Gene Mapping,Transcriptome Sequencing And Cloning Of Two Lignin Biosynthetic Genes Of A Stem Lodging Mutant In Soybean

Soybean [Glycine max (L.) Merr.], originated in China, is not only the traditional crop, but also 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. Lodging is a common problem in crop production. Lodging can cause longer stem internode with soft and creeping characters, and influences the formation, transport and storage of photosynthetic products in soybean, so it has become one of the most important factors limiting the high and stable yield. The resistance to lodging is a necessary trait for high-yielding varieties of soybean, but there are only a few studies on the genetic regulation of soybean lodging, which restricts the efficiency to develop the lodging-resistant soybean. Exploration and use of some soybean stem morphological mutants can provide insights into the developmental and genetic mechanisms of stem lodging. In this study, an EMS-induced stem lodging mutant NT82was identified and crossed with three wild-type parents, Williams82, Nannong1138-2and Kefeng1Hao, to obtain genetic population to study the inheritance and gene mapping of the mutant trait, and the transcriptome sequencing between NT82and its wild-type NN1138-2was conducted. The major objective is to screen out some important genes related to soybean stem lodging according to the results of transcriptome and gene mapping analyses. The main results are as follows:All F1plants of three crosses were erect without lodging. According to the results of Chi-test of goodness-of-fit for segregation ratios of normal to stem lodging plants in F2and F2：3generations, the ratios in F2of Williams82x NT82and Kefeng1Hao x NT82crosses fitted the expected3normal:1mutant, the ratio in a pooled F2.3heterozygous lines from Williams82x NT82also fitted3normal:1mutant. The segregation ratio of (NG94-156*NT82) F2population did not fit3normal plants:1mutant, but that in the pooled F2:3heterozygous lines fitted the expected3normal:1mutant ratio. So it inferred that NT82stem lodging trait might be controlled by a pair of recessive gene, tentatively designated as sl (stem lodging). The stem lodging plants had adverse performance in plant height, node numbers on main stem, pod number per plant and grain number per plant in the F2population of Williams82xNT82and NG94-156xNT82. Using NG94-156x NT82F2mapping population, the sl locus was roughly located in the region between Satt614and Satt440on linkage group I (Chromosome20), with a genetic distance80.8cM; and the sl gene was located in the region between Satt270and Satt292on linkage group I, with a genetic distance32.67cM by using the Williams82x NT82population.Transcriptome sequencing technology was used to detect the gene transcription differences of shoot tissues between wild-type and NT82mutant. After mutant plants appeared stem lodging at R1-R2stage, the sixth internode and the ninth to tenth internodes of NT82plant stems were collected and named as NTM-1and NTM-2representing different growth stages of the stem, and the ninth to tenth internodes of wild-type plants were also obtained and named as NTW. The results showed that there were2177differentially expressed genes between NTM-1and NTW,2812genes between NTM-2and NTW, and only1006differentially expressed genes between NTM-1and NTM-2. Significant levels of GO were calculated by elimGO algorithm. The main functions related to stem lodging between NTM-1and NTM-2samples included calcium binding, response to stress, lipoxygenase activity, oxidoreducatase activity, xyloglucan: xyloglucosyl transferase activity, cell wall. The main functions related to stem lodging between NTM-1and NTW included calcium binding, xyloglucan: xyloglucosyl transferase activity, cell wall, response to stress, regulation of carbohydrate metabolic process, secondary cell wall biogenesis, lignin catabolic process, phenylpropanoid metabolic process, cellulose biosynthetic process, phenylalanine ammonia-lyase activity. Four genes related to ligin biosynthetic pathway, GLyma18g02690, Glyma06g14200, G1yma20g16100, G1yma05g03780, were screened out for further study. The transcript splice sites of NT82were investigated and seven kinds of alternative splicing types, including intron is the most popular event, were identified using MATS software.Full-length cDNA of GmCAD4and GmCOMT was cloned from NT82, Nannong1138-2and Nannong94-156. Sequence analyses showed that GmCAD4was composed of 1077bp and358amino acids, coding a39.15KDalton protein with isoelectric point of5.81. It had five exons and two introns. The GmCOMT had1098bp with four exons and three introns, it coded a39.90KDalton protein with isoelectric point of5.77, which is composed of365amino acids. GmCAD4and GmCOMT were localized in the cytoplasm and the endoplasmic reticulum, respectively. Multiple alignments showed GmCAD4and GmCOMT had the highest homology with those of common bean. We designed a pair of primers that contained attB sites. After BP reaction we got the entry vector pDONR/Zeo-GmCAD4and pDONR/Zeo-GmCOMT. Then by doing LR reaction we got the destination vectors pMDC83-GmCAD4and pMDC83-GmCOMT were also obtained through the Gateway technique.