ICRISAT Archival Report 2006 - The seedlings of success in the ...

Milestone D.2.1.2: DNA extracts of mini core and reference sets of groundnut and sorghum conserved
(HDU/RKV/CTH/DH/CLLG, 2009)
Milestone D.2.1.3: DNA extracts of mini core set of pigeonpea conserved (HDU/RKV/CTH/DH/CLLG/KBS,
2010)
Milestone D.2.1.4: DNA extracts of mini core of finger millet and reference sets of pigeonpea, pearl millet and
finger millet conserved (HDU/RKV/CTH/DH/CLLG/KBS, 2011)
Milestone D.2.1.5: Requested DNA samples of specific accessions distributed for utilization
(HDU/RPT/RKV/DH/NBPGR, Annual)
Output target D.3: Allele specific sequence diversity in the reference sets staple crops studied (2011)
Activity D.3.1: Study allele specific sequence diversity in the reference sets of staple crops
Milestone D.3.1.1: Allele specific sequence diversity in the reference set of chickpea studied (RKV/HDU/DH,
2010)
To identify the candidate genes for analyzing the sequence diversity, bioinformatics analyses in the first
instance, a total of 20 DREB1A and 34 DREB2A non-redundant protein sequences were obtained through an in
depth analysis of databases. The output alignment files from both ClustalW and MUSCLE were used for
phylogenetic analysis and consensus primer design. The primers could be designed by CODEHOP for five
blocks (A, B, C, D and E) in case of DREB1A and only for one block (A) in case of DREB2A. A total of 22 (9
forward and 13 reverse) primers for DREB1A and 13 (5 forward and 8 reverse) primers for DREB2A were
designed and synthesized. All possible combinations (90 combinations in DREB1A and 40 combinations of
DREB2A) were checked for amplification using 55-50 0 C touch down PCR profile. In terms of getting
homologous sequences in both species, single ‘putative’ amplicon was obtained with only 5 primer
combinations; however, all these amplicons were smaller (