QTLs related to eating quality in rice 339 reported that a stable gene cluster controlling AC and eating quality across 8 environments was located in the interval R727–G1149 on chromosome 8. In a following study using the same genetic material, Wan et al. (2005b) mapped a major QTL for the percentage of grains with chalkiness (PGWC), qPGWC-8, in the same chromosomal interval. Pre- vious studies demonstrated that grain chalkiness was signif- icantly correlated with AC and most of RVA parameters (Li et al. 2009, Sui et al. 2005). Liu et al. (2010) also found that endosperm chalkiness was closely related to starch metabo- lism by analyzing gene expression profiles of Asominori and CSSL50 carrying the QTL cluster on chromosome 8. The objectives of this study were (i) to dissect the stable gene cluster and define the candidate genomic regions of QTLs for the AC and RVA profiles on chromosome 8, and (ii) select genes related to chalkiness formation and starch biosynthesis for expression analysis in the target region. Materials and Methods Genetic population and field experiment The IR24/Asominori CSSL population consisted of 66 lines (CSSL1–CSSL66) derived from backcrossing F7 recom- bination inbred lines of Asominori × IR24 to Asominori (Kubo et al. 1999). Among the 66 lines, CSSL50 carried the target QTL cluster on chromosome 8. To further reduce the substituted segment on non-target chromosomes, CSSL50 was backcrossed to Asominori followed by self-pollination until BC4F1. At the same time, every generation was detect- ed by simple sequence repeat (SSR) markers on non-target chromosomes to eliminate the background interference (Guo et al. 2011). BC4F1 individuals, designated as CSSL50-1, were selected on the target chromosome by 12 SSR markers, which harbored a small segment spanning R727-XNpb397. The progenies of CSSL50-1 and Asominori were em- ployed for further study as shown in Fig. 1. In the summer of 2008, a total of 395 BC4F2 plants were developed from the progenies of BC4F1 individuals. In the process of selecting candidate secondary subsitututed lines, 12 simple sequence repeat (SSR) markers were used and a total of 8 sub-CSSLs were obtained from 395 BC4F2 plants. Heterozygous indi- viduals in BC4F2 populations, derived from the former 8 sub-CSSLs, were self-pollinated to produce the next segre- gation populations (BC4F3). In the winter of 2008, a total of 1,162 BC4F3 individuals from these 8 sub-CSSLs were planted. To determine the size of subsitututed segments, we developed 17 new SSR markers and identified a total of 8 BC4F3 sub-CSSLs among which the majority of genomic regions was homozygous for Asominori alleles. Similarly, heterozygous individuals were used to produce the next self- pollinated populations (BC4F4). 1,800 BC4F4 individuals from these 8 heterozygous BC4F3 sub-CSSLs were planted in the summer of 2009. By MAS with the newly developed 12 SSR markers, 14 sub-CSSLs were isolated. In the winter of 2009, a total of 3,320 homozygous progenies of the 14 Fig. 1. Breeding scheme for the development of the CSSLs and proge- ny selection. To obtain the substituted segments on target chromo- some, CSSL50 was backcrossed to Asominori followed by self- pollination until BC4F1, and the genetic background was selected by SSR markers on target/ non-target chromosome. The BC4F1 individu- als, designated as CSSL50-1, harbored a small segment spanning R727–X397. Sub-CSSLs, the progenies of CSSL50-1, were employed for further study. AA type and II type, represent lines homozygous for Asominori (AA) and IR24 (II) alleles, respectively, were used to iden- tify the phenotype. And IA type, represents lines heterozygous for Asominori and IR24 alleles, was used to further shorten the segments on target interval. BC4F4 sub-CSSLs were grown. All the data were used to analyze QTLs. Field experiments were conducted in the summers of 2008 and 2009 at the Experimental Station of Nanjing Agri- cultural University, Jiangsu. In the winters of 2008 and 2009 the CSSLs were grown in Lingshui County, Hainan. Ten plants per line with two replications were transplanted in single rows with 13.3 cm between plants and 25 cm between rows. Field management followed normal agricultural prac- tice. These materials were harvested individually at maturity to prevent over-ripening. Trait measurement and starch analysis Approximately 40 days after heading, the plants were har- vested in bulk and the hulled grain was air-dried and stored at room temperature for 3 months before milling. The grains were dried to a moisture content of 13.0% with a range of 12.5–13.7%. All samples were dehulled in an electrical de- huller (Model JNMJ3, China) and milled by a sample miller (Model JFS-13A, China). Milled rice was ground to flour sieved through a 100-mesh sieve. Samples were refrigerated until analyzed (Tan et al. 1999). The assay for amylose and paste viscosity was conducted with two replications.