Gene Mapping, Expression Profile And Candidate Gene Analyses Of Zigzag Stem In Soybean

Soybean [Glycine max （L.） Merr.] originated in China, which is the most important source of plant protein and edible vegetable oil in the modern world. The soybean yield per unit area is relatively low in China, so the high-yield breakthrough is always the main breeding objective. Ideotype can improve utilization efficiency of light energy. It is important to develop an ideotype with good adaptation to local environment at both individual and population level. Soybean zigzag stem has zigzag and short internodes and can be used to increase soybean lodging resistance under high-yield environment, so it is a specific trait with potential values in soybean breeding. The trait can be combined with good genetic background to develop an ideal plant type to explore yield breakthrough in soybean. On the other hand, soybean zigzag stem performs as an abnormal growth mutant, isolation of the target mutant gene can provide new insights into understanding of the genetic mechanisms of shoot growth and development in plant. In the thesis, quite a few F2populations were used to study the inheritance of soybean zigzag stem according to the previous research. A set of NIL material was also used to analyze different genes through the gene chip technique. Finally, some important genes were identified according to the results of gene mapping and microarray analyses, and their expression and function was analyzed. The main results are as follows:Total20F2populations from the crosses between zigzag stem materials and normal stem parents were used to test the expected3norma1:1mutant or15norma1:1mutant segregation ratio by using chi square test. Among them, ten fitted3:1ratio, two fitted15:1ratio, indicating that zigzag shoot might be controlled by two pairs of recessive genes. However, eight crosses didn’t fit any of the above ratios, it might be related with the genetic background of parents or some environment factors. In the F2of Zhechun3RH×NG94-156, the means of plant height, internode number on main stem, total pods and total seed number per plant of mutants were lower than those of normal plants, while in （NN1138-2M×NG94-156） F2population, there were not differences in internode number on the main stem and total seed number per plant between two stem types. It is possible to develop elite zigzag stem soybean with good agronomic characters from hybrid progenies of some crosses. In order to verify the previous results, two reported regions （one is Gm14:30058525…33087558, the other is the Sat₄24to the end of chromosome） on B2linkage group were further mapped with70SSR makers using a F2population of NN1138-2M×NG94-156. The sb gene was located between BARCSOYSSR₁4₁415and BARCSOYSSR₁4₁424, with a50bp physical distance （Gm14:47661103...47901399）. There were17predicted candidate genes in the regions.Transcript differences of shoot tissues of a set of near isogenic line TN01were detected by using Affymetrix gene chip. Total1384different genes, of which899genes were up-regulated and485genes down-regulated, were detected with the standard of fold change>2and P<0.01. Significant levels of GO were calculated by elimGO algorithm, and57significant up-GO and38significant down-GO were identified. The main functions related to zigzag stem included response to gibberellin stimulus, oxidation reduction, cellular response to gravity, gibberellic acid mediated signaling pathway, regulation of short-day photoperiodism and flowering, response to salicylic acid stimulus, systemic acquired resistance, salicylic acid mediated signaling pathway, regulation of salicylic acid biosynthetic process. Six genes, Glyma14g38580, Glyma04g09350, Glyma20g2728, Glyma11g04750, Glymal5g01500, and Glyma09g27490, were screened out. Ten significant up-pathways and six significant down-pathways were also identified. The six selected genes performed significantly in the synthesis of secondary metabolites, phenylalanine metabolism, flavonoids biosynthesis, diterpenoid biosynthesis. They displayed significant changes and relatively active according to the analysis of co-expression network.Glymal4g38580（cinnamic acid4-hydroxylase, C4H）, was identified as an important gene regulating the development of zigzag stem according to the results of gene mapping and microarray chip analyses. C4H is the second key enzyme and catalyzed the second step of the phenylpropanoid pathway. cDNA sequences of GmC4H in ten materials were obtained, and there was some base differences among them. However, no amino difference was found between wild and mutant materials. The gene was down-regulated in the mutant type of TN01NIL material by qRT-PCR, which was in accorded with the chip result. Sixteen genes were co-expression with Glyma14g38580in mutant plants while only5ones in the wild type.The PPR gene family was a newly discovered gene family, which was closely related to the function of two important organelles, mitochondria and chloroplast. GmPPR1gene was identified as a important candidate gene for zigzag stem in previous study. We constructed the GmPPRl gene vector and transformed it into Arabidopsis thaliana. Twelve transgenic plants were produced after the detection of hygromycin, GUS staining and PCR analysis. Transgenic plants had earlier maturity, their shoots were thin with bending and crawl characters in compared with the wild type. However, the function and regulation mechanism of GmPPR1to zigzag stem remained to be studied.