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MARKER ASSISTED
SELECTION: RICE
Presented by:
Elizabeth Philip
INTRODUCTION
 MARKERS
Genetic markers represent genetic differences between individual
organisms or species. Generally, they do not represent the target
genes themselves but act as ‘signs’ or ‘flags’.
TYPES:
Morphological: height, grain color
Biochemical: isozymes
Cytological: G banding
DNA-based and/or molecular: RFLP, RAPD
 QTL: Quantitative Trait Loci:
o The regions within genomes that contain genes associated with a particular quantitative trait are
known as quantitative trait loci (QTLs).
o Quantitative traits are also called polygenic traits, because their phenotypes are controlled by
the combined activity of many genes.
GENE VS MARKER
 The gene of interest directly cause production of
protein or RNA that produce a desired trait or
phenotype.
 Markers are genetically linked to the gene of interest.
 If the gene of interest is not known, markers linked to
the gene of interest can still be used to select for
individuals with desirable alleles of gene of interest
MARKER ASSISTED SELECTION
◦ Process whereby a marker 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).
◦ Trait of interest is selected not based on the trait itself but on a marker linked to it.
◦ The assumption is that linked allele associates with the gene and/or quantitative trait locus (QTL)
of interest. MAS can be useful for traits that are difficult to measure, exhibit low heritability,
and/or are expressed late in development.
◦ Pre-Requisites: Two pre-requisites for marker assisted selection are: (i) a tight linkage between
molecular marker and gene of interest, and (ii) high heritability of the gene of interest.
◦ Markers Used: The most commonly used molecular markers include amplified fragment length
polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), random amplified
polymorphic DNA (RAPD), simple sequence repeats (SSR) or micro satellites, single nucleotide
polymorphisms (SNP), etc. The use of molecular markers differs from species to species also.
Conventional plant breeding is primarily based on phenotypic selection of superior
individuals among segregating progenies resulting from hybridization.
Breeding for specific traits in plants is expensive and time consuming.
The progeny often need to reach maturity before a determination of the success of
the cross can be made.
The greater the complexity of the trait, the more time and effort needed to achieve a
desirable result.
 The goal to MAS is to reduce the time needed to
determine if the progeny have trait.
 The second goal is to reduce costs associated
with screening for traits.
 If we can detect the distinguishing trait at the
DNA level we can identify positive selection very
early.
Marker Assisted Selection in comparison to Conventional Plant
Breeding
Steps in Marker Assisted Selection (MAS):
1. Selection of parents and development of breeding populations:
Parents with contrasting characters are chosen to get usable level of polymorphisms in RFLP
markers.
Parents should be pure homozygous.
Selected plants are crossed to obtain F1plants.
F2 progeny is required for study of segregation patterns of marker.
2. DNA extraction:
DNA is isolated from each plant of F2 population.
The isolated DNA is digested with specific restriction enzymes to obtain fragments of DNA.
3. QTL MAPPING:
The next step is to map the gene or quantitative trait locus (QTL) of interest by using different techniques and
then use this information for marker assisted selection.
The markers to be used should be close to gene of interest (<5 recombination unit or cM) in order to ensure
that only minor fraction of the selected individuals will be recombinants.
Two markers are used in order to reduce the chances of an error due to homologous recombination. For e.g.,
if two flanking markers are used at same time with an interval between them of approximately 20cM, there is
higher probability(99%) for recovery of the target gene.
 Markers must be tightly-linked to target loci!
Ideally markers should be <5 cM from a gene or QTL.
4. QTL confirmation
 QTL mapping studies should be independently confirmed or verified; testing the accuracy
of results from the primary QTL mapping study
5. QTL validation
 Generally refers to the verification that a QTL is effective in different genetic backgrounds
6. Marker validation
Testing the level of polymorphism of most tightly-linked markers within a narrow window
(say 5 - 10 cM) spanning a target locus and also testing the reliability of markers to predict
phenotype.
Steps in Marker
Assisted
Selection (MAS):
MAS SCHEMES:
1. MARKER ASSISTED BACKCROSSING (MAB):
 To incorporate a major gene from an agronomically inferior source (the donor parent) into
an elite cultivar or breeding line (the recurrent parent).
 The desired outcome is a line containing only the major gene from the donor parent, with the recurrent
parent genotype present everywhere else in the genome. Two types of selection are recognized:
• Foreground selection: the breeder selects plants having the marker allele of the donor parent at the
target locus. The objective is to maintain the target locus in a heterozygous state (one donor allele and
one recurrent parent allele) until the final backcross is completed. Then, the selected plants are self-
pollinated and progeny plants identified that are homozygous for the donor allele
• Background selection: the breeder selects for recurrent parent marker alleles in all genomic regions
except the target locus, and the target locus is selected based on phenotype. Background selection is
important in order to eliminate potentially deleterious genes introduced from the donor. So-called
' linkage drag ', the inheritance of unwanted donor alleles in the same genomic region as the target
locus, is difficult to overcome with conventional backcrossing, but can be addressed efficiently with the
use of markers.
