All the 3000 accessions of the chickpea composite collection including the two internal controls, Annigeri (ICC 4918) and ICCV 2, were grown in the field. ICCV 2 is an early maturing (flowers about two weeks earlier and matures one week earlier than Annigeri) kabuli chickpea with resistance to wilt (Fusarium oxysporum f. sp. ciceri race 1) [40 ], and released for cultivation in India (as Swetha), Sudan (as Wad Hamid) and Myanmar (as Yezin 3) [41 ]. Annigeri belongs to desi chickpea and was released for its earliness and wide adaptation for cultivation in the peninsular India [42 ]. A single plant from each accession was harvested and the seeds obtained from such plants were used to raise seedlings for DNA extraction. Young leaf tissues of each accession from the greenhouse grown plants were harvested and immediately stored in 96-well plate that consists of 94 accessions and two controls (Annigeri and ICCV 2). The two controls were added to each set of 94 accessions placed in 96-well plates for DNA extraction. DNA isolation for all 3000 accessions was carried out at ICRISAT.
A high-throughput DNA isolation protocol [43 ] was adopted to isolate DNA from the leaf tissues in 96-well format. DNA quantification, quality check and normalization to 5 ng/μl were done on agarose gel (0.8%) using lambda DNA standard (MBI Fermentas, USA). DNA isolated for all the 3000 accessions at ICRISAT was supplied to ICARDA for genotyping with 15 SSR markers.
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