Seed purity test

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


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

Seed purity test

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Seed purity test

Similar to Seed purity test (20)

More from Senthil Natesan

More from Senthil Natesan (20)

Recently uploaded

Recently uploaded (20)

Seed purity test