Topic Role of Marker Assisted Selection in Plant Resistance is described in detail including some case studies. Types of markers used in genetic engineering and biotechnology are described in detail. Marker assisted selection is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant of a trait of interest. Since the first reported linkage of an agronomically important trait (a quantitative trait locus affecting seed weight) to a simply controlled gene (seed colour) in common bean by Sax (1923), it has taken more than 60 years for genetic markers to become a qualified tool for plant breeding programs. In rice, the Xieyou 218 hybrid was the first to be developed through MAS to select individuals carrying a bacterial blight-resistant gene. Marker-assisted selection (MAS) can be applied at the seedling stage, with high precision and reductions in cost. Genetic mapping of major genes and quantitative traits loci (QTLs) for agricultural traits is increasing the integration of biotechnology with the conventional breeding process. Traits related to disease resistance to pathogens and to the quality of some crop products are offering some important examples of a possible routinary application of MAS. For more complex traits, like yield and abiotic stress tolerance, a number of constraints have severe limitations on an efficient utilization of MAS in plant breeding. However, the economic and biological constraints such as a low return of investment in small-grain cereal breeding, lack of diagnostic markers, and the prevalence of QTL-background effects hinder the broad implementation of MAS but over the past 2 decades, a number of R-genes conferring resistance to a diverse range of pathogens have been mapped in many crops using molecular markers.

1. Role of Marker Assisted Selection (MAS) in Plant Resistance
Presented by
Randeep Choudhary
Credit Seminar
On
Seminar In charge
Dr. Pokhar Rawal
Major Advisor
Dr. N.L. Meena
Date & Time
25/03/2022 , 03:30 PM
Department of Plant Pathology
Rajasthan College of Agriculture
Maharana Pratap University of Agriculture & Technology
Udaipur-313001

2. OVERVIEW
Definition
Types of markers
Properties of ideal marker for MAS
Importance of Molecular Markers
Use of DNA Markers in MAS
MAS Schemes in Plant Breeding
Steps for MAS
Process of MAS
Advantages of MAS Over Conventional Methods
Case Studies
Gene-Marker Association for Disease Resistance in Different Crops
Conclusion

3. WHAT IS MAS ?
• Marker assisted selection or marker aided selection (MAS) is a process whereby a marker
(morphological, biochemical or one based on DNA/RNA variation) is used for
indirect selection of a genetic determinant or determinants of a trait of interest (i.e.
productivity, disease resistance, abiotic stress tolerance, and/or quality). This process is used
in plant and animal breeding.
• MAS refers to the use of DNA markers tightly linked to the target loci as a substitute for
phenotypic screening or to assist it.
• Marker assisted selection (MAS) is indirect selection process where a trait of interest is
selected not based on the trait itself but on a marker linked to it.
Ribaut et al., 1998

4. WHAT IS MAS ?

5. PROCESS OF MAS

6. HISTORY
1. Botstein et al. (1980) – Developed the first DNA-based genetic markers Restriction fragment length
polymorphisms (RFLPs).
2. Bernatzky & Tanksley (1986) – Constructed first linkage map in a crop plant (tomato) based on RFLPs.
3. Paterson et al. (1988) - first who used a complete RFLP linkage map to resolve quantitative traits into discrete
Mendelian factors.
4. Williams et al. (1990) – Developed Random-amplified polymorphic DNAs (RAPDs).
5. Vos et al. (1995) – Developed Amplified Fragment Length Polymorphism (AFLPs).
6. Powell et al. (1996) – Developed Microsatellites, also termed Simple Sequence Repeats (SSRs).
7. Gupta et al. (2001) - Single Nucleotide Polymorphisms (SNPs).
Brumlop, S. And Finckh, M.R.,2010

7. TYPES OF MARKERS
Positive selectable marker
Positive selectable markers are
selectable markers that confers
selective advantage to its host
organism. An example would be
antibiotic resistance, which allows
the host organism to survive
antibiotics selection.
Negative selectable marker
Negative selectable markers are
selectable markers that would
eliminate its host organism upon
selection. An example would be
thymidine kinase, which would
make the host sensitive to
ganciclovir selection.
Positive / Negative selectable marker

