QTLs related to eating quality in rice Table 2. Location and additive effects of QTL for AC and RVA in CSSLs in four environments 2008 Nanjing 2008 Hainan 2009 Nanjing 2009 Hainan Locus Location qSBV-8 RM7580–RM7356 −25.31 qCSV-8 RM5351–RM23200 93.87 qAC-8-1 RM7356–RM7556 15.8 qAC-8-2 RM23510–RM23579 17.76 Meana Aa −5.91 −3.71 −0.71 0.56 PVE (%)a 30.06 19.88 18.91 20.72 Mean A 115.12 13.81 NDb −4.15 16.70 ND 0.52 18.04 PVE (%) Mean 91.95 16.00 PVE (%) A ND −3.88 14.48 ND 0.48 19.71 Mean 13.00 A ND ND ND 0.51 a A, additive effect; PVE, phenotypic variation explained. Mean values were calculated from all sub-CSSLs with highly significant difference and the same orientation of additive effects. b ND, not detected. 341 PVE (%) 17.57 chromosome segment from IR24. The length of substituted segments in the 14 sub-CSSLs ranged from 2.7 to 19.2 cM, with an average of 9.5 cM. The major agronomic characters of sub-CSSLs were not significantly different from those of Asominori (see Supplemental Table 1). RVA profile characteristics and Amylose content of sub- CSSLs In the summer of 2008 in Nanjing, three sub-CSSLs (Lines 1, 2 and 4) had significantly higher SBV values than Asominori. These sub-CSSLs overlapped in the interval RM5351–RM23200. However, no significant differences in SBV were found between the three sub-CSSLs and Asominori in the other three environments (Fig. 3A). In addition, the CSV values of the four sub-CSSLs (Lines 1, 2, 3 and 4) were significantly lower than those of Asominori across three environments (Fig. 3A). The AC values of the four sub-CSSLs (Lines 1, 2, 3 and 4) were higher in Nanjing than in Hainan, and also higher in 2008 than in 2009 (Fig. 3A). In the summer of 2008 in Nanjing, the AC of three sub-CSSLs (Lines 1, 2 and 4) was lower than those of Asominori. Meanwhile, the means of three sub-CSSLs (Lines 11, 13 and 14) harboring the target segment in the interval of RM5485–RM23653 were signifi- cantly higher than those of Asominori. In other environ- ments, significant differences were observed between the phenotypic values of the four sub-CSSLs (Lines 11, 12, 13 and 14) and Asominori (Fig. 3B). Substitution mapping of QTLs for starch properties using sub-CSSLs In the summer of 2008, two QTLs for AC, qAC-8-1 and qAC-8-2, were identified using six sub-CSSLs (Lines 1, 2, 4, 11, 13 and 14), and mapped in the marker intervals RM7356–RM7556 (Table 2 and Fig. 2B) and RM447– RM6485, respectively (see Supplemental Fig. 1). The ef- fects of qAC-8-1 and qAC-8-2 were nearly equal, but in reverse orientation. qAC-8-1 was detected in only one year, with the percentage of phenotypic variation explained (PVE) being 18.91% and an increasing additive effect of about 0.71 from the Asominori allele (Table 2). qAC-8-2 was expressed stably across four environments using stepwise constructed CSSLs, and the QTL was located in RM23510–RM23579 (3.10 cM, 776 kb), with a PVE of 17.57–20.72% and an increasing additive effect of 0.52 from the IR24 allele (Table 2 and Fig. 2B). Two QTLs for RVA profile, qSBV-8 and qCSV-8, were identified (Table 2 and Fig. 2B). qSBV-8 was located in the region of RM7580-RM7356 using three sub-CSSLs (Lines 1, 2 and 4), with average PVEs of 30.06% and an increasing additive effect of 5.91 from the Asominori allele. This locus was detected only in the summer of 2008. Whereas qCSV-8 was mapped in the marker interval RM5351–RM23200 cor- responding to 3.07 cM (779 kb) using three sub-CSSLs in three environments, with PVE of 14.48–19.88% and an increasing additive effect of about 3.71 from the Asominori allele (Table 2 and Fig. 2B). Taken together the significant genetic effects demonstrat- ed by t-test analysis and the same additive effect direction among sub-CSSLs confirmed the presence of QTLs for AC and eating quality as reported by Wan et al. (2004). Expression of genes at the qAC-8-2 locus Across four environments qAC-8-2 was consistently de- tected and delimited to a 776 kb of genomic region, contain- ing ten BACs (Fig. 4). Based on gene expression profiles reported by Liu et al. (2010), eight up-regulated genes and seven down-regulated genes were concentrated on seven BACs in the approximate location of qAC-8-2. Among them, two remarkably up-regulated genes (Os08g0534900 and Os08g0536000) were selected for further study (Fig. 5). These genes were involved in cell rescue/defense and carbo- hydrate metabolism, respectively. According to previous studies, these genes are believed to influence the occurrence of chalkiness or starch metabolism. A total of seven sub- CSSLs, including Lines 1, 3, 6 and 11 carrying no target qAC-8-2 alleles and Lines 12, 13 and 14 carrying target qAC-8-2 alleles, were used to analyze the expression of the three up-regulated genes. The expression profiles of the two genes were highly vari- able during seed development. Os08g0534900 was highly expressed at the early stage of grain development (Fig. 5A). Compared to Asominori, expression of Os08g0534900 was higher in sub-CSSLs carrying the target qAC-8-2 allele at most measured stages, but was indistinctive in sub-CSSLs not carrying the target qAC-8-2 allele and CSSL50-1.