2. MARKER ASSISTED GENE PYRAMIDING (MAGP):
• Aims to assemble multiple desirable genes
into a single genotype; used to improve
qualitative traits such as disease and insect
resistance.
3. COMBINED APPROACHES:
A combination of phenotypic screening and MAS
approach may be useful
1.To maximize genetic gain
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 ASSISTED SELECTION (MAS): AN
ADVANCED MOLECULAR TOOL IN RICE BREEDING
 Severe yield loss is observed in rice cultivation due to a number of abiotic and biotic stresses
 Several environmental factors have threatened sustainable agricultural production in emerging countries, with the
main variables affecting the future of agricultural production being higher incidence of extreme weather and a
number of environmental problems
 Biotic stresses - pathogens, pests and weeds
 Abiotic stresses - drought and periodic cycles of submergence, extreme cold, soil salinity,
 Crop losses caused by major biotic stressors such as bacterial
blight and blast disease, and due to insect pests are quite
high
 The occurrence of new stresses necessitates development of
highly improved and novel approaches to enhance the
capability of various rice varieties that can survive attacks
caused by several pathogens at once while also surviving in
unfavourable environments with high level of grain quality.
Marker assisted introgression of bacterial blight resistance
genes into Samba mahsuri
◦ Samba mahsuri (BPT5204) is a medium slender grain indica rice variety
that is very popular with farmers and consumers across India because of
its high yield and excellent cooking quality. However, the variety is
susceptible to several diseases and pests, including bacterial blight (BB).
◦ Bacterial blight (BB):
◦ BB caused by Xanthomonas oryzae pv. Oryzae (Xoo) is wide spread in
irrigated and low land rice ecosystem.
◦ It causes wilting of seedlings and yellowing and drying of leaves
◦ Growing resistant varieties is the only possible way to tackle the disease.
◦ More than 30 genes (Xa, Xa2........Xa30)characterized.
◦ Genotypes carrying single resistant gene have shown susceptibility at
different locations
• The xa21,xa13 and xa 5 genes were found to provide resistance against strains
within the Xoo lineage
• A rice line called SS1113 contains Xa21, xa13 and xa5 genes but scores low on
other characteristics
MARKERS USED FOR BACKGROUND SELECTION
• At each backcross generation, markers
closely linked to the three genes were used to
select plants possessing these resistance
genes (foreground selection) and
microsatellite markers polymorphic between
donor and recurrent parent were used to
select plants that have maximum contribution
from the recurrent parent genome
(background selection).
• A selected BC4F1 - selfed to generate
homozygous BC4F2 plants with different
combinations of BB resistance genes.
• The three-gene pyramid and two-gene
pyramid lines exhibited high levels of
resistance against the BB pathogen.
• Using PCR based molecular markers in a backcross-breeding program, 3 major BB resistance genes (Xa21,
xa13 and xa5)were introgressed into Samba Mahsuri from a donor line (SS1113) in which all the three genes are
present in a homozygous condition.
ADVANTAGES LIMITATIONS
Simpler method compared to phenotypic screening:
Especially for traits with laborious screening
Costly method
Selection at seedling stage: Important for traits such as grain
quality; Can select before transplanting in rice
Detection of various linked DNA markers is a difficult
and time consuming task.
Increased reliability: No environmental effect; Can
discriminate between homozygotes and heterozygotes and
select single plants
May become less efficient than phenotypic selection
in the long term.
APPLICATION:
Useful in gene pyramiding for disease resistance and insect resistance
Used for improvement of quality characters in different crops
Used in in both plants and animals
REFERENCES:
 Das, G. (2017). Insight into MAS: A Molecular Tool for Development
of Stress Resistant and Quality of Rice through Gene Stacking.
Frontiers.
https://www.frontiersin.org/articles/10.3389/fpls.2017.00985/full
 5.1 Marker assisted breeding. (2006). International Rice Research
Institute.
http://www.knowledgebank.irri.org/ricebreedingcourse/Marker_a
ssisted_breeding.html
 Marker assisted breeding of bioticstress resistance in Rice(2012).
