DNA Markers Techniques for Plant Varietal Identification

Genomics and Proteomics lab - www.tnaugenomics.com

DNA Markers Techniques
for Plant Varietal
Identification
Dr.N.Senthil
Associate Professor ( Biotechnology)
Genomics and proteomics lab
N. Senthil, 2P.Tamilkumar , 3M. Raveendran, 4R. Jerlin and 5R. Umarani
&3
Centres for Plant Molecular Biology, TNAU, Coimbatore-3
2
Department of Seed Science and Technology, TNAU, Coimbatore-3.
4&5
.Seed Centre, Tamil Nadu Agricultural University, Coimbatore-3


Genomics time line


Comparing genomes: Example from the grasses
This is now one of the most well-known figures in plant
comparative genomics.
This consensus comparative map of 7 grasses
shows how the genomes can be aligned in
terms of “rice linkage blocks” (Gale and Devos
1998). Any radial line starting at rice, the
smallest genome and innermost circle, will pass
through regions of similar gene content in each
of the other species.

Therefore a gene on the chromosome of one
grass species can be anticipated to be present
in a predicted location on a specific
chromosome of a number of other grass family
species. This has facilitated much sharing
among researchers working on any of these
species and others that may be also related
(Phillips & Freeling 1998).

Automated sequencing reactions - each reaction can resolve 600 to 750 bp

(labeled with fluorescent dyes)


SNP discovery- Early methods

• Re-sequencing of PCR amplicons with or without pre-screening
• Direct sequencing of DNA segments amplified by PCR)from several individuals is the most direct way to
identify SNP polymorphisms
• Alternatively, an allele-specific-PCR or primer-extension assay may be developed relatively straightforwardly.
Rafalski 2002 Curr Opin Plant Biol 5 :94-100


DNA sequencing output
If you have DNA sequence produced from a PCR product or a library of
ESTs, the sequence of your DNA segment(s) will be given to or, more
usually, emailed or electronically transferred to you..

If the data is in the chromatogram form, you will need to manually generate
a text file such as the one below (by “reading” the bases yourself) or, more
typically, use one of the many software programs available to do this for you.

If you retrieve a sequence from a public database, it will already be in this
format for you.

The first 480 bases of the DNA sequence of GAN, a drought tolerance
related gene in Arabidopsis (GenBank Accession AY986818).

Background information : Markers
• The identification of varieties of agricultural crops is
important at every stage of the seed production chain
(Cooke, 1995).

• The only legally recognized method in our country for
genetic purity assessment based on field plot grow out
tests, which include only the morphological traits

• But morphological traits may not be sufficient for
discrimination of all new varieties and hybrids, they also
subjected to environmental influences and requires one
full season .


What are molecular markers?
• A marker, in this context, is an identifier (sometimes called a
“tag”) of a particular aspect of phenotype and/or genotype; its
inheritance can easily be followed from generation to
generation.

• Markers can be:

Morphological: phenotypic variation which is scorable on the
basis of a single plants (e.g. flowering time)
• Biochemical: variants in the size or net charge of a protein (eg
isozymes) or in the chemical composition of a metabolite (e.g.
sugar)
• Molecular: variants in the DNA sequence (eg
microsatellites)


Molecular markers : Costs

• Using molecular markers requires the use of
specific laboratory equipment, at the very least a
PCR (polymerase chain reaction) thermalcycler
and electrophoresis and visualization
equipment.
• So start-up costs can be high, although these
may be compensated for by later savings (and
prices of the necessary equipment and reagents
have been decreasing over time).

Molecular markers :
Technical skills needed

• Along with the equipment required for
molecular marker work comes the need for the
technical skills and knowledge of how to do the
work and understand the results.
• These are not difficult skills to learn, but are not
always part of a classical plant breeder’s
education.


Considerations in selection of marker
type
• There are many types of molecular markers
available. Which type you select to use for your
project will depend on:
• What the goals of the project are
• How variable the germplasm is
• What sort of population is being analyzed
• What level of resolution is needed
• Whether or not there is previous work you can
take advantage of (ie. Marker development)


Classification of DNA markers
Probe Based (eg. RFLP)
 Restriction fragment length polymorphism (RFLP)

Amplification Based (eg. RAPD, SSR,ISSR,SCAR)
 Random amplified polymorphic DNA (RAPD)
 Simple sequence repeats (SSR)
 Inter Simple Sequence Repeats (ISSR)
 Sequenced Characterized Amplified Region Markers (SCAR)

Combination of probe based and PCR based markers (eg. AFLP)
 Amplified fragment length polymorphism (AFLP)

New Generation markers
 Single Nucleotide Polymorphism (SNP)


Relative importance of molecular
markers (Christian Schlotterer. 2004 )


RFLP(Restriction Fragment Length
Polymorphism)

PCR

Schematic drawing of the different steps of polymerase chain reaction (PCR): (a) denaturing step at
92-95°C; (b) primer annealing step (37-68°C depending of the technique); (c) extension step at 72°C
(P=Taq DNA polymerase), and (d) end of the first cycle with two copies of DNA strands. The two resulting
DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for
each new cycle.


AFLP(Amplified Fragment Length
Polymorphisms)
Developed in the early 1990’s by Keygene
Combination of both RFLP and PCR techniques
Four steps
 DNA is digested with two different restrictionenzymes

 Oligonucleotide adapters are ligated to the endsof the
DNA fragments

 Specific subsets of DNA digestion products are amplified,
using combinations of selective primers

 Polymorphism detection is possible with radioisotopes,
fluorescent dyes or silver staining


Genome organization

• It is important to remember that only part
(sometimes a very small part!) of the DNA
sequence is composed of genes.
• The rest is non-coding sequence, including lots
of repetitive sequences, microsatellites and
transposons. In some species, the genic fraction
of the genome may be <10% of the total.


