Genomic DNA Extraction and PCR Amplification

Genomic DNA was isolated from well-expanded leaves from the field grown plants following the protocol of Rogers and Bendich [22 ]. All PCR amplifications were carried out in a 20 μl reaction volume containing 50 ng of template DNA, 800 μM of dNTPs, 2 mM of MgCl₂, 10 pmol each of forward and reverse primers and enzyme Taq polymerase (1 Unit, i-Taq DNA polymerase, Intron Biotechnology, Inc.). The PCR profile included initial denaturation at 94°C for 5 min followed by 36 cycles of 94°C for 30 sec, annealing at 62°C for 30 sec and 72°C for 1 min 30 sec and a final extension at 72°C for 12 min. For the purpose of sequencing, amplified fragments were cloned in the pGEM-T Easy TA cloning vector (Promega, Madison, USA). Sequencing was done by capillary electrophoresis on Applied Biosystems 3730 Genetic Analyzer. Sequencing of each sample was done in a minimum of 3 independent replicates to remove chances of any PCR related error. Sequence files were assembled and analysed using EditSeq, SeqMan and MegAlign software packages of DNAStar (DNAStar Inc.). For mapping, marker genotyping data of the mapping population were added to the existing map of B. juncea [21 (link)] using the program JoinMap version 4.0 [23 ]. Genome walk was performed using PCR genomic libraries based on the method described by GenomeWalker Universal Kit (Clontech Ltd.).