8. TYPES OF MARKERS
Genetic Markers : Genetic markers are the biological features of the individual organism that are determined by the
allelic form of the gene or genetic loci and can be transferred from one generation to another.
These do not represent the target genes, but act as their escort or flags; however, they are located near the target gene
(tightly linked) but have no effect on the phenotypes of the trait of interest and occupy a specific position in the
genome within chromosomes called the loci (or locus).
Xu classified genetic markers into two categories: 1. Classical markers
2. DNA-Based / Molecular markers
1. Classical Markers : It includes Morphological, Cytological and Biochemical markers.
Morphological and biochemical markers are affected by environmental factors and the developmental stages of the
plant. They are also limited in numbers and hence have not proved effective in molecular breeding.
Pathania et al., 2017

9. TYPES OF MARKERS

10. MORPHOLOGICAL MARKERS
• First markers loci available that have obvious impact on morphology of plant. Genes that affect form,
coloration, male sterility or resistance among others have been analyzed in many plant species. Examples of
this type of marker may include the presence or absence of awn, leaf sheath coloration, height, grain color,
aroma of rice etc.
• Generally correspond to the qualitative traits that can be scored visually.
• The major limitations with these markers are; high dependency on environmental factors, undesirable
features such as dwarfism or albinism, time consuming, labour intensive and requirement of large plant
population.
Stuber et al.,1999

11. BIOCHEMICAL MARKERS
• A gene that encodes a protein that can be extracted and observed; for example, isozymes and storage proteins.
• Biochemical markers are superior to morphological markers in that they are generally independent of
environmental growth conditions. The only problem with isozymes in MAS is that most cultivars (commercial
breeds of plants) are genetically very similar and isozymes do not produce a great amount of polymorphism
and polymorphism in the protein primary structure may still cause an alteration in protein function or expression.
• Isozymes – different molecular forms of the same enzyme which are identified by their activities. They are products
of the various alleles of one or several genes.
• Proteins – product of gene expression.
• Both, can be isolated and identified by electrophoresis and staining.

12. CYTOLOGICAL MARKERS
The chromosomal banding produced by different strains; for example, G banding.
Structural and numerical variations are identified.
Structural : Deletion, Insertion etc.
Numerical : Trisomy, Monosomy, Nullisomy

13. DNA-Based Markers
DNA-Based Markers : DNA-based markers represent variations in genomic DNA sequences of different individuals.
They are detected as the differential mobility of fragments in a gel, hybridization with an array or PCR amplification,
or as DNA sequence variation.
Development of these markers began in 1974, when the analysis of fragments generated by restriction enzyme
digestion of adenovirus DNA was used for physical mapping of a gene. The variation generated by such fragments was
later called restriction fragment-length polymorphism (RFLP), and was used as the first DNA-based marker.
Pathania et al., 2017

14. MOLECULAR/DNA-Based MARKERS
1. Hybridization-Based Markers : Hybridization-based markers include RFLP, diversity array technology, VNTRs,
single feature polymorphisms, and restriction site-associated DNA markers.
E.g.: RFLP- RFLP is a variation in the DNA sequence of a genome that can be detected by digesting the DNA
with restriction enzymes into small fragments and resolving them by gel electrophoresis.
RFLP is the most widely used hybridization-based molecular marker.

15. MOLECULAR/ DNA-based MARKERS
2. Polymerase Chain Reaction Based Markers:
A. Random Amplified Polymorphic DNA: RAPDs are the most common PCR-based markers of a dominant
nature. These consist of a 10-base pair (bp) DNA fragment as a single decamer primer sequence.
B. Amplified Fragment-Length Polymorphism: The selective amplification of DNA fragments obtained by
restriction enzyme digestion resulted in the generation of AFLP markers. Two restriction enzymes,
one hexa cutter (e.g., EcoRI) and one tetra cutter (e.g., MseI), are used to digest high molecular weigh
DNA.

