Fine Mapping And Candidate Gene Analysis Of A QTL Controlling Hull Silicon Content In Rice

Silicon plays an important role during the process of rice growth and development. Three genes controlling silicon transportation and accumulation in rice have been cloned. With the advance of molecular quantitative genetics, gene identification through QTL mapping approach has been performed. Studies about QTL mapping for rice silicon traits have also been performed in the last 4 years.In our previous study, a QTL named qHUS6 controlling hull silicon content was located on the short arm of rice chromosome 6 by using the RIL population crossed by zhenshan97B and Milyang46 and it can explain 17.4% of the phenotypic variance. In present study, an F2:3 population containing 221 lines derived from an individual, which was selected from the RIL population, was used for QTL validation and dissection. This population has a segregate region RM587-RM19784 on the short arm of chromosome 6 but homozygous genetic background. A series of individuals containing smaller heterozygous target region were selected from this population for QTL dissection, validation and fine mapping and then the candidate gene analysis was performed. The results were summarized as follow:1. In the winter of 2004, the RHL6 F2:3 population containing 221 lines was planted in Hainan Province. With this population, two QTLs controlling hull silicon content were detected in the region RM587-RM19784 on the short arm of rice chromosome, of which qHUS6-1 was located in the region RM510-RM204 with the additive effect of 1.50 and dominance effects of 1.17 and qHUS6-2 was located in the region RM19715-RM19784 with the additive effects of -1.12 and dominance effects of 0.03. They can explain 20.23% and 23.75% of phenotypic variance, respectively. The alleles from the Milyang46 and Zhenshan97B increased hull silicon content, respectively. qHUS6-1 acted as a positive dominant QTL but qHUS6-2 as an additive QTL; in the background, a QTL with minor effect was detected on the chromosome 2. The allele from Zhenshan97B increased hull silicon content and it can explain 4.1% of the phenotypic variance.2. Three individuals carrying smaller heterozygous segments were selected from RHL6 F2:3 population, of which two individuals covered the qHUS6-1 region and another covered the qHUS6-2 region. Two F2:3 populations were derived from the selfed seeds of the two individuals covered the qHUS6-1. QTL mapping was performed by using these two populations which were planted in Zhejiang Province in the summer of 2006 and qHUS6-1 was delimitated to a 147.0-kb region flanked by RM510 and RM19417. It can explain as high as 65.26% and 76.3% of the phenotypic variance in the two populations respectively and the allele from Milyang46 increased hull silicon content in both of these two populations. Five groups of F3 lines with different genotypic compositions in the qHUS6-2 region were selected from the other F2:3 population and were planted in Zhejiang Province in the summer of 2006. Two QTLs were separated with two-way ANOVA, of which qHUS6-2a was located in interval RM19706-RM19795 and qHUS6-2b in interval RM314-RM19665. The allele from Milyang46 increased hull silicon content for both of these two QTLs. 3. Two individuals named TF6-15 and TF6-17 carrying smaller heterozygous segments covering the qHUS6-1 region were selected from FM4 F3 population and two F2 populations was derived from these two individuals, respectively. 10, 10 and 20 plants that were maternal homozygous, paternal homozygous and heterozygous in the target interval were selected from one of the F2 populations respectively. And another F2 population was also performed as above. An F3 population was derived with these materials and was planted in Zhejiang Province in the summer of 2007. Two–way ANOVA was performed for the lines from the same individual in order to detect whether there was QTL in the target interval and then compared the result from two series of NILs. There was significant difference between the NILs derived from TF6-15 and no difference between NILs from TF6-17. The qHUS6-1 was delimitated to a 62.4-kb region flanked by RM3414 and Si2944. The allele from Milyang46 increased hull silicon content.4. Based on the result of fine mapping of qHUS6-1, candidate genes search was performed and 10 ORFs were found in the fine mapping region. Through bioinformatic analysis, one of the candidate genes was thought to be the target gene. By comparing the sequences of the genomic DNA of the target gene from the two parents, there was no difference in the coding region between two parents and the full length of the gene was 2,592bp; by comparing the sequences of the cDNA of the target gene from the two parents, we found that there was no intron in this gene and the cDNA was 2,592-bp long and can produce a protein of 863 amino acid long. There was a RNA editing occurred at 104th nucleotide in the cDNA from Zhenshan 97B, resulting a change from tyrosine of Milyang46 to cysteine of Zhenshan97B at the 35th amino acid.