SSR Single Sequence Repeat

flanking region I
flanking region II
microsatellite
plant A

plant B

specific primers were designed corresponding to
flanking sequence of microsatellite

plant A

plant B

PCR analysis and analyze on 6 %denaturing
polyacrylamide gel with silver staining

Schematic of SSR A B Repetitive sequence
primer I

assay primer II


SSR

•


Inter-Simple Sequence Repeat (ISSR)
• Relies on one primer for PCR that anneals to an SSR
region and amplifies region between inversely
oriented adjacent SSRs
• Can be undertaken for any species that contains a
sufficient number and distribution of SSR motifs
• Genomic sequence data not required
• Amplifies large numbers of DNA fragments per
reaction representing multiple loci from across the
genome
• Ideal method for fingerprinting varieties


ISSR


Genetic Diversity of
Indian Jatropha Species


SCAR Marker


Sequence Characterized Amplified Regions
(SCAR)


Cleaved Amplified Polymorphic Sequence
(CAPS)
• Primers used to amplify the genome segment of
interest
• Amplification product digested with selected
restriction enzyme
• Digest is subjected to agarose gel electrophoresis
to detect polymorphism in the lengths of
fragments generated
• Codominant marker
• Quick assay


Purity test of Rice parents and hybrids
using SSR markers


Microsatellite markers polymorphism between
parental lines and rice hybrids

Tamilkumar et al.,2009


Microsatellite markers polymorphism
between parental lines and rice hybrids

• Five microsatellite markers RM276, RM 234, RM 258, RM202 and
RM 204 together differentiated 5 hybrids and the parental lines at
least with a single marker allele difference.

• The microsatellite marker, RM234 amplified alleles specific to
differentiate parental lines of CORH3 likewise RM276 for KRH2,
RM258 for PRH10, RM202 for AJAY and RM204 for RAJALAXMI
used to differentiate parental lines of respective hybrids.



Amplification pattern of the parental
lines obtained using the SSR marker
RM202


Testing genetic purity of hybrid seeds of
CORH3 using the SSR marker RM 234
• Genomic DNA was isolated from 50 seedlings of the CORH3
hybrid (random sample)

• PCR analysis was performed by means of the RM234 out of
50 random samples microsatellite marker identified presents
of single pollen shedder (B line) seed, which had a CMS line
specific fragment

• This amounts to 2% off types in the hybrid seed produced
.
• The results were confirmed using 400 seeds from the same
seed lot through Grow out test (GOT).


Testing genetic purity of hybrid seeds of CORH3 using the
SSR marker RM 234

Lane 2 = TNAUCMS2A (CMS line), Lane 3 = CB87R (restorer line). DNA
was isolated from single seedlings of the CORH3 hybrid, PCR analysis was
performed and genotype assessed (Lanes 4–12) Off type in Lanes 8.


Seed Purity Assessment Of Rice Hybrid Using
Microsatellite Markers
Arrow indicates contaminants Yashitola et al.,2002

Detection of impurities in the Indian rice hybrid-KRH2
Through multiplex PCR using the microsatellite markers RM164 and
RM206. M—50 bp ladder, A—CMS line (IR58025A), H— Hybrid
(KRH2), R—Restorer line (KMR3), 221 to 240— Samples of hybrid
KRH2 collected from a commercial seed-lot.

SSR (Multiplex)

Multiplex PCR assay for distinguishing rice hybrids
using three SSR markers

Lane C1-IR58025A, lane R1-IR40750R, lane H1-DRRH1, lane C2-
IR58025A, lane R2-KMR3R, lane H2-KRH2, lane C3-IR58025A, lane
R3-C20R, lane H3-CORH2, lane C4- IR58025A, lane R4-BR827-35R,
lane H4-Sahyadri

(Sundaram et al., 2007)

SSR markers utilization in seed purity assessment of
IR58025A Sundaram et al., 2007

Two-dimension assay involving a 20 *20 grow-out matrix for assessment of purity of
IR58025A with the help of SSR markers RM202 and RM276. (a) Row-wise lanes 6 & 8 and
Column-wise lanes 3 & 18 (indicated by arrows) represent contaminants. (b) Schematic
representation of the 20 *20 matrix based method for rapid identification of contaminants in
IR58025A. Plants at intersections of 6th row 18th column and 8th row and 3rd column (indicated
by arrow) were identified as contaminants


Comparison of the most used marker systems
Feature RFLPs RAPDs AFLPs SSRs SNPs

DNA required 10 0.02 0.5-1.0 0.05 0.05
(μg)
DNA quality High High Moderate Moderate High
PCR based No Yes Yes Yes Yes
Number of 1.0-3.0 1.5-50 20-100 1.0-3.0 1.0
polymorph
Loci analysed

Ease of use Not easy Easy Easy Easy Easy
Amenable to Low Moderat Moderate High High
automation e
Reproducibilit High Unreliab High High High
y le
Development Low Low Moderate High High
cost
Cost per High Low Moderate Low Low
analysis
(Korzun et al.,2001)


Conclusion
• DNA profiling could be used now for the
verification or confirmation of varietal identity
and in some quality control situations.

• DNA profiling methods for statutory variety
registration is still under discussion between
UPOV and other interested parties.


Thanks to
• Dr R.Umarani
• Dr Jerlin
• Mr Tamil Kumar
• Ms Padma
Seed centre , TNAU

DNA Markers Techniques for Plant Varietal Identification

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