1. The document discusses a seminar on applying molecular markers in plant breeding. It defines different types of markers including morphological, cytological, biochemical, and DNA/molecular markers. 2. It describes various molecular marker techniques like RFLP, RAPD, AFLP, and SSR. The techniques differ in characteristics like being dominant or co-dominant. 3. Molecular markers have important applications in plant breeding like marker-assisted selection, genetic diversity analysis, germplasm characterization, variety identification, and gene pyramiding. 4. Two case studies demonstrate the use of SSR markers to study genetic diversity in aromatic rice accessions and identify hybrids in sunflower. Specific markers were

1. SEMINAR ON
APPLICATIONOF MOLECULAR MARKERS IN PLANT BREEDING
PRESENTED BY
SUNIL.L
Reg .No:K-19/061
Research guide SEMINAR INCHARGE
Dr. M. S. Mote Dr.A.G.Bhoite
Asst. Prof. of Agril ,Botany Asso.Prof.of Agril, Botany
Division of Agril, Botany Division of Agril, Botany
RCSM College of Agriculture, Kolhapur RCSM College of Agriculture ,Kolhapur

2. What is a marker
Classification of markers
Types of molecular /DNA marker
Application of molecular markers
Case study
Conclusion
Future thrust
CONTENTS

3. INTRODUCTION

4. What is a Marker ???
Any genetic element (locus,allele or DNA sequence) which
can be readily detected by phenotype ,cytological or
molecular techniques and whose inheritance can be easily
followed.

5. MARKERS

6. Morphological markers
Disadvantage:
• low polymorphism
• Generally dominant
• Limited in number
• Unstable to environmental
effect
Markers that are related to shape, size, colour, and surface of
various plant parts. Such characters are used for varietal
identification.

7. Cytological markers
Markers that are related with variations present in the
numbers, banding patterns, size, shape, order and position of
chromosomes are known as cytological markers.
Disadvantage: low polymorphism and need special technique

8. Biochemical markers
• Markers that related to variations in protein and amino acid
banding pattern are known as biochemical markers.
• These are detected by electrophoresis and specific staining.
• Disadvantages: limited in
number, and are influenced
by environmental factors or
the developmental stage of
the plant (Winter & Kahl,
1995).

9. DNA/Molecular markers
• The DNA markers are related to variation in genomic DNA
sequences of different individuals.
• They are based on naturally occurring polymorphism in DNA
sequence i.e. base pair addition, deletion, substitution.
• DNA markers are unlimited in number and are not affected
by environmental factors and the developmental stage of the
plant(Winter&Kahl,1995).
• These markers are selectively neutral because they are usually
located in non-coding regions of DNA.

10. • Markers that are close together or tightly-linked will be
transmitted together from parent to progeny more frequently.
• markers that are located in close proximity to genes
(i.e.tightly linked) may be referred as gene‘tags’.
• They are used to flag the position of a particular gene or trait
of interest.

12. Co-dominant marker
• It indicate or differentiate
whether individual is
homozygous and
heterozyous
• Ex: RFLP,SSR
Dominant marker
• It does not differentiate
• Ex:AFLP,RAPD

13. Restriction Fragment Length Polymorphism (RFLP)
• It refers to variations found within a species in the length of DNA fragment
generated by specific endonuclease.
Advantages :
• Simple technique
• Co dominant
• highly reproducible
disadvantages:
• Requires large quantities of high
molecular weight DNA.
• Expensive, time consuming and
labor intensive .
Uses:
• Used in determining paternity
cases.
• Diagnosis of pathogen in plants.
• gene mapping ,germplasm
characterization.

14. Randomly Amplified Polymorphic DNA (RAPD)
• RAPD refers to variations found within a species in the randomly amplified
fragments of DNA generated by using a single oligonucleotide as a primer of
arbitrary sequence.
Advantages
• Simple and quick technique
• Primers are readily available
• very small DNA samples required
Disadvantages:
• polymorphism is limited
• dominant marker
• Reproducibility of results is low or
inconsistent
Uses:
• used for DNA fingerprinting of
cultivars
• Varietal identification ,construction of
linkage maps

15. Amplified Fragment Length Polymorphism (AFLP)
advantages:
• Requires small quantities of DNA
• highly reproducible
• Less labour intensive and faster
technique
disadvantages:
• Dominant markers
• Requires very high quality DNA
Uses:
• Used in germplasm characterization
• Varietal identification and MAS
• It refers to variations observed within a species in the length of amplified DNA
fragments made by restriction endonuclease.
• AFLP shares features of both RFLP and RAPD . it uses restriction enzyme
digested genomic DNA as template for PCR amplification.

16. Simple Sequence Repeat(SSR)
• SSR markers are microsatellite based markers analysed by PCR
amplification of a short genomic region containing the repeated sequence.
Advantages :
• Co-dominant
• Highly polymorphic
• Requires very small amounts of DNA
Disadvantages:
• The cost of developing SSR markers is
very high
• labour intensive
Uses:
• Used for genetic mapping of
eukaryotes.

17. Overview of important markers

18. Application of molecular markers

19. Generally co-dominant markers (SSR,RFLP etc) are used for the
identification/confirmation of hybridity

20. Marker assisted selection(MAS)
• MAS is an indirect selection for trait of interest(gene)based on
molecular markers linked to that trait of interest .
(Or)
• Selection for specific gene (which affect a trait of interest)
using genetic markers is referred as MAS
• Marker-assisted selection may greatly increase the efficiency
and effectiveness in plant breeding compared to conventional
breeding methods (Witcombe &Virk, 2001).
• MAS is done with the help of molecular markers or molecular
techniques Example: RFLP, SSR,RAPD,CAPS,SSCP, SNP etc.

