Phenotypic Analysis And Gene Mapping Of Dwarf Mutants Gmdwf11/18 In Soybean

Mutagenic breeding is one of the principal ways for genetic improvement of crops.A large number of mutants were obtained by EMS mutagenesis technology,which can be utilized for subsequently concerned functional genes identify and molecular breeding in soybean.So far,some genes related to plant morphology of soybean have been cloned,few is known on the isolation functional genes related to plant morphology by using of mutants in soybean.In this paper,soybean cultivar Hedou 12(H12)is used as experimental material.A large number of dwarf mutants were obtained by EMS mutagenesis,and phenotypic and genetic analysis of the two dwarf mutants Gmdwf11 and Gmdwf18 which were selected from it.Map-based cloning and BSA re-sequencing were used for locating the candidate genes of the two mutants,and the candidate gene of Gmdwf11 was cloned successfully.All these work laid a good foundation for further revealing the molecular mechanism of functional genes,cultivating high-yielding and lodging resistant in soybean.The main process and results of this research were as follows:1.Screening and identification of soybean dwarf mutants Gmdwf11/18Through phenotype observation on M2 generation population of EMS mutagenic Hedou 12,twenty dwarf mutant lines were obtained,which divided into two types:Dwarf mutants with plant height were less than half of the Hedou 12,which exhibited dwarfism,tiny leaves,compacted and shortened internodes,including seven mutants(such as:Gmdwf11 and Gmdwf18).Semi-dwarf mutants with the plant height was between dwarf mutants and Hedou 12,which exhibited semi-dwarf,shrinkage leaves,including 13 mutants.2.Phenotype analysis,gene mapping and cloning a dwarf mutant Gmdwf11Analysis of the test data showed that compared with the wild type,the plant height of the dwarf mutant Gmdwf11 was significantly stuntedness,with shortened internodes and tiny leaves,and its plant morphology is more compact,less pod numbers.Paraffin section results show that there was little difference in the morphology of shoot apical meristem between Gmdwf11 and Hedou 12,but the cell length of the central stem pith parenchyma in the third shoot is distinctly short and the cell number is significantly increased.So we speculated that the dwarfing phenotype was caused by the rapid proliferation and hampered elongation growth of the cells in internodes.Exogenous GA3 can restore the dwarf phenotype of Gmdwf11,indicating that the gibberellin biosynthesis pathway of the Gmdwf11 was blocked.Both transcriptome sequencing and gene expression analysis confirmed that the expression of gibberellin signal pathway related genes in Gmdwf11 were aberrant.We finally come to the conclusion that the candated gene of Gmdwf11 can regulate soybean stem elongation through influencing GA3 synthesis and signal transduction genes expression.A cross between the dwarf mutant Gmdwfll and Williams 82 was made,and an F2 population plants was obtained.The wild types and mutants were isolated from the F2 population by phenotype observation and genetic analysis.The result of χ2 tests showed that Gmdwf11 fitting a 3:1 ratio.This indicates that the phenotypic changes were controlled by a single recessive nuclear gene.The F2 generation obtained from hybridization was used as a map clone population,and the dwarf mutant Gmdwf11 was initially located by polymorphic Indel markers.It was found that the Gmdwf11 mutation site was closely linked to the molecular marker Gm0069.By expanding the F2 population and designing new molecular markers,the mutation site was located in a region between Gm1008 and Gm1009 on chromosome 11,which is 0.72 Mb.Continue to expand the separation group,the mutation site was finally located in a 76 kb region on chromosome 11,which harbors two annotated genes.By cloning and sequencing of the two candidate genes in Gmdwf11 and wild type,we found that the CDS and promoter sequences of gene 2 in the mutant and wild type are identical.However,the sequence of gene 1 in Gmdwf11 had a base substitution of G to A at the 222 bp downstream of ATG,which is the recognition and alternative splicing site of the first intron in gene 1.Analysis of the CDS sequence of the gene 1,we found that the first intron sequence was not cut off,unable to correctly encode its functional protein.It is speculated that gene 1 is GmDWF11,a key candidate gene for regulating plant height.RNA sequencing and gene expression analysis verified that the gene 1 was down-regulated in Gmdwf11.An overexpression vector and the CRISPR/Cas9 double mutation vector were constructed,which laid the foundation for the study of the function of the gene 1.3.Phenotypic analysis and gene mapping of a dwarf mutant Gmdwf 18Through phenotype identification,we found that the other mutant Gmdwf18 showed phenotype with dwarfism,leaf shrinkage and sterility,which compared with the wild type Hedou 12.A cross between Williams 82 and the Gmdwf18 mutant was made,and an F2 population plants was obtained.Phenotype observation and genetic analysis reveal that wild types and mutants were isolated from the F2 population.The result of χ2 tests showed that Gmdwf18 fitting a 3:1 ratio.This indicates that the trait of the dwarf mutant Gmdwf18 is also controlled by a single recessive nuclear gene.High quality sequencing data were obtained via BSA re-sequencing technology.Two related regions with a total length of 0.83 Mb which located in the chromosome 18 were obtained by correlation analysis of SNP and Indel,and there are 28 genes in these two regions.The F2 population was constructed by hybridization experiment for gene mapping.Using Indel markers on chromosome 18,a candidate region was mapped to a genomic region on chromosome 18.The results of gene mapping were also showed that the mutant site of the dwarf mutant Gmdwf18 were located on chromosome 18.