Slideshare. https://www.slideshare.net/TNAUgenomics/marker-
assisted-breeding-of-biotic-stress-resistance-in-rice
 Marker Assisted Selection in Crop Breeding. Slideshare
https://www.slideshare.net/PawanChauhan16/marker-assisted-
selection-in-crop-breeding
THANK
YOU

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Marker assisted selection (rice).pptx

  • 2. INTRODUCTION  MARKERS Genetic markers represent genetic differences between individual organisms or species. Generally, they do not represent the target genes themselves but act as ‘signs’ or ‘flags’. TYPES: Morphological: height, grain color Biochemical: isozymes Cytological: G banding DNA-based and/or molecular: RFLP, RAPD  QTL: Quantitative Trait Loci: o The regions within genomes that contain genes associated with a particular quantitative trait are known as quantitative trait loci (QTLs). o Quantitative traits are also called polygenic traits, because their phenotypes are controlled by the combined activity of many genes.
  • 3. GENE VS MARKER  The gene of interest directly cause production of protein or RNA that produce a desired trait or phenotype.  Markers are genetically linked to the gene of interest.  If the gene of interest is not known, markers linked to the gene of interest can still be used to select for individuals with desirable alleles of gene of interest
  • 4. MARKER ASSISTED SELECTION ◦ Process whereby a marker 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). ◦ Trait of interest is selected not based on the trait itself but on a marker linked to it. ◦ The assumption is that linked allele associates with the gene and/or quantitative trait locus (QTL) of interest. MAS can be useful for traits that are difficult to measure, exhibit low heritability, and/or are expressed late in development. ◦ Pre-Requisites: Two pre-requisites for marker assisted selection are: (i) a tight linkage between molecular marker and gene of interest, and (ii) high heritability of the gene of interest. ◦ Markers Used: The most commonly used molecular markers include amplified fragment length polymorphisms (AFLP), restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), simple sequence repeats (SSR) or micro satellites, single nucleotide polymorphisms (SNP), etc. The use of molecular markers differs from species to species also.
  • 5. Conventional plant breeding is primarily based on phenotypic selection of superior individuals among segregating progenies resulting from hybridization. Breeding for specific traits in plants is expensive and time consuming. The progeny often need to reach maturity before a determination of the success of the cross can be made. The greater the complexity of the trait, the more time and effort needed to achieve a desirable result.  The goal to MAS is to reduce the time needed to determine if the progeny have trait.  The second goal is to reduce costs associated with screening for traits.  If we can detect the distinguishing trait at the DNA level we can identify positive selection very early. Marker Assisted Selection in comparison to Conventional Plant Breeding
  • 6.
  • 7. Steps in Marker Assisted Selection (MAS): 1. Selection of parents and development of breeding populations: Parents with contrasting characters are chosen to get usable level of polymorphisms in RFLP markers. Parents should be pure homozygous. Selected plants are crossed to obtain F1plants. F2 progeny is required for study of segregation patterns of marker. 2. DNA extraction: DNA is isolated from each plant of F2 population. The isolated DNA is digested with specific restriction enzymes to obtain fragments of DNA.
  • 8. 3. QTL MAPPING: The next step is to map the gene or quantitative trait locus (QTL) of interest by using different techniques and then use this information for marker assisted selection. The markers to be used should be close to gene of interest (<5 recombination unit or cM) in order to ensure that only minor fraction of the selected individuals will be recombinants. Two markers are used in order to reduce the chances of an error due to homologous recombination. For e.g., if two flanking markers are used at same time with an interval between them of approximately 20cM, there is higher probability(99%) for recovery of the target gene.  Markers must be tightly-linked to target loci! Ideally markers should be <5 cM from a gene or QTL.
  • 9. 4. QTL confirmation  QTL mapping studies should be independently confirmed or verified; testing the accuracy of results from the primary QTL mapping study 5. QTL validation  Generally refers to the verification that a QTL is effective in different genetic backgrounds 6. Marker validation Testing the level of polymorphism of most tightly-linked markers within a narrow window (say 5 - 10 cM) spanning a target locus and also testing the reliability of markers to predict phenotype.
  • 11. MAS SCHEMES: 1. MARKER ASSISTED BACKCROSSING (MAB):  To incorporate a major gene from an agronomically inferior source (the donor parent) into an elite cultivar or breeding line (the recurrent parent).  The desired outcome is a line containing only the major gene from the donor parent, with the recurrent parent genotype present everywhere else in the genome. Two types of selection are recognized: • Foreground selection: the breeder selects plants having the marker allele of the donor parent at the target locus. The objective is to maintain the target locus in a heterozygous state (one donor allele and one recurrent parent allele) until the final backcross is completed. Then, the selected plants are self- pollinated and progeny plants identified that are homozygous for the donor allele • Background selection: the breeder selects for recurrent parent marker alleles in all genomic regions except the target locus, and the target locus is selected based on phenotype. Background selection is important in order to eliminate potentially deleterious genes introduced from the donor. So-called ' linkage drag ', the inheritance of unwanted donor alleles in the same genomic region as the target locus, is difficult to overcome with conventional backcrossing, but can be addressed efficiently with the use of markers.
