Genetic Analysis And MAP Cloning Of Restorer Gene Of ZD Type Cytoplasmic Male Sterile Lines Of Soybean

Soybean?Glycine max?is a self-pollinated crop.The process of hybridization requires a tedious process.The improper operation may cause stigma damage.The main pathway of soybean heterosis is three-line hybridization.Screening and cloning excellent fertility restorer genes are the basis for heterosis utilization.The fertility restorer genes have been cloned in a variety of crops.However,due to the large number of repeat sequences and complex structure of soybean genome,the current research on fertility restorer genes of Soybean only proceeds to basic genetic analysis and molecular localization.There are no fertility restorer gene have been cloned.The mechanism of the soybean fertility restorer gene is still unclear,which greatly hinders the cross-breeding of soybean and not conducive to the reproduction of soybean excellent traits.Therefore,cloning the fertility restorer gene is beneficial to detecting the restorer line at the genetic level,and reducing the complexity of the screening work.In addition,it also lays the foundation for understanding the mechanism of soybean restorer genes and the interaction between soybean cytoplasm and nucleus.In this study,the progenies,F₂,F_2:3,F_3:4,F_4:5:5 and F_5:6,derived from the cross between W931A?rfrf[S]?and WR016?RfRf[N]?were used as experimental materials.The molecular markers,combined with pollen microscopic results and seed setting rate,were used to analyze the fertility restorer genes.Then the fertility restorer genes were analyzed by genome-wide sequencing,and the differences between the restorer line and the sterile line and maintainer line were compared.The candidate genes for the fertility restorer gene were identified and were subcellularly localized.It attempts to reveal the genetic characteristics between nuclear and cytoplasm,and provide a theoretical basis for the genetic breeding of soybean.The results obtained in this study are as follows:?1?Linkage analysis results:The restorer gene was located between the two molecular markers,BARCSOYSSR₁6₀907 and BARCSOYSSR₁6₀933,of the Chromosome16?Gm 16?.With reference to Williams 82?Wm 82?,a total of 512 kb in this interval,containing44 genes.?2?Whole genome sequencing results:For SMRT,a total of 31.7 Gb was obtained,952.6 Mb was spliced,and 1390 Contigs were obtained.Contig N50 is 1.95 Mb.For Hiseq,the total data volume is 168.58Gbp,and Q30 reaches 93.39%.There are some differences among WR016,Wm 82 and ZhongHuang 13?ZH13?genome.Combining the restorer gene's ability to enter the function of mitochondria,through protein prediction,it was found that a total of 21genes in the target range can enter the mitochondria,10 of which have amino acid differences in the coding regions of the WR016 and W931A/W931B genomes,removing 4 disease resistance protein,The remaining 6 genes other than the medicinal protein can be used as candidate genes for the Rf for subsequent research.?3?Subcellular localization:Among the 6 candidate genes,Glyma.16g139800 gene encodes PPR protein.Cloning MTS of the PPR gene and constructing 35S promoter-driven MTS-GFP?35S::MTS-GFP?fusion expression vector?pCAMBIA3300-2b-MTS-GFP?.Then transferred to tobacco in order to transient expression.The results showed that GFP signal distribution in the nucleus and cytoplasm,While W931A signal intensity is much lower than several other varieties,indicating that their protein do have differences.