1. An Assignment on Marker Assisted Selection
Submitted to:
Submitted By: Vadodariya Gopal D. M.Sc.(Agri.) 2nd Sem. Genetics & Plant Breeding

2. Marker Assisted Selection
Marker assisted selection (MAS) is a combined product of traditional genetics and molecular biology. MAS allows for the selection of genes that control traits of interest. Combined with traditional selection techniques, MAS has become a valuable tool in selecting organisms for traits of interest, such as color, meat quality, or disease resistance.

3. BACKGROUND INFORMATION
Deoxyribonucleic acid (DNA) is a molecule made up of pairs of building blocks called nucleotides. The four kinds of nucleotides that make up DNA are adenine (abbreviated as the single letter A), guanine (G), cytosine (C), and thymine (T). The DNA molecule has the shape of two intertwined spirals, referred to as a double helix.
The sequence of nucleotides that make up a gene can differ among individuals. The different forms of a gene are called alleles. The alleles are the result of nucle­otide differences in a gene that affect an amino acid sequence of a protein. This can result in a change, addition, or deletion of a protein that can affect the phenotype.

4. Differences among alleles caused by a single nucleotide, called SNPs, can be the basis of genotyping tests. Genotyping means using laboratory methods to determine the sequence of nucleotides in the DNA from an individual, usually a specific gene. Genetic tests based on SNPs utilize DNA derived from an individual to determine the nucleotide in the gene of interest.
Marker assisted selection is the process of using the results of DNA testing in the selection of individuals to become parents for the next generations. The information from the DNA testing, combined with the observed performance records for individuals, is intended to improve the accuracy of selection and increase the possibility of identifying organisms carrying desirable and undesirable traits at an earlier stage of development.
It is important to combine DNA results with perfor­mance and phenotype information to maximize the effectiveness of selection for traits of interest. Combin­ing information from performance records and genetic tests into the selection process will be better than using performance, phenotype, and markers separately.

5. Molecular Markers
Until recently, researchers relied on information about how animals, plants, and their relatives perform to make observations about the genes they possess. Today, researchers can use molecular markers to find genes of interest that control how plants and animals perform. Some molecular markers are pieces of DNA that have no known function or impact on animal and plant performance. Other markers may involve the gene of interest itself.

6. Linked Markers One type of molecular marker is called a linked marker.
Using well-designed experiments, scientists can find molecular markers that are located very close to major genes of interest. The molecular marker is said to be linked to that gene. Linked markers are only near the gene of interest on the chromosome and are not part of the DNA of the gene of interest.
Suppose that scientists are trying to locate a certain gene in an animal species. Choosing animals randomly from a population and studying them would give the scientists no clues about whether a marker is associated with the gene. However, if scientists studied the progeny (offspring) of the mating of male and female animals through many generations, they may determine the presence of a useful molecular marker.

7. Marker-Assisted Selection
Direct Markers
A second kind of molecular marker is one that is part of the gene of interest. Direct markers are easier to work with after they are found, but they often are more difficult to find than linked markers
Marker-Assisted Selection
Three common technologies used as molecular markers are: restriction fragment length polymor­phisms, simple sequence repeats, and single nucleotide polymorphisms.

8. Restriction Fragment Length Polymorphisms (RFLPs)
Restriction fragment length polymorphisms (RFLPs) were the first molecular markers used to diagnose genetic variability in organisms. RFLP uses restriction enzymes to digest (cut) the DNA molecule and identify regions linked to a trait. The number of DNA frag­ments generated by one restriction enzyme digest can be in the millions, with many being several thousand nucleotides long. This makes it difficult to determine specific DNA fragments that are associated with the trait of interest on an electrophoresis gel. To help visualize specific DNA fragments, a technique called Southern blotting was developed.
Southern blotting uses a porous membrane containing specific radioactive DNA probes for one or more DNA fragments. Probes are very short pieces of DNA used to find specific sequences of A, C, T, and G in very long pieces of DNA from a chromosome. The probe hybridizes (attaches) to the membrane at a unique DNA band on an electrophoresis gel. The membrane containing the probe is developed on X-ray film and analyzed.

9. Simple Sequence Repeats or Microsatellites
Simple sequence repeats (SSRs), also called microsatellites, are repeated units of two to six nucle-otides that occur throughout an organism's genome. The sequence ATATATAT is one example of a microsatellite. The sequence GATGATGAT is another example. SSRs are useful as molecular markers because they are highly polymorphic (have many forms). SSRs have been used successfully as markers in a wide range of analysis, particularly those involving disease diagno­sis and forensics.

