A brief outlook on Molecular markers

1. Molecular MarkersMolecular Markers
Their applications in crop improvementTheir applications in crop improvement
Presented By :
Mrinali M.
M.Sc 1st
Year
Dept of Seed Science and
Technology

2. What is a Marker?What is a Marker?
• Marker is an allelic difference or variation at a given locus in
the DNA that can be observed at morphological, biochemical
or molecular level
• Molecular marker are based on naturally occurring changes
or polymorphism in DNA sequence (deletion, substitution,
addition, tandem repeat or duplication)
• All molecular markers occupy specific genomic positions
within the chromosome k/as ‘loci’
• Markers located in close proximity to desirable genes
(tightly linked) are k/as ‘gene tags’
• Agriculturally important traits are governed by many genes
‘Polygenic’ (Quantitative traits)
• Regions in genome containing genes associated with a
particular quantitative trait are k/as Quantitative Trait Loci
(QTL)

3. Why Molecular Marker ?Why Molecular Marker ?
• Because they are selectively neutral as they
are present in the non coding region of the
genome.
• Makers are co-segregating with the trait of
interest.
• They follow exactly the Mendelian pattern
of inheritance.
• Free from epistatic interaction or
pleiotropic effect

4. A Perfect Molecular MarkerA Perfect Molecular Marker
• Polymorphic
• Co-dominant
• Reproducible
• Robust
• Cost effective
• Easy to use
• High throughput
• Closely linked to the trait of interest
Marker
Trait Marker

5. Level of analysis of markersLevel of analysis of markers
Class of Marker Level of Analysis
1. Morphological Markers Phenotype
2. Biochemical Markers Gene Product
3. DNA markers / genetic
markers
DNA Sequence

6. 1. Morphological Markers1. Morphological Markers
• Are botanical descriptors of plant which are visually or
phenotypically characterized
• K/as DUS descriptors or universal markers
1. D- Distinctiveness
2. U- Uniformity
3. S- Stability
• Seed colour, seed shape, seed size,
flower colour, growth habits,
plant pigmentation

7. 2. Biochemical Markers2. Biochemical Markers
• Are of two types:
1. Protein based
2. Enzyme based known as Isozyme
• First true biochemical marker was an allelic variant
of enzyme pyruvate dehydrogenase
• Isoforms can be resolved by gel electrophoresis
based on their size, shape and amino acid
differences
• Can be easily assayed and detected
• They are low in polymorphism as compared to DNA
markers

8. 3. DNA based Markers3. DNA based Markers
A. On the basis of ability to discriminate between
same or different species
1. Co-dominant: discriminate between homo and
heterozygotes
2. Dominant: which do not discriminate between
homo and heterozygotes
• They can be visualized by:
a. Gel electrophoresis
b. Ethidium bromide or silver staining
c. Radioactive or colorimetric probes

9. Comparison between co-dominant &Comparison between co-dominant &
dominant markersdominant markers
P1 P2 F1
AA aa Aa
P1 P2 F1
BB bb Bb

10. Classification ofClassification of
Molecular MarkersMolecular Markers
Hybridization
based
markers

11. • RAPD :Random Amplified Polymorphic DNA
• RFLP: Restriction Fragment Length Polymorphism
• AFLP: Amplified Fragment Length Polymorphism
• SSR :Simple Sequence Repeat
• ISSR: Inter Simple Sequence Repeat
• SNP : Single Nucleotide Polymorphisms
• SCAR: Sequence Characterized Amplified Region
• CAPS :Cleaved Amplified Polymorphic Sequence
• TRAP: Target Region Amplification Polymorphism
• DArT : Diversity Arrays Technology
• DAF: DNA Amplification Fingerprinting
• SRAP: Sequence Related Amplified Polymorphism
Markers : Full FormsMarkers : Full Forms

12. • On the basis of principles and methods of detection
Collard B.C.Y. et al , 2005. (Euphytica)

13. A. Hybridization based markersA. Hybridization based markers
• RFLP – Restriction Fragment Length Polymorphism
• Based on polymorphism arising due to chromosomal
aberrations occurred in the specific regions of DNA
• Are co-dominant. Can identify a unique locus as c- DNA of
known function are used as probe, chromosomal positions
of the specific gene are identified
• Restriction enzyme can recognize and cut DNA wherever a
specific short sequence occurs
• Molecular probe for Southern
hybridization may be radioactive
or non radioactive
• Size of defined restriction
fragments can be compared
electrophoretically

