Breeding Of New Cytoplasmic-nuclear Male Sterile Lines And Study On Differential Transcriptomics And Proteomics Between NJCMS1A And NJCMS1B In Soybean(Glycine Max(L.)Merr.)

The utilization of heterosis has been proved to be one of the potential approaches to improve crop yield.Cytoplasmic-nuclear male sterility plays an important role in the production of hybrid seeds.Soybean(Glycine max(L.)Merr.)is an important source of plant protein and oil.But,low yield is a key factor restricting its development.So far,extensive research has been conducted on soybean cytoplasmic-nuclear male sterility including three-lines breeding,genetics,cytology,and molecular biology in China and abroad.But,it is necessary to continue the breeding of new cytoplasmic-nuclear male-sterile line and study on the molecular mechanism related to male sterility in soybean.The soybean cytoplasmic-nuclear male sterile line NJCMS1A was developed through consecutive backcross procedures with the cultivar N8855 as donor parent and N2899(designated as NJCMS1B afterwards)as recurrent parent by National Center for Soybean Improvement.In this study,the soybean cytoplasmic-nuclear male sterile line NJCMS1A was used as the plant material to carry out the breeding of new cytoplasmic-nuclear male sterile line.Meanwhile,the differential transcriptome and proteome analysis were conducted between the male sterile line NJCMS1A and its maintainer NJCMS1B in soybean by RNA-Seq and iTRAQ technology,respectively.The main results were as follows:1.Breeding of new cytoplasmic-nuclear male sterile lines and identification of maintainers and restorers of the male sterile line NJCMS1A in soybeanOn the basis of previous studies of National Center for Soybean Improvement,using soybean cytoplasmic-nuclear male sterile line NJCMS1A as female parent and the conventional cultivars as male parent,4 new stable cytoplasmic-nuclear male sterile lines were breeded by continuous backcross method and named as NJCMS6A,NJCMS7A,NJCMS8A and NJCMS9A respectively.The identification results of maintainers and restorers of the male sterile line NJCMS1A showed that 11 cultivars such as Youbian30 etc.were identified as the maintainers of NJCMS1A,46 cultivars such as Jidou17 etc.were identified as the restorers of NJCMS1A,but,other 17 cultivars such as Hedou18 etc.weren't be determined as the maintainers or restorers of NJCMS1A.2.Study on differential transcriptomics between the male sterile line NJCMS1A and its maintainer NJCMS1B in soybeanThe differential transcriptomic analysis between flower buds of cytoplasmic-nuclear male sterile line NJCMS1A and its maintainer NJCMS1B in soybean was conducted using Illumina sequencing technology(RNA-Seq).As a result,365 differential expressed genes(DEGs)between NJCMS1A and NJCMS1B were screened by "FDR ? 0.05 and |log2Ratio(FC)| ? 1" as the threshold,among which,339 DEGs down-regulated and 26 DEGs up-regulated in NJCMS1A compared to in NJCMS1B.Gene Ontology(GO)annotation showed that 242 DEGs(66.3%)were annotated to 19 functional categories.Clusters of Orthologous Groups of proteins(COG)annotation showed that 265 DEGs(72.6%)were classified into 19 categories.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that 46 DEGs(12.6%)were assigned to 33 metabolic pathways.qRT-PCR analysis validated that the gene expression pattern in RNA-Seq was reliable.According to the transcriptome analysis results combined with reported literