12 XNpb248

resistanceGm1 Gall midge resistance OPK-7Fgr Fragrance 8 RG28


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Essential Requirements

- marker(s) should cosegregate or be closely linked ( 1 cM orless) with the desired trait

- Efficient screening of mol marker for large population ( easyanalysis, cost, etc.)

- Highly reproducible scoring technique- Choice of Markers- Ease of marker assay technique- Extent of polymorphism- Reproducibility


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Advantages of MAS:-Segregants can be scored at the seedling stage for traitsexpressed late in the development

-Difficult ,expensive and time-consuming traits( e.g. droughttolerance) can be scored

-Several selection for many traits can be carried outsimultaneously

-Heterozygotes are easily identified-Hasten the BC breeding programme-MAS is well-suited for introgressing exotic germplasm


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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


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Use of MAS:

1. Gene I ntrogression:

markers are used to hasten the recovery of the recipientgenome during an introgression breeding program

Gene flow from cultivated to landraces, from transgenics tocultivated and wild species

Wild relatives of crops constitute a source of genetic variationwhich can be efficiently utilized for improvement of bothqualitative and quantitative traits


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I t is often observed that the desirable genes such as those fordisease resistance remain linked with undesirable weedy

characteristics of the alien species

During gene introgression by backcrossing the linkedundesirable gene also gets transferred to the recipient parent.T his is referred to as linkage drag

Molecular markers can reduce linkage drag at least 10-foldin a fraction of the time needed by the traditional breeding


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1.Gene I ntrogression:

I ntrogression is the repeated crossing of a hybrid derivedfrom a donor and recipient, back to the recipient, which is

referred to as the recurrent parent.

T ypically, in each generation, the individuals with the desiredtrait are selected, and crossed back to the recurrent parent.

T o approximate 100% return to the recurrent parentgenetic background, approximately 6 to 8 backcrossgenerations are required.


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1.Gene I ntrogression:

T he key observation is to make is that when you say youwant to breed a gene into some genetic background that hasall the important quality traits you want, you are reallyasking to maximize the number of chromosomal segmentsfrom the recurrent parent, while at the same time bringingalong the gene of interest.

I f you get most of the recurrent parent genome back, you

automatically expect to get back all of its qualities.

T his strategy is sometimes referred to as genomic selection.(GS)


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T he pyramided lines having three or four genes incombination showed an increased and wider spectrum of resistance to bacterial blight than those having a singleresistance gene.

T he pyramided lines showed a wider spectrum and ahigher level of resistance than lines with only a single gene

Sequence-tagged site (S T S) markers were used to pyramidthree genes for BB resistance in an elite breeding line of rice.


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2.Gene Pyramiding

Pyramiding of genes has been suggested as an effective way toprovide durable form of disease and insect resistance in cropplants

MAS was applied for pyramiding four genes for BB resistancee.g. X a4 , xa5 , xa13 , and X a21 .

Breeding lines with two or three genes were also developed.


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3.Development of Heterotic hybrids:

Molecular markers based on genetic analysis can be used toidentify specific genomic regions containing Q TL s for yieldshowing dominance or overdominance gene action in the F2 of a cross between two inbred lines

T his will enable analysis of existing cross in forms of the no. of Q TL involved and magnitude of their effect on the measuredtrait

Subsequently additional inbred lines can be tested againststandard lines for the presence of additional dominant oroverdominant Q TL s


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T his could be then incorporated into standard line s bymarker aided introgression

I n this way the existing inbreds can be improved first andthen used to develop hybrids for realizing higher heterosisthan in the existing ones

e.g. maize and rice

Genetic markers can be used for the prediction of hybridperformance

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12 Marker-Assisted Selection Lec