2.3. Marker genotyping
DNA was extracted from 6–8 individuals in each accession following the method of Zheng et al. [18]. To facilitate marker-assisted selection (MAS), sequence tagged site (STS) markers, rather than single nucleotide polymorphism (SNP) markers, were developed based on Insertion/Deletions (InDels) between the Nipponbare and 9311 genome sequences at the SKC1 (Chr. 1) [6], SalT (Chr. 1) [8] and DST (Chr. 3) [19] loci. Primers ( Table 1) were designed using Oligo 7.0 software. Eight STS markers were developed, 2 for SKC1, 3 for SalT and 3 for DST ( Table 1). Wn11463 and Wn11466 were designed based on 4 bp and 17 bp InDels downstream of SKC1 (LOC_Os01g20160); Wn13900 was based on a 4 bp InDel upstream of SalT (LOC_Os01g24710); Wn13902 and Wn13903 were based on 7 bp and 8 bp InDels in the SalT coding region; Th32637 was based on a 3 bp InDel upstream in DST (LOC_Os03g57240); and Th32638 and Th32369 were based on 12 bp and 18 bp InDels in the coding region of DST.