16. MOLECULAR/ DNA-based MARKERS
C. Single-Nucleotide Polymorphisms: A single-nucleotide base difference between two DNA sequences or
individuals is an SNP.
• SNPs result from transitions (C/T or G/A) or transversions (C/G, A/T, C/A, or T/G) according to nucleotide
substitutions.
• A single-nucleotide base is the smallest unit of inheritance; thus SNPs provide the simplest form of molecular
marker. Typically, SNP frequencies are in the range of one SNP for every 100-300 bp in plants.

17. Properties of Molecular Markers
1. Moderately to high polymorphic.
2. Frequent occurrence in the genome.
3. Even distribution throughout the genome.
4. Easy access.
5. Easy and fast assay.
6. High reproducibility.
7. Easy exchange of data between laboratories.
8. Low cost for both marker development and assay.

18. • Five main considerations for the use of DNA markers in MAS (Mohler and Singrun, 2004) are –
1. Reliability: Molecular markers should co-segregate or tightly linked to traits of interest, preferably less than 5
cM genetic distance. The use of flanking markers or intragenic markers will greatly increase the reliability of the
markers to predict phenotype.
2. DNA quantity and quality: Some marker techniques require large amounts and high quality DNA, which may
sometimes be difficult to obtain in practice, and this adds to the cost of the procedures.
3. Technical procedure: Molecular markers should have high reproducibility across laboratories and transferability
between researchers. The level of simplicity and time required for the technique are critical considerations. High-
throughput simple and quick methods are highly desirable.
4. Level of polymorphism: Ideally, the marker should be highly polymorphic in breeding material and it should be
co-dominant for differentiation of homozygous and heterozygous individuals in segregating progenies.
5. Cost: Molecular markers should be user-friendly, cheap and easy to use for efficient screening of large
populations. The marker assay must be cost-effective in order for MAS to be feasible.
USE OF DNA MARKERS IN MAS
Mohler and Singrun, 2004

19. MAS Schemes
1. Early generation marker assisted selection: MAS has great advantage in early generation selections by
eliminating undesirable gene combinations especially those that lack essential disease resistance genes.
• MAS-based early generation selection not only selects suitable gene combinations but also ensure a high
probability of retaining superior breeding lines. (Eathington et al. 1997)
• An important prerequisite for successful early-generation selection with MAS are large populations and low
heritability of the selected traits. The relative efficiency of MAS is greatest for characters with low heritability.
(Lande and Thompson 1990)
2. Marker-assisted backcrossing (MABC): Backcrossing is used in plant breeding to transfer favourable traits
from a donor plant into an elite genotype (recurrent parent).
• In repeated crossings the original cross is backcrossed with the recurrent parent until most of the genes stemming
from the donor are eliminated. (Becker 1993)
• There are three levels of selection in which markers may be applied in backcross breeding. Markers can be used in
the context of MABC to either control the target gene (foreground selection) or to accelerate the reconstruction of
the recurrent parent genotype (background selection) and to select backcross progeny having the target gene with
tightly linked flanking markers in order to minimize linkage drag (recombinant selection).

20. MAS Schemes in Plant Breeding
3. Marker-assisted recurrent selection (MARS): The improvement of complex traits via phenotypic recurrent
selection is generally possible, but the long selection cycles impose restrictions on the practicability of this breeding
method.
• With the use of markers, recurrent selection can be accelerated considerably and several selection-cycles are possible
within one year, accumulating favorable QTL alleles in the breeding population.
(Eathington et al. 2007).
4. Marker assisted pyramiding: Pyramiding is the process of simultaneously combining multiple genes/QTLs together
into a single genotype. This is possible through conventional breeding but extremely difficult or impossible at early
generations.
• The Barley Yellow Mosaic Virus (BaYMV) complex as an example is a major threat to winter barley cultivation in
Europe. As the disease is caused by various strains of BaYMV and Barley Mild Mosaic Virus (BaMMV), pyramiding
resistance genes seems an intelligent strategy.

21. MAS Schemes in Plant Breeding
5. Combined marker-assisted selection: The strategic combination of MAS with phenotypic screening is known as
‘combined MAS’ (coined by Moreau et al. 2004).
• It may have advantages over phenotypic screening or MAS alone in order to maximize genetic gain (Lande and
Thompson 1990). This approach could be adopted when additional QTLs controlling a trait remain unidentified or
when a large number of QTLs need to be manipulated.
• Zhou et al. (2003) observed in wheat that, MAS combined with phenotypic screening was more effective than
phenotypic screening alone for a major QTL on chromosome 3BS for Fusarium head blight resistance.

