molecular markers and their types and applications in vegetables

1. Role of molecular markers in vegetable crops
Presented by,
Vanisree Padmanabhan
2019534005

2. MARKERS
• 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 as ‘loci’
• Markers located in close proximity to desirable genes (tightly linked) are
known 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 known as Quantitative Trait Loci (QTL)

3. 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. • Random marker- located at random sites in the genome and their
relevance to plant function is not known.
• Gene based marker- located within genes
• Functional marker – such gene based marker whose alleles reliably
reflect the functions of the alleles of concerned genes

5. Marker closed linked to the trait of interest will be inherited
together and rarely will be separated by recombination
• Laws of Inheritance- Genetic
Linkage
• ‘QTL mapping’ is based on the
principle that genes and markers
segregate via chromosome
recombination (called crossing-over)
during meiosis (i.e. sexual
reproduction), thus allowing their
analysis in the progeny (Paterson,
1996)

6. A good molecular marker
be polymorphic and evenly distributed throughout the genome,
 provide adequate resolution of genetic differences
generate multiple, independent and reliable markers
simple, quick and inexpensive
need small amounts of tissue and DNA samples
have linkage to distinct phenotypes, no epistasis
require no prior information about the genome of an organism
Co- dominant in nature
 Frequent occurrence in genome
Selective neutral behavior (the DNA sequences of any organism are
neutral to environmental conditions or management practices)

8. 1.Morphological Markers
These are botanical descriptors of plant which are visually or
phenotypically characterized.
First to be used and easy to score.
Male sterile identification:
Bright green hypocotyls – Broccoli
Glossy foliage - Brussels sprouts
Potato leaf, green stem – Tomato
Brown seed coat colour - Onion

9. 2.Protein-Based markers
• Proteins (Isozymes) - dependent on environmental factors, superior
to morphological markers
• less polymorphic differences (problem in commercial breeds of
plants)
• variants of an enzyme or non-enzyme protein that differ from each
other in electrophoretic mobility.
• Isozymes are codominant markers.

10. 3.DNA markers
Major molecular techniques were used to reveal the genetic
variation.
i) Amplification by PCR
ii)Electrophoresis
iii)Hybridization
iv)Sequencing

11. POLYMERASE CHAIN REACTION
PCR can produce millions of copies of a DNA segement in a matter of few
hours.
Developed by Mullis.
Uses a pair of primers complementary to the end sequence of the DNA –
AMPLICON
Thermostable DNA polymerase- “Taq polymerase”
Steps involved :
steps Temperature ⁰C Duration Objective achieved
Denaturation 98 First cycle 2min
Later cycles 1min
Separation of two strands of template DNA
Annealing 40-60 1 min Pairing between the primers and the target DNA
Prime
extension
72 2min; 7min in final
cycle
Replication of the target segment of template DNA

13. ELECTRPHORESIS
• Electrophoresis enables to distinguish DNA
fragments of different lengths.
• DNA is negatively charged, therefore, when an
electric current is applied to the gel, DNA will
migrate towards the positively charged
electrode.
• Shorter strands of DNA move more quickly
through the gel than longer strands resulting in
the fragments being arranged in order of size.

14. HYBRIDIZATION
One of the most commonly
used nucleic acid
hybridization techniques is
Southern blot hybridization
Southern blotting was
named after Edward M.
Southern who developed
this procedure at Edinburgh
University in the 1975

15. SEQUENCING
The process of determining the order of the nucleotide bases along a DNA
strand is called sequencing.
Two methods for sequencing DNA was developed;
Sanger Sequencing method- chain termination
Maxam-Gilbert sequencing method-chemical termination
Chain elongation terminated using
dideoxynucleotides produced by
DNA polymerase
the purines(A+G) are depurinated
using formic acid, the guanines
(and to some extent the adenines)
are methylated by dimethyl sulfate,
and the pyrimidines (C+T) are
hydrolysed using hydrazine. The
addition of salt (sodium chloride)
to the hydrazine reaction inhibits
the reaction of thymine for the
Conly reaction.

16. TYPES OF MOLECULAR MARKERS
Based on the way of detection;
Sequencing based
SNP
PCR Based
RAPD,AFLP,CAPS SCAR,SSR,ISSR etc.
Hybridization based
RFLP DArT
• Linked marker - located very close to major
genes of interest
• Direct marker - it is part of gene of interest
• cis marker - linked with the trait of interest
(dominant genes)
• trans marker - linked with the opposite allele
(recessive traits )

17. • CO DOMINANCE AND DOMINANCE
Dominant marker: A marker shows dominant inheritance with homozygous dominant individuals
indistinguishable from heterozygous individuals
Codominant marker: A marker in which both alleles are expressed, thus heterozygous individuals
can be distinguished from either homozygous state

18. Restriction fragment length polymorphism
• Genomic DNA digested with Restriction Enzymes
• DNA fragments separated via electrophoresis and transfer to nylon
membrane
• Membranes exposed to probes labelled with P32 via southern
hybridization
• Film exposed to X-Ray

19. Restriction fragment length polymorphism

20. RFLP
Advantages
co-dominant
sequence specific
Reproducible
Disadvantages
High quality and large amount of
DNA
Radiolabeled probe
Not automated
Tedious, time consuming, costly

21. Randomly Amplified Polymorphic DNA
10 nts. long arbitrary primers that PCR amplify various
complementary regions in the genome.

