Marker Assisted Selection

Prof. Dina El-Khishin

Agricultural Genetic Engineering Research Institute

(AGERI)

Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes Aug. 31st- Sept. 4th, 2014

Marker-Assisted Selection 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. A tool that can help plant breeders select more efficiently for desirable crop traits. MAS is not always advantageous, so careful analysis of the costs and benefits relative to conventional breeding methods is necessary.

F2

P2

F1

P1 x

large populations consisting of thousands of plants

PHENOTYPIC SELECTION

Field trials Glasshouse trials

Donor Recipient

CONVENTIONAL PLANT BREEDING

Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot

F2

P2

F1

P1 x

large populations consisting of thousands of plants

Resistant Susceptible

MARKER-ASSISTED SELECTION (MAS)

MARKER-ASSISTED BREEDING

Method whereby phenotypic selection is based on DNA markers

Prerequisites for an efficient marker-assisted

selection program

High throughput DNA extraction

Markers Markers (morphological, protein, cytological) can also be used in MAS programs. RFLP, SSR, RAPD, AFLP, SCAR, and SNP For efficient MAS: Ease of use Small amount of DNA required Low cost Repeatability of results High rate of polymorphism Occurrence throughout the genome Codominance

Genetic maps. Linkage maps provide a framework for detecting marker-trait associations and for choosing markers to employ in MAS. Once a marker is found to be associated with a trait in a given population, a dense molecular marker map in a standard reference population will help identify markers that are closer to, or that flank, the target gene.

Knowledge of associations between molecular markers and traits of interest.

The most crucial ingredient for MAS is knowledge of markers that are associated with traits important to a breeding program.

Data management system

Large numbers of samples are handled in a MAS program, with each sample potentially evaluated for multiple markers. This situation requires an efficient system for labeling, storing, retrieving, and analyzing large data sets, and producing reports useful to the breeder.

Potential advantages of MAS

It can be performed on seedling material thus reducing the time required before a plant’s genotype is known. In contrast, many important plant traits are observable only when the plant has reached flowering or harvest maturity. Knowing a plant’s genotype before flowering can be particularly useful in order to plan the appropriate crosses between selected individuals.

MAS is not affected by environmental conditions. Some crop production constraints (such as disease, insect pests, temperature and moisture stress) occur sporadically or non-uniformly. Therefore, evaluating resistance to those constraints may not be possible in a given year or location. MAS offers the chance to determine a plant’s resistance level independent of environment.

When recessive alleles determine traits of interest they cannot be detected through phenotypic evaluation of heterozygous plants, because their presence is masked by the dominant allele. In a traditional backcross program, plants with recessive alleles are identified by progeny evaluation after self-pollination or testcrossing to a recessive tester. This time-consuming step can be eliminated in a MAS program, because recessive alleles are identified by linked markers.

when multiple resistance genes are pyramided together in the same variety or breeding line,

the presence of each individual gene is difficult to verify phenotypically. The presence of one resistance gene may conceal the effect of additional genes. This problem can be overcome if markers are available for each of the resistance genes.

Environmental variation in the field reduces a trait’s heritability , the proportion of phenotypic variation that is due to genetics. In a low heritability situation, progress from phenotypic selection will be slow, because so much of the variation for the trait is due to environmental variation, experimental error, or genotype x environment interaction, and will not be passed on to the next generation. If a reliable marker for a trait is available, MAS can result in greater progress than phenotypic selection in such a situation.

MAS may be cheaper and faster than conventional phenotypic assays, depending on the trait. e.g., evaluating nematode resistance is usually an expensive operation because it requires artificial inoculation of plants with nematode eggs, followed by a labor-intensive technique to count the number of nematodes present. Selecting on the basis of a reliable marker would probably be cost-effective in this case. On the other hand, plant height is cheap and easy to measure, so there may not be an economic advantage in using markers for that trait.

A consideration that may affect cost effectiveness of MAS is that multiple markers can be evaluated using the same DNA sample. Extraction of DNA from plant tissue is one of the bottlenecks of MAS. Once DNA is extracted and purified, it may be used for multiple markers, for the same or different traits, thus reducing the time and cost per marker.

Potential drawbacks of MAS

Linkage maps of two chromosomes showing positions of

two resistance genes and nearby markers.

MAS may be more expensive than conventional techniques, especially for startup expenses and labor costs.

Recombination between the marker and the gene of interest may occur, leading to false positives.

e.g., if the marker and the gene of interest are separated by 5 cM and selection is based on the marker pattern, there is an approximately 5% chance of selecting the wrong plant. This is based on the general guideline that across short distances, 1 cM of genetic distance is approximately equal to 1% recombination. The breeder will need to decide the error rate that is acceptable in the MAS program, keeping in mind that errors are also usually involved in phenotypic evaluation.

Markers must be tightly-linked to target loci!

• Ideally markers should be <5 cM from a gene or QTL

• Using a pair of flanking markers can greatly improve reliability but increases time and cost

Marker A

QTL 5 cM

RELIABILITY FOR SELECTION

Using marker A only:

1 – rA = ~95%

Marker A

QTL

Marker B

5 cM 5 cM

Using markers A and B:

1 - 2 rArB = ~99.5%

To avoid this last problem it may be necessary to use flanking markers on either side of the locus of interest to increase the probability that the desired gene is selected. Sometimes markers that were used to detect a locus must be converted to 'breeder-friendly' markers that are more reliable and easier to use.

Examples : RFLP markers converted to STS markers RFLP requires several steps and a large quantity of highly purified DNA. STS can be detected via PCR using primers developed from RFLP probe sequences. Thus the same locus can be detected with the two types of marker, but the STS marker is far more efficient. RAPD markers converted to SCAR markers Results of RAPD reactions may vary from lab to lab, and may be considered less reliable for MAS. SCAR markers are developed by sequencing RAPD bands and designing more specific 18-25 base PCR primers to amplify the same DNA segment more reliably.

