Cloning Of Soybean Curly Leaf And Dwarf Mutant Gene GmCLD1 And Identify Of Candidate Genes Regulated By Polyadenylation Sites

With the publication of the soybean reference genome,the use of genetic maps and physical maps for mapping and cloning of genes has been greatly accelerated.In the study of map-based cloning of soybean agronomic traits,natural populations are used to locate a large number of QTL related to various agronomic traits.However,the research of mapped and cloned of soybean EMS-mutated leaf type and plant height genes are rarely reported.In this study,the mapping and cloning of the mutant gene of soybean curly leaf and dwarf mutant Gmcldl were carried out,which laid the foundation for revealing the regulation mechanism of leaf type and plant height.Polyadenylation is a process of post-transcriptional modification.Poly(A)is formed by polyadenylation of the pretranscript,which is a key step in transcript maturation and is related to the complete of transcript structure of genes.At present,relevant research has been reported in rice,Arabidopsis and other plants,but there is no report on the candidate genes related to the characteristics of soybean genome polyadenylation sites and their regulation.Therefore,studying the characteristics of soybean gene polyadenylation sites and identify candidate genes regulated by alternative polyadenylation sites are of great significance for revealing the regulatory mechanism of soybean gene transcription and functions.The main process and results of this research were as follows:1.Forward genetic cloning of Glycine max curly leaf and dwarf 1 geneA short,wrinkled and curly leaf M4 generation mutant Gmcld1(Glycine max curly leaf and dwarf 1)was selected from the mutant library created by soybean EMS mutagenesis of "Hedou12".The compound leaves of Gmcld1 showed wrinkled,narrow,slender leaves,and the leaves curled toward the proximal end.During the vegetative growth period,Gmcldl was slower than the wild type,so the plant was shorter.After entering the R1 stage of reproductive growth,its leaf shrinkage and dwarf phenotype weakened,and the leaf phenotype returned to close to normal,and the plant height was similar to that of the wild type.The analysis of seeding data show that compared with the wild type,the difference in the number of main stem nodes of Gmcldl is small,the number of seeds per plant,the grain weight per plant,and the number of seeds per pod are significantly reduced.The seed is partially hypogenesis.Paraffin sectioning of the shoot apical meristem of Gmcld1 showed that the size was not significantly different from that of the wild type,and the structure of the flower organs was normal.Genetic analysis was carried out through the hybrid population constructed with"Williams82" and the BC1F3 population constructed with wild type "Hedou 12".The separation ratio of wild-type and Gmcld in the F2 hybrid population was close to 3:1.Chi-square test showed that Gmcld1 was regulated by a single nuclear gene.Using 120 pairs of Indel marker primers,the 21 mutants isolated from the F2 generation hybrid population were used for primary map-based cloning,and the candidate loci was located in the 7.84M region of Chr09:0-Chr09:7840485 on chromosome 9.By expanding the F2-F4 generation population for fine mapping,the mutation region was finally located in the 242.599 kb interval of Chr09:2168654-Chr09:2411253.Using BC1F3 backcross population mutant single plants and normal plants for BSA sequencing,the mutation site was also located within the Chr09:2953-Chr09:3683468 interval on chromosome 9,and it was found that the map-based cloning interval was included.There are 140 polymorphic SNP and Indel in the fine mapping region,including 32 genes.The homozygous SNP were sequenced and it was found that only Chr09:2342753 had a G to A mutation.Located in the coding region of the GmCLD1 gene,and the gene is identified as Gmcld1 mutant gene which encodes helicase protein.In order to further verify the function of the candidate gene,its RNAi interference vector was constructed and transformed into soybean,and 20 To generation positive plants were obtained.At the same time,a subcellular localization vector,a yeast two-hybrid vector,a CRISPR/Cas9 vector and an overexpression vector were constructed,which laid the foundation for further verification of the function of the gene and revealing the molecular mechanism of its regulation.RNA-Seq sequencing of Gmcldl and wild-type "H12" were performed.GO and KEGG enrichment analysis found that differentially expressed genes identified in mitochondria have largest genes number,and the most enriched genes are related to carbon metabolism pathways.Tissue-specific expression analysis found that the gene was expressed in all organs of the plant,with the highest expression in leaves.Finally,most of GO-enriched genes in mitochondria are up-regulated.2.Search for polyadenylation sites in the whole soybean genomeAnalyzed RNA-seq data of 410 soybean samples,and identified 53,934 soybean gene polyadenylation sites.The polyadenylation sites within 24 bp were defined as a polyadenylation cluster,and 23493 polyadenylation clusters were obtained.It was found that among the genes containing polyadenylation sites,3715 genes have alternative polyadenylation sites.It is speculated that these genes are regulated by alternative polyadenylation sites.Further analysis found that the CDS and intron regions containing polyadenylation sites were longer than those without polyadenylation sites,and 1043 sites were identified to be located in gene introns or CDS regions.The enrichment analysis of sites located in CDS and Intron with higher relative expression levels revealed that genes are mainly involved in biological functions such as starch biosynthesis and reducing pentose-phosphate cycle pathways.Co-localization analysis of these genes containing alternative polyadenylation sites and some published QTLs in soybeans identified 115 candidate genes that are regulated by polyadenylation in soybean agronomic traits.