14. Major events in RFLP Analysis

15. B. PCR based markersB. PCR based markers
• Reduce time, efforts and expenses
• Based on use of pair of primers (reverse and forward)
• Designed either on the basis of random sequences or on
specific sequences flanking the DNA segment that needs to
be amplified
1. Single primer used as forward and reverse primer - AP-
PCR(~20nt), RAPD(~10nt), DAF (~6-8nt) (same random
primer is inversed within an amplifiable distance)
2. A pair of primers used – STSs, SCARs or STARs

16. PrimerPrimer
• Primer are short DNA sequences having free 3’-OH (~ 20bp)
usually used to amplify the marker loci

17. i. RAPDi. RAPD
• RAPD – Random Amplified Polymorphic DNA
• It was the 1st
PCR based marker technique and it is by far the
simplest. 1st
time reported by Welsh and McClelland (AP-
PCR) & Williams et al. in 1990
• Short PCR primers (approx. 10 bp) are randomly selected to
amplify random DNA segments throughout the genome
• The resulting amplification product is generated at the
region flanking a part of the 10 bp priming sites in the
appropriate orientation
• RAPD is a dominant marker
• RAPD products are usually visualized on Agarose gels
stained with ethidium bromide
• Modified approach of RAPD is DNA Amplification
Fingerprinting (DAF)

18. Major events in RAPD

19. ii. AFLPii. AFLP
• AFLP - Amplified Fragment Length Polymorphism
• Are differences in restriction fragment lengths caused by
SNPs and INDELs that create /abolish restriction sites
• Based on selective PCR amplification of restriction fragments
from a total digest of genomic DNA
• Oligonucletotide adapters (~20 bp) are ligated at the end of
DNA fragments
• Adapted DNA fragments are amplified by PCR

20. Major events in AFLP

21. SCARsSCARs
• SCAR – Sequence Characterized Amplified Region
• Based on sequence of polymorphic bands from
RAPD/RFLP/AFLP linked to trait of interest
• Longer primers (15-30 bp) are
designed for specific amplification
of particular locus
• Higher in reproducibility than
RAPD/RFLP
• Are co-dominant

22. iii. Single Nucleotide Polymorphism (SNPs)iii. Single Nucleotide Polymorphism (SNPs)
• SNP is a DNA sequence variation
occurring when a single nucleotide
(A, T, C, or G) in the genome differs
between members of a species
• Eg. AAGCCTA and AAGCTTA (called
alleles C and T)
•Can be known only after DNA
sequencing of several genomes
• Used in biomedical research, crop
and livestock breeding programs

23. iv. Simple Sequence Repeats (SSRs)iv. Simple Sequence Repeats (SSRs)
• Also k/as Short Tandem Repeat (STRs)/microsatellites which
are repeating sequences of 2-6 nt of DNA (motifs).
• Are co-dominant, abundant, dispersed throughout the genome
• Occur in DNA when a pattern of two or more nucleotides are
repeated which are adjacent to each other
• Eg. (CATG)n in a genomic region is typically in the non-coding
intron region
• Of four types: EST-SSR, Genomic-SSRs, Mitochondrial-SSRs,
Chloroplastic-SSRs
• Expressed Sequence Tags are the expressed regions in a DNA
sequence from a c-DNA clone that corresponds to m RNA. As
they represent the transcribed part of genome they show
higher transferability. Used for developing STSs, SSR, SNPs
• Used extensively in Rice genome (www.gramene.org)

24. Major events in SSR Analysis

25. Applications of MarkerApplications of Marker
1. Confirmation of hybridity
2. Linkage Mapping
3. Marker Assisted Selection
4. Trait based Selection
5. Saturated maps
6. Orthologous gene mapping
7. Gene tagging
8. Heterosis breeding
9. Haplotype mapping
10.DNA fingerprinting for varietal identification
11.Phylogenetic and evolutionary studies
• Interspecific hybridization

26. Confirmation of HybridityConfirmation of Hybridity
• Heterozygosity of F1 can be detected

27. Linkage MappingLinkage Mapping
• For linkage mapping we want mapping population which is
immortal, universal, homozygous (true breeding type) and
does not fluctuate
• BC1F2, F2, DH, F2:F3, RILs, NILs

28. LinkageLinkage
MapMap

29. Marker Assisted Selection (MAS)Marker Assisted Selection (MAS)
• MAS consists of identifying association between molecular
markers and genes controlling agronomic traits (major
genes) , and using these to improve plant populations
• Selection is made on genotype rather than phenotype, which
increases the speed and efficiency of selection
• It is used for manipulating both qualitative (disease
resistance) and quantitative (yield) traits
• Molecular markers are used to increase the probability of
identifying truly superior genotypes, by early elimination of
inferior genotypes