10. Single Nucleotide Polymorphisms (SNPs)
On average, SNPs will occur in an organism's DNA more than 1% of the time. Because only about 3% to 5% of an organism's DNA codes for proteins, most SNPs are found outside the regions of genes of interest. SNPs found in a gene of interest are of particular interest to researchers because they are directly associated with a desired trait. Because of the recent advances in technol­ogy, SNPs are playing a greater role in selection and diagnosis of genetic traits.

11. MARKER-ASSISTED BREEDINGP1 x P2
Susceptible
Resistant F1
large populations consisting of thousands of plants F2
MARKER-ASSISTED SELECTION (MAS)
Method whereby phenotypic selection is based on DNA markers

12. Overview of ‘marker genotyping’ (1) LEAF TISSUE SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS

13. Considerations for using DNA markers in plant breeding
Technical methodology
simple or complicated?
Reliability
Degree of polymorphism
DNA quality and quantity required
Cost**
Available resources
Equipment, technical expertise

14. Markers must be tightly-linked to target loci!
Ideally markers should be <5 cM from a gene or QTL
Marker A
QTL
5 cM
RELIABILITY FOR SELECTION
Using marker A only:
1 – rA = ~95%
Marker A
QTL
Marker B
5 cM
Using markers A and B:
1 - 2 rArB = ~99.5%
Using a pair of flanking markers can greatly improve reliability but increases time and cost

15. Markers must be polymorphic
RM84
RM296
P1 P2
P1 P2
Not polymorphic
Polymorphic!

16. DNA extractions LEAF SAMPLING DNA EXTRACTIONS Mortar and pestles
Porcelain grinding plates
LEAF SAMPLING
Wheat seedling tissue sampling in Southern Queensland, Australia.
High throughput DNA extractions “Geno-Grinder”
DNA EXTRACTIONS

17. Agarose or Acrylamide gels
PCR-based DNA markers
Generated by using Polymerase Chain Reaction
Preferred markers due to technical simplicity and cost
PCR Buffer +
MgCl2 +
dNTPS +
Taq +
Primers +
DNA template
PCR
THERMAL CYCLING
GEL ELECTROPHORESIS
Agarose or Acrylamide gels

18. Advantage of Molecular Markers
Simpler method compared to phenotypic screening
Especially for traits with laborious screening
May save time and resources
Selection at seedling stage
Important for traits such as grain quality
Can select before transplanting in rice
Increased reliability
No environmental effects
Can discriminate between homozygotes and heterozygotes and select single plants

19. Potential benefits from MAS
more accurate and efficient selection of specific genotypes - May lead to accelerated variety development
more efficient use of resources - Especially field trials

20. MAS BREEDING SCHEMES Marker-assisted backcrossing Pyramiding
Early generation selection
‘Combined’ approaches

21. 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
RECOMBINANT SELECTION
BACKGROUND SELECTION
TARGET LOCUS SELECTION
FOREGROUND SELECTION
BACKGROUND SELECTION

22. 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,

23. Select F2 plants that have Gene A and Gene B
Process of combining several genes, usually from 2 different parents, together into a single genotype
Breeding plan
Genotypes P1
Gene A x P1
Gene B
P1: AAbb x
P2: aaBB F1
Gene A + B
F1: AaBb F2 F2 AB Ab aB ab
AABB
AABb
AaBB
AaBb
AAbb
Aabb
aaBB
aaBb
aabb
MAS
Select F2 plants that have Gene A and Gene B
Hittalmani et al. (2000). Fine mapping and DNA marker-assisted pyramiding of the three major genes for blast resistance in riceTheor. Appl. Genet. 100:
Liu et al. (2000). Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat. Plant Breeding 119:

24. SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS)
PEDIGREE METHOD
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
P1 x P2 F1
Phenotypic screening F2
Plants space-planted in rows for individual plant selection F3
Families grown in progeny rows for selection. F4 F5
Preliminary yield trials. Select single plants. F6
Further yield trials F7
Multi-location testing, licensing, seed increase and cultivar release
F8 – F12
Benefits: breeding program can be efficiently scaled down to focus on fewer lines

25. CONVENTIONAL BACKCROSSING
BC2
MARKER-ASSISTED BACKCROSSING
P1 x F1
P1 x P2
BC1
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST % OF DONOR GENOME
P1 x P2
P1 x F1
BC1
VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT PARENT
BC2

26. Combined approaches
In some cases, a combination of phenotypic screening and MAS approach may be useful
To maximize genetic gain (when some QTLs have been unidentified from QTL mapping)
Level of recombination between marker and QTL (in other words marker is not 100% accurate)
To reduce population sizes for traits where marker genotyping is cheaper or easier than phenotypic screening.