Genetics Analysis And Cloning Of Associated Gene Fragments For Large Grain Size Mutant In Wheat

The traditional method of chemical mutagenesis can produce high-density induced mutations with similar genetic background. This can not only solve the problem of poor germplasm in wheat breeding, but also provides a research platform for the fine positioning, cloning, and functional analysis of related genes. In this study, we have got the mutant called large grain size and long-culm line 8008 by treated the wheat variety"Yannong 15"with EMS (ethyl methane sulfonate). Taking the mutant line 8008 as experiment material, we went on the genetic analysis of mutant character including grain longth, grain width and grain weight, tried to map these mutant gene and clone associated gene fragment, and this could lay a foundation for forther revealing the genetic mechanism of grain weight heredity in wheat.(1) Cross the large grain size and long-culm line 8008 with Yannong15 to construct the population for genetic analysis. Through the phenotype analysis of F2 and F3 population, we found that the traits high grain weight & low grain weight, wide grain & narrow grain, long-culm & short-culm showed a 3:1 ratio of the separation, and differences by chi-square test was significant, which shows that all these three pairs of traits are controlled by single dominant gene. Correlation analysis showed that grain width and grain weight, grain length and weight, long-culm and weight are among the most significant correlation(2) We look for polymorphic markers from 860 EST-SSR and 960 SSR primer pairs by amplying in line 8008 and Yannong 15. These polymorphic markers were amplied in F2 population for forther analysis. Finally, a total of 99 polymorphic fragments were detected from 59 primer pairs, which were mainly distributed over 16 chromosomes. Most of these markers (27 primer pairs) were mapped on genome B; and there were 7, 7 and 6 primer pairs which were mapped on chromosome 1B, 2B and 7B, respectively.(3) The mutant line 8008 and Yannong 15 are used as tester and driver respectively for the structure of forward and reverse suppression subtractive hybridization cDNA libraries. Positive clones are randomly chosen from each of the two libraries and then sequenced, and propose sequence alignment in GenBank by BlastN in GenBank. A total of 12 ESTs which was highly homologous with the known functional genes were obtained. These functional genes could be grouped into 3 parts., include genes associated with photosynthesis, such as RuBP carboxylase, phytochrome, chloroplast, etc.; and material metabolism-related genes, such as homocysteine hydrolase, cysteine protease, fructosyltransferase, sucrose synthase type I, etc.; and genes of RNA function such as RNA-binding protein, ribosomal protein, and so on. There are 5 EST sequences which are highly homologous with unknown function of Gramineae crops cDNA fragments,and seven ESTs fail to find homology EST match.