22. Advantages of MAS :
• Simpler than phenotypic screening especially in the case of
complex traits.
• High efficiency( screening of progeny in early stage allows a
breeder to reject undesirable genotypes from the programme
more quickly
• Time saving (field trails ,disease test/screening)
• Selection of genotypes can be carried out at the seedling
stage
Disadvantages of MAS :
• Requires technical skill
• Expensive technique

23. Genetic diversity analysis
• Analysis of genetic diversity is important for determination of
phylogenetic relationships among the lines.
• Molecular markers can be used for assessment of genetic
diversity in cultivars.
• Generally diversity analysis is based on phenotypes of number
of characters. but phenotypic variation is expected to reflect
only a part of the variation present at the genotypic level.
• Molecular markers may be expected to capture the genetic
variation more effectively than the trait phenotypes.

24. Germplasm characterisation and conservation
• Molecular markers can be gainfully used in the different
activities of germplasm conservation
• The germplasm accessions of a crop species may be
genotyped for a set of molecular markers
• The marker data can be used for grouping of the accessions
• Marker genotype data would allow identification and removal
of duplications in the collection ,which would reduce the cost
of managing the collection.

25. Identification of varieties and hybrids
• Molecular markers genotypes can be used to develop DNA
fingerprints of different individuals /lines/varieties/hybrids.
• SSR markers are widely used for DNA fingerprinting.
• Marker data helps in
unequivocal
identification of
different varieties and
hybrids ,and can be
useful for registration of
varieties by serving as a
tools for their
identification.

26. Determination of genetic purity
• Molecular markers provide the sensitive and comprehensive
test for cultivar purity.
• Marker data of DNA is extracted from individual seedlings
used in the detection/ identification of given variety from
other contaminated variety/hybrids/weeds.

27. Gene pyramiding
• Combining two or more different genes conferring
resistance to the pathogen/insect in the single lines/variety.
• Pyramiding is extremely difficult to achieve using
conventional methods( because it requires progeny test
,repeated disease tests)
• The use of molecular markers greatly facilitates gene
pyramiding as it minimize the need for disease tests and
progeny tests.

28. 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
F2
F1
Gene A + B
P1
Gene A
x P2
Gene B
MAS
Select F2 plants that have Gene
A and Gene B
Genotypes
P1: AAbb P2: aaBB
F1: AaBb
• Process of combining several genes, usually from 2 different parents,
together into a single genotype
x
Breeding plan

29. Case study

30. CASE STUDY -1
objectives :
• To quantify the genetic divergence of aromatic rice accessions using SSR
markers and
• to identify the potential accessions for introgression into the rice breeding
program.
Material and methods:
• This research was carried out to study the genetic diversity among the 50
aromatic rice accessions including three local check varieties using the 32
simple sequence repeat (SSR) markers.
• Seeds of the 53 rice accessions were sown in the different pots using
randomized complete block design (RCBD) with three replications.

32. • The dendrogram based on UPGMA and Nei’s genetic distance
classified the 53 rice accessions into 10 clusters.
• Analysis of molecular variance (AMOVA) revealed that 89% of
the total variation observed in this germplasm came from
within the populations, while 11% of the variation emanated
among the populations.
• Using all these criteria and indices, seven accessions
(Acc9993, Acc6288, Acc6893, Acc7580, Acc6009, Acc9956,
and Acc11816) from three populations have been identified
and selected for further evaluation before introgression into
the breeding program and for future aromatic rice varietal
development.

33. CASE STUDY -2
Objective:
• To identify the pure hybrid or confirmation of hybridity in sunflower
hybrids
• To identify the specific marker associated with each hybrid and
parental lines.
Material and methods :
• The study included five hybrids, four female lines and two male
lines( which are listed in table 1)

34. • 58 primer pairs SSR markers were screened to identify the
specific marker associated with each hybrid and parental
lines.
• Among the five hybrids studied, hybrids KBSH-44 and KBSH-
53 could be distinguishable from their parental lines using a
specific SSR marker.

35. • Based on the complementary banding patterns between the
hybrids and their parents, the SSR marker ORS 309 and ORS
170 was identified as the two specific markers distinguish F1
hybrid KBSH-44 form their parental lines.

36. Similarly, hybrid of sunflower KBSH-53, could be identified and
distinguished from their parental lines by the SSR marker ORS 811

37. CASE STUDY-3
Objectives:
• This study is to develop wheat lines with inbuilt tolerance to
drought stress using marker assisted backcross breeding (MABB)
approach employing three linked quantitative trait loci (QTLs).
Material and methods:
• The high-yielding wheat cultivar ‘HD2733’ sensitive to drought and
is used as the recurrent parent.
• ‘HI1500’ released for water-limiting conditions and carrying
drought-tolerant QTLs (Xbarc68-101+ Xgdm93+ Xgwm165) was
used as donor parent.

39. Grain yield in five improved lines of ‘HD2733’
• The three QTLs combined through MABC led to the development of
29 improved lines that are tolerant to drought stress.
• The improved lines have desirable morpho-physiological characters,
in tandem with high chlorophyll content, low canopy temperature,
high normalized difference vegetation index and at par grain yield
compared to the original parent ‘HD2733’.
• Out of 29 lines five drought stress-tolerant lines selected to be the
future products for release as new improved wheat cultivars.

40. FUTURE THRUST
• With the highly advanced molecular genetic techniques, we
are still not achieving our goals due to inaccurate
phenotyping.
• High-throughput phenotyping techniques solve these
problems.
• There is a need to make the molecular marker technology
more precise, productive and cost effective in order to
investigate the underlying biology of various traits of interest.

41. CONCLUSION