30. X
Back crossings
Wild P1
with
resistant
gene
Cultivated
P2 with
susceptible
gene
F1
F2
F3 F4 F5 F6 F7
Plant breeder aims to
improve the resistance of
a cultivated sp. Thus, he
performs a cross
between susceptible
cultivated sp. with a wild
sp. that possess the
required resistance. At
least 6 BC steps are
necessary to get 99% of
genome of recurrent
parent

31. Collard B.C.Y. et al , 2005. (Euphytica)
A robust marker is
developed for a major
QTL controlling disease
resistance (arrow). By
using this marker plant
breeder may substitute
large field trials and
rough out many
unwanted plants (75%)

32. Trait based selectionTrait based selection
• Earliest demonstrations of power of molecular markers was
provided by Beckman and Soller (1986) for indirect selection
of gene in breeding program
• In South Australian Barley, a single dominant resistance
gene to cereal cyst nematode (Ha2) was transferred from a
resistance to susceptible variety through 3 cycles of marker-
assisted backcrossing using a single molecular marker

33. Orthologous gene mappingOrthologous gene mapping
• Also known as “Comparitive mapping”
• c DNA clones of one crop plant are being mapped onto the
linkage maps of other crops
• More saturated maps can be generated by mapping marker
clones
• 1st
comparative maps in plants were generated by Tanksley
in Tomato & potato in 1988
• Generated especially in grasses

34. Gene TaggingGene Tagging
• Refers to mapping of genes of agricultural importance
present close to known markers
• A Molecular marker very closely linked to a gene can act as a
‘tag’
• Gene tagging is the pre requisite for MAS and Map based
gene cloning
• Eg. Cereal cyst nematode were tagged in wheat, Leaf rust
resistance genes Lr9, Lr10, Lr19, Fertility restorer genes for
Polima and Ogura cytoplasm in Brassica

35. Heterosis BreedingHeterosis Breeding
• Lee et al (1989) in corn suggested that RFLP analysis provides
an alternative to field testing
• Since then several attempts were made to correlate
heterosis with variability at molecular level
• Melchinger et al (1991) analyzed 32 maize inbred lines for
heterosis
• Stuber et al (1992) mapped QTLs contributing to heterosis in
the cross between elite maize inbred lines B73 and Mo17
• Xio et al (1995) mapped QTLs for heterosis in one of the
highest yielding indica x japonica hybrids and proposed
domiance as the major cause of heterosis in rice

36. DNA fingerprinting for varietalDNA fingerprinting for varietal
identificationidentification
• Smith and Smith (1992) have presented comprehensive
review on need of fingerprinting of crop varieties
• RAPD, microsatellite and AFLPs are the markers of choice for
purpose because all are PCR based markers and does not
require any prior information on nucleotides
• It is useful in protection of proprietry germplasm especially
the CMS lines, characterization of accessions in plant
germplasm collections
• Eg. Sex identification in dioecious plants by Parasnis et al
(1999) using microsatellite markers (GATA)4

37. Phylogenetic and evolutionary studiesPhylogenetic and evolutionary studies
• To study the evolutionary relationships within and between
species, genera or larger taxonomic groupings
• Studies are made on similarities and differences among taxa
using numerous genetic markers especially RAPD and RFLP
(Paterson et al, 1991)
• Eg. Two lines of rice Azueena and PR 304 were classified as
indicas using morphological characters while these behaved
as japonicas in crossing studies

38. ReferencesReferences
• Collard B.C.Y., Jahufer M.Z.Z., Brouwer J.B., Pang E.C.K.
(2005) An introduction to markers, quantitative trait loci
(QTL) mapping and marker-assisted selection for crop
improvement: The basic concepts. Euphytica 142: 169–196
• Xu, Y. and J.H. Crouch. (2008) Marker-assisted selection in
plant breeding: from publication to practice. Crop Sci. 48:
391-407
• Gupta, P.K., Balyan, H.S., Sharma, P.C., Ramesh, B. 1996.
Microsatellites in Plants: A New Class of Molecular Markers.
Current Science, 70:45-54
• Dudley, J.W. 1993. Molecular Markers in Plant
Improvement: Manipulation of Genes Affecting Quantitative
Traits. Crop Science, 33(4): 660-668
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