22. Advantages of MAS Over Conventional Methods
1. Gene stacking for a single trait: MAS allows breeders to identify the presence of multiple genes/alleles related
to a single trait, when the alleles do not exert individually detectable effects on the expression of the trait.
E.g.: when one gene confers resistance to a specific disease, breeders would be unable to use traditional
phenotypic screening to add another gene to the same cultivar in order to increase the durability of resistance.
In such cases, MAS would be the only feasible option.
2. Recessive genes: MAS allows breeders to identify heterozygous plants that carry a recessive allele of interest whose
presence cannot be detected phenotypically. In traditional breeding approaches, an extra step of selfing is required to
detect phenotypes associated with recessive genes.
3. Heritability of traits: MAS is mainly useful in selection for traits with low heritability up to a point, gains from
MAS increase with decreasing heritability.
4. Seasonal & Geographical considerations: Using molecular markers, at any time of the year / In one location,
breeders can screen for the presence of an allele (or alleles) associated with traits that are expressed only during
certain growing seasons / In other Locations.
5. Multiple genes, multiple traits: MAS offers potential savings when there is a need to select for multiple traits
simultaneously. With conventional methods, it is often necessary to conduct separate trials to screen for individual
traits.

23. Examples of MAS Applications in Different Crops
Wheat :
Ragimekula et al., 2013

24. Examples of MAS Applications in Different Crops
Barley :
Ragimekula et al., 2013

25. Examples of MAS Applications in Different Crops
Soybean and White Bean :
Ragimekula et al., 2013

26. CASE STUDY-1
Bacterial Leaf Blight of Rice: Samba Mahsuri (BPT5204) is a rice variety that is popular in India. Despite
its popularity among Indian farmers, the variety is susceptible to many insects and diseases, including BLB
caused by Xanthomonas oryzae pv oryzae. This disease is responsible for up to 50% losses in the yield of
rice crops in India. Sundaram et al. applied MAS for the introgression of three resistance genes (Xa21,
xa13, and xa5) into Samba Mahsuri from a donor line SS1113 possessing all three genes in a homozygous state.
The donor line was crossed with Samba Mahsuri and the F1 thus obtained was backcrossed with the recipient
parent Samba Mahsuri.
Sundaram et al., 2008

28. CASE STUDY-2
1. Stripe Rust of Barley: In barley, MAS has been employed for the management of stripe rust caused by Puccinia
striiformis Westend. f. sp. hordei, an important disease of barley worldwide.
• Several qualitative and quantitative genes conferring resistant against barley stripe rust have been reported by many
workers. Three QTLs (QTL4, QTL5, and QTL7) were identified on chromosomes 4, 5, and 7 respectively.
• Castro et al. pyramided these three QTLs and studied their effect on resistance against the disease at the seedling
stage. The parents used in the study were Orca, Harrington, and D1-72.
• Orca, a two row barley cultivar derived from the cross between Calicuchima-sib and Bowman, was the source of
QTL4 and QTL7.
• Harrington is a two-row malting barley cultivar. D1-72 is a line derived from the cross between Shyri and Galena
population that has a resistance allele at QTL5 tracing to Shyri.
Castro et al., 2002

30. Gene-Marker Association for Disease Resistance in Different Crops

31. CONCLUSION
• Among all the developments during the past 3 decades in genetic engineering and molecular biology, One of the
most fascinating development is the birth of MAS after the discovery of DNA marker technology.
• The MAS concept has proved a boon to plant breeders and plant pathologists because it is associated with disease
resistance and provides solutions to problems faced in applying conventional crop improvement programmes.
• The advent of advances in DNA marker technology, together with the concept of MAS, provides new solutions
for selecting and maintaining desirable genotypes.
• MAS has made it possible to carry out selection in early-segregation generations and at early stages of plant
development for pyramiding R-genes, thus obtaining the end product in a short time.

32. THANKYOU