Markers must be polymorphic

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

RM84 RM296

P1 P2

P1 P2

Not polymorphic Polymorphic!

Imprecise estimates of QTL locations and effects may result in slower progress than expected. Many QTLs have large confidence intervals of 20 cM or more or their relative importance in explaining trait inheritance has been over-estimated. Markers developed for MAS in one population may not be transferrable to other populations, either due to lack of marker polymorphism or the absence of a marker-trait association.

(1) TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Conducting a MAS program

MAS BREEDING SCHEMES

1. Marker-assisted backcrossing

2. Pyramiding

3. Early generation selection

4. ‘Combined’ approaches

Marker-assisted backcrossing (MAB)

• MAB has several advantages over conventional backcrossing: – Effective selection of target loci

– Minimize linkage drag

– Accelerated recovery of recurrent parent

1 2 3 4

Target locus

1 2 3 4

RECOMBINANT SELECTION

1 2 3 4

BACKGROUND SELECTION

TARGET LOCUS SELECTION

FOREGROUND SELECTION BACKGROUND SELECTION

Pyramiding • Widely used for combining multiple disease

resistance genes for specific races of a pathogen

• Pyramiding is extremely difficult to achieve using conventional methods – Consider: phenotyping a single plant for multiple

forms of seedling resistance – almost impossible

• Important to develop ‘durable’ disease resistance against different races

• Process of combining several genes, usually from 2 different parents, together into a single genotype

F2

F1

Gene A + B

P1

Gene A x P1

Gene B

MAS

Select F2 plants that have Gene A and Gene B

Genotypes

P1: AAbb P2: aaBB

F1: AaBb

F2 AB Ab aB ab

AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

x

Breeding plan

Hittalmani et al. (2000). Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in riceTheor. Appl. Genet. 100: 1121-1128

Liu et al. (2000). Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat. Plant Breeding 119: 21-24.

Early generation MAS • MAS conducted at F2 or F3 stage

• Plants with desirable genes/QTLs are selected and alleles can be ‘fixed’ in the homozygous state – plants with undesirable gene combinations can

be discarded

• Advantage for later stages of breeding program because resources can be used to focus on fewer lines

References:

Ribaut & Betran (1999). Single large-scale marker assisted selection (SLS-MAS). Mol Breeding 5: 21-24.

F2

P2

F1

P1 x

large populations (e.g. 2000 plants)

Resistant Susceptible

MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes

MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes

P1 x P2

F1

PEDIGREE METHOD

F2

F3

F4

F5

F6

F7

F8 – F12

Phenotypic screening

Plants space-planted in rows for individual plant selection

Families grown in progeny rows for selection.

Preliminary yield trials. Select single plants.

Further yield trials

Multi-location testing, licensing, seed increase and cultivar release

P1 x P2

F1

F2

F3

MAS

SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS)

F4 Families grown in progeny rows for selection.

Pedigree selection based on local needs

F6

F7

F5

F8 – F12 Multi-location testing, licensing, seed increase and cultivar release

Only desirable F3 lines planted in field

breeding program can be efficiently scaled down to focus on fewer lines

Combined approaches In some cases, a combination of phenotypic screening and MAS approach may be useful

1. To maximize genetic gain (when some QTLs have been unidentified from QTL mapping)

2. Level of recombination between marker and QTL (in other words marker is not 100% accurate)

3. To reduce population sizes for traits where marker genotyping is cheaper or easier than phenotypic screening

‘Marker-directed’ phenotyping

BC1F1 phenotypes: R and S

P1 (S) x P2 (R)

F1 (R) x P1 (S)

Recurrent Parent

Donor Parent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 …

SAVE TIME & REDUCE COSTS

*Especially for quality traits*

MARKER-ASSISTED SELECTION (MAS)

PHENOTYPIC SELECTION

(Also called ‘tandem selection’)

• Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping

References:

Han et al (1997). Molecular marker-assisted selection for malting quality traits in barley. Mol Breeding 6: 427-437.

MAS: MARKER-ASSISTED SELECTION - Plants are selected for one or more (up to 8-10) alleles

MABC: MARKER-ASSISTED BACKCROSSING

One or more (up to 6-8) donor alleles are transferred to an elite line

MARS: MARKER-ASSISTED RECURRENT SELECTION

Selection for several (up to 20-30) mapped QTLs relies on index (genetic) values computed for each individual based on its haplotype at target QTLs

GWS: GENOME-WIDE SELECTION Selection of genome-wide several loci that confer tolerance/resistance/ superiority to traits of interest

using GEBVs based on genome-wide marker profiling

A variety of approaches

Conclusion MAS is a methodology that has already proved its value. It is likely to become more valuable as a larger number of genes are identified and their functions and interactions elucidated. Reduced costs and optimized strategies for integrating MAS with phenotypic selection are needed before the technology can reach its full potential.

References •Marker-Assisted Selection - Objectives and Overview Patrick Byrne Department of Soil and Crop Sciences at Colorado State University, USA Kelley Richardson Department of Crop and Soil Sciences at Oregon State University, USA •MARKER-ASSISTED BREEDING FOR RICE IMPROVEMENT Bert Collard & David Mackill Plant Breeding, Genetics and Biotechnology (PBGB) Division, IRRI bcycollard@hotmail.com & d.mackill@cgiar.org •Towards utilization of genome sequence information for pigeonpea improvement By ICAR institutes, SAUs and ICRISAT •MAS Breeding University of Nebraska Institute of Agriculture and Natural Resources This presentation has been compiled from those references