22. Some variations in RAPD
• DNA amplification fingerprinting (DAF)
5bp single arbitrary primers-less specific
Identification of sex in papaya using OPA 06 primer (Somsri et al,
2007)
• Arbitrary primed Polymerase chain reaction (AP-PCR)
10-50 bp- Number of fragments amplified are less but more
specific.

23. RAPD
Advantage
Fast and easy assay
Sequencing information not
required
Few nanograms of DNA
Disadvantage
Low reproducibility
Dominant
High quality of DNA template

24. Sequence Characterized Amplified Region (SCAR)
Longer primers (15-30 bp) are
designed for specific amplification
of particular locus
SCAR markers linked to the gene
inducing beta-carotene accumulation
in Chinese cabbage.

25. Amplified Fragment Length Polymorphism (PCR +
RFLP)
selectively amplifying a subset of restriction fragments restriction
endonucleases.
Procedures
Digestion
Adapter ligation
Amplified
Electrophoresis

26. AFLP
• Involves cleavage of DNA with two different enzymes
• Involves ligation of specific linker pairs to the digested DNA
• Subsets of the DNA are then amplified by PCR
• The PCR products are then separated on acrylamide gel
AFLPs have stable amplification and good repeatability
An additional advantage over RAPD is their reproducibility.
- Involves the use of RFLP and PCR techniques
- Compared with the widely used RFLP, AFLP is faster, less labor
intensive and provide more information.

27. Simple sequence repeats
SSR are short tandem repeats of 1-6 base pairs present in eukaryotic
genome
Also called as
STR(Short tandem repeats)
Microsatellites
SSLP( simple sequence length polymorphism)
Types based on repeats:
1.Mono repeat
2.di-repeat
3.tri-repeats
4.Tetra-repeats

29. Single nucleotide polymorphism
These are positions in a genome where some individuals have one
nucleotide (eg. G) and others have a different nucleotide (eg. C)
Although each SNP could potentially
have four alleles,(because there are
four nucleotide)
• Any two unrelated individuals
differ by one base pair every
1,000 or so, referred to as SNPs.
• Many SNPs have no effect on cell
function and therefore can be used
as molecular markers.

30. Disadvantage
• There is a high possibility that
SNA does not display
variability in the family that is
being studied .
Advantages
• They are abundant in number
• They can be typed by methods that do not involve gel
electrophoresis
• SNP detection is more rapid because it is based on
oligonucleotide hybridization analysis.

31. Markers differ based on :
• Genomic abundance
• Polymorphism level
• Locus specificity
• Reproducibility
• Technical requirements
• Financial investment
Other markers:
 Cleaved Amplified Polymorphic Sequence
(CAPS/PCR-RFLP)
 Inter Simple Sequence Repeat (ISSR)
 Single-strand conformation Polymorphism (SSCP)

33. 1.Assesment of genetic variability:
 Dominant markers-RAPD used for the analysis of pepper breeding
lines (Heras et al., 1996) revealed very narrow genetic base with
more than 50% of the DNA bands being common among all the
lines.
RAPD and SSR markers are effective in differentiating among the
genotypes of Solanum aethiopicum and Solanum melongena.

34. 2. Gene tagging:
Genetic markers that are located in close proximity to genes (i.e.
tightly linked) may be referred to as gene ‘tags’
• mapping of genes of economic significance close to known markers.
• pre-requisite for MAS and map based gene cloning.
• Tomato TMV resistance Tm-2 locus
• nematode resistanc- Mi gene
• powdery mildew resistance gene ol-1 on chromosome 6 of tomato-
RAPD and SCAR markers.

35. 3.DNA finger printing for varietal identification:
• Identification and ascertaining variability in germplasm.
• Useful for characterization of accessions in plants.
F1 Chilli hybrids was determined using two molecular techniques
RAPD and ISSR.
Genome sequenced crops
Cucumber - 367mb
Potato - 844mb
Chinese cabbage - 283.8mb
Tomato - 900mb
Melon -450mb
Watermelon - 375mb

36. 4.Breeding lines and accession identification
Accessions are difficult to distinguish as they differ in few
morphological traits.
Molecular markers can easily distinguish such closely related
genotypes.
By using RAPD markers, Waycott and Fort successfully
differentiated nearly identical germplasm lines of bitterhead lettuce.

37. 5.Sex identification
Dioecious species-early identification of male and female plants.
 SCAR markers in the asparagus were developed by Jiang and Sink.
These were linked to the sex locus at a distance of 1.6 cM.
To facilitate the differentiation of XY from YY males in asparagus.

38. 6.Identification of cultivar
Microsatellites were developed to allow vastly reliable identification
of cultivars.
In tetraploid potato, the comparative assessment of different DNA
fingerprinting techniques showed that the AFLP have the maximum
discrimination power.
Breeder rights : DUS + molecular profiles

40. 7.Marker assisted selection:
• 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

41. Marker assisted back crossing

42. Advantages of MAS
• It can be performed on seedling material
• It is not affected by environmental conditions.
• Determination of recessive alleles.
• Gene pyramiding.
• Selecting traits with low heritability.
• Testing specific traits (quarantine).
• It is cheaper and faster

43. Gene pyramiding
Assembling multiple desirable genes from multiple parents into a
single genotype
Genotype with all target gene
Objective
1. Enhance trait performance
2. Increase durability
3. Broadening genetic base

44. The success of gene pyramiding are the inheritance model of the
genes for the target traits.
Problem
• Linkage drag
• Target gene tightly linked to gene with large negative effects on other
traits.