  • 12.
  • 13. 2. MARKER ASSISTED GENE PYRAMIDING (MAGP): • Aims to assemble multiple desirable genes into a single genotype; used to improve qualitative traits such as disease and insect resistance. 3. COMBINED APPROACHES: A combination of phenotypic screening and MAS approach may be useful 1.To maximize genetic gain 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
  • 14. MARKER ASSISTED SELECTION (MAS): AN ADVANCED MOLECULAR TOOL IN RICE BREEDING  Severe yield loss is observed in rice cultivation due to a number of abiotic and biotic stresses  Several environmental factors have threatened sustainable agricultural production in emerging countries, with the main variables affecting the future of agricultural production being higher incidence of extreme weather and a number of environmental problems  Biotic stresses - pathogens, pests and weeds  Abiotic stresses - drought and periodic cycles of submergence, extreme cold, soil salinity,  Crop losses caused by major biotic stressors such as bacterial blight and blast disease, and due to insect pests are quite high  The occurrence of new stresses necessitates development of highly improved and novel approaches to enhance the capability of various rice varieties that can survive attacks caused by several pathogens at once while also surviving in unfavourable environments with high level of grain quality.
  • 15. Marker assisted introgression of bacterial blight resistance genes into Samba mahsuri ◦ Samba mahsuri (BPT5204) is a medium slender grain indica rice variety that is very popular with farmers and consumers across India because of its high yield and excellent cooking quality. However, the variety is susceptible to several diseases and pests, including bacterial blight (BB). ◦ Bacterial blight (BB): ◦ BB caused by Xanthomonas oryzae pv. Oryzae (Xoo) is wide spread in irrigated and low land rice ecosystem. ◦ It causes wilting of seedlings and yellowing and drying of leaves ◦ Growing resistant varieties is the only possible way to tackle the disease. ◦ More than 30 genes (Xa, Xa2........Xa30)characterized. ◦ Genotypes carrying single resistant gene have shown susceptibility at different locations
  • 16. • The xa21,xa13 and xa 5 genes were found to provide resistance against strains within the Xoo lineage • A rice line called SS1113 contains Xa21, xa13 and xa5 genes but scores low on other characteristics
  • 17. MARKERS USED FOR BACKGROUND SELECTION
  • 18. • At each backcross generation, markers closely linked to the three genes were used to select plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were used to select plants that have maximum contribution from the recurrent parent genome (background selection). • A selected BC4F1 - selfed to generate homozygous BC4F2 plants with different combinations of BB resistance genes. • The three-gene pyramid and two-gene pyramid lines exhibited high levels of resistance against the BB pathogen. • Using PCR based molecular markers in a backcross-breeding program, 3 major BB resistance genes (Xa21, xa13 and xa5)were introgressed into Samba Mahsuri from a donor line (SS1113) in which all the three genes are present in a homozygous condition.
  • 19. ADVANTAGES LIMITATIONS Simpler method compared to phenotypic screening: Especially for traits with laborious screening Costly method Selection at seedling stage: Important for traits such as grain quality; Can select before transplanting in rice Detection of various linked DNA markers is a difficult and time consuming task. Increased reliability: No environmental effect; Can discriminate between homozygotes and heterozygotes and select single plants May become less efficient than phenotypic selection in the long term. APPLICATION: Useful in gene pyramiding for disease resistance and insect resistance Used for improvement of quality characters in different crops Used in in both plants and animals
  • 20. REFERENCES:  Das, G. (2017). Insight into MAS: A Molecular Tool for Development of Stress Resistant and Quality of Rice through Gene Stacking. Frontiers. https://www.frontiersin.org/articles/10.3389/fpls.2017.00985/full  5.1 Marker assisted breeding. (2006). International Rice Research Institute. http://www.knowledgebank.irri.org/ricebreedingcourse/Marker_a ssisted_breeding.html  Marker assisted breeding of bioticstress resistance in Rice(2012). Slideshare. https://www.slideshare.net/TNAUgenomics/marker- assisted-breeding-of-biotic-stress-resistance-in-rice  Marker Assisted Selection in Crop Breeding. Slideshare https://www.slideshare.net/PawanChauhan16/marker-assisted- selection-in-crop-breeding THANK YOU