Fine Mapping And Candidate Gene Exploration Of Loci Corresponding To Brachytic And Fasciation Stem In Soybean

Soybean [Glycine max (L.) Merr.] originated in China and has been cultivated more than five thousand years, which is one of the most important grain and oil crops in the world. The soybean yield per unit area is relatively low, so the high-yield breakthrough is always the main breeding objective. It is important to develop an ideotype with good adaptation to local environment at both individual and population level. Brachytic stem and fasciated stem are soybean stem morphological mutants, which show great potentials to improve lodging resistance and develop new lines suitable for increasing population density. In this study, two F2 populations between NT-1 (a brachytic and fasciated stem line) and two normal stem parents NN1138-2 and Forrest were utilized for fine mapping of sb and f gene of two stem traits, and candidate gene analysis through bioinformatics and molecular cloning strategies was also conducted to elucidate the developmental mechanism and breeding potentials of brachytic stem and fasciated stem in soybean. The main results were as follows:1. Fine mapping and candidate genes analysis of sb for brachytic stemThe results of genetic analysis from the progenies of NN1138-2×NT-1 and Forrest×NT-1 confirmed that the brachytic stem was controlled by one pair of recessive gene sbsb. In 2009,92 and 43 F2 brachytic stem individuals from the two crosses were randomly selected and used for rough mapping of sb gene respectively, and the gene was located on the long arm of chromosome 14, with the genetic distance of 14.1cM and 9.5cM from the Sat₄24 marker respectively. In 2010, two fine mapping populations consisting of 315 and 153 mutant F2 individuals were established, and finally fine-mapped the sb locus within a 67kb (Gml4:47,901,370..47,968,346) interval flanked by two SSR markers BARCSOY SSR 14 1424 and BARCSOYSSR 14 1428.The candidate genes in the delimited region was predicted and annotated, and four genes (Glyma14g38750, Glyma14g38760, Glyma14g38770 and Glyma14g38790) were identified according to the Glyma1.0 annotations. Among them, Glyma14g38770 and Glyma14g38790 belong to the low-confidence genes. Therefore, Glyma1438750 and Glyma1438760 were regarded as the candidate genes of sb. According to Glymal.0 gene models, primers were designed to amplify these predicted genes from NT-1, NN1138-2 and Forrest. The Glyma1438750-encoded product is referred to as an FAD NAD binding oxidoreductase (FNBO), however, the FNBO protein sequence does not vary between NT-1 and the wild types. Glyma14g38760 belongs to the PPR family and was named as GmPPR1. There is one amino acid variation, i.e. an arginine (Arg) replaced by a cysteine (Cys), in Glyma14g38760 between mutant and wild parents, this mutation might cause the sb phenotype.2. Fine mapping and candidate gene analysis of f for fasciated stemAccording to the results of genetic analysis, it was confirmed that the fascinated stem in soybean was controlled by a recessive gene f.In 2009, two F2 populations with 82 and 67 recessive plants from NN1138-2×NT-1 and Forrest×NT-1 were used for rough mapping f gene respectively, and the gene was located between Satt546 and Satt644 with genetic distance 8.8 cM and an interval flanked by Satt041 and Satt141 with genetic distance 9.8 cM respectively. More polymorphic SSR markers were identified and the f locus was further flanked by two markers BARCSOYSSR₀2₁303 and BARCSOY SSR₀2₁313 with the physical distance of about a 257kb interval by using 82 fascinated stem individuals of the NN1138-2×NT-1 cross.In 2010, we expanded the fine mapping populations to prove the mapping result. The f locus was also localized to a region between BARCSOYSSR₀2₁303 and BARCSOYSSR₀2₁314 according to the 269 recessive individuals from Forrest×NT-1. Twenty-one new SSR markers in the delimited region were developed by SSRIT and SSR HUNTER softwares. Two markers (SOYSSR6 and SOYSSR17) were proved to be polymorphic between NT-1 and NN1138-2. Then, fine mapping of f were performed by using 408 fasciated stem individuals of NN1138-2×NT-1 cross, and f gene was finally narrowed down to a 194 kb interval (Gm02:42,270,235..42,464,093).Different gene prediction and annotation programs were used to search for candidate genes in the 194kb region. Seven candidate genes for fascinated stem were identified by combining with the latest research result. Among them, Glyma02g36940 is an ortholog of CLV1 which controls the fasciation stem in Arabidopsis thaliana, while there were no variances in the protein sequence between NT-1 and normal parent Williams 82.