Fine Mapping A Main Effect QTL For Brown Rice Protein Content And Construction Of Near Isogenic Line

Rice protein is one of the most important nutritional quality, and also is the second most abundant constituent of milled rice, following starch. As one of the staple food, rice protein is one of the main sources of protein intake for people. Rice protein content is typical quantitative traits, With the advent of molecular marker, introgression of genes controlling good quality by marker-assisted selection has been proved to be an effective choice to improve the quality of cultivars. However, the genetic basis of protein is complex and often controlled by quantitative trait loci. Thus, fine mapping and cloning those QTLs underlying protein is needed for rice genetic improvement. Near Infrared Spectroscopy is used from 1980s as a new technology. It is the use of chemical substances in the Near-Infrared Spectroscopy Determination of the optical properties of the region in some samples of one or more chemical ingredients and characteristics of the physical determination of new technologies. In recent years, Near Infrared Reflectance Spectroscopy (NIRS) has become an active area of research in plant breeding.Through Marker-assisted selection, we analysis the effect of QTLs and constructed Several NILs for protein content and amino acids content. All of these NILs are used Zhenshan 97B as their background and One of the QTLs was selected for fine mapping.1. NILs Construction:From 2007 to 2009,7 NILs were constructed in Wuhan and Hainan, Zhenshan 97B as the background. There are 4 NILs for Zhenshan97B/Delong 208, whose target section were located on chromosomes 1,2,7,9; for Zhenshan97B/Nanyangzhan, we constructed 3 NILs, which located on chromosomes 1,2, 8. These NILs were backcrossed above 3 times, then self crossing once.2. Analysis of random population:The 1241 NIR quality analyzer and the flow analyzer were used as two methods for measuring protein content. The phenotype of random population separated between the two phenotypes, showed the distribution system of quantitative traits. Chi-square test hypothesis indicated that low protein content to high protein content of the segregation ratio is 3:1, consistent with Mendelian segregation ratio, indicating that the background of near-isogenic lines have basically pure, large population can be used to develop for screening recombinant plant, with in the fine mapping. At the same time the results of both methods have good correlation, correlation coefficient is 0.85, at p=0.01 level, reached a highly significant. So indicated that NIRs such easy method can replace the traditional flow analysis method.3. Local QTL linkage analysis:In the interval RM472-RM1387, we found out some new polymorphic markers to re-analyze the genetic effects of QTL. While the results of two methods both located QTL Bqpcl between the interval RM11961-RM6333, LOD value for 20.19 and 13.97, the additive effect for 4.463 and 15.79, and each of the phenotypic variation for 57.83% and 35.97%. The range of genetic distance is 2.1cM, and the physical distance is about 180.2kb.4. Fine mapping the QTL:We planted 6000 and 4000 plants in Hainan and Wuhan as large populations for selecting recombination. There were 87 recombination plants, and then were used as progeny test. The Bqpcl locus was delimited to a 88kb region flanked bv SSR marker SHL4 and InDel marker HL13 by overlapping mapping. Across the region, there are two BAC, AP003297 and AP003261. Through the Rice Genome Annotation (Osal) Release 6.1, we search and analysis the sequence between markers SHL4 and HL13 and found this area containing Twelve ORFs, of which eight are predicted function, and four are unknowned proteins. These eight ORFs include:a RNA recognition motif containing protein, a snRKl-interacting protein 1, a mitotic checkpoint family protein, a galactosyltransferase, a PHD-finger domain containing protein, a receptor-like protein kinase HAIKU2 precursor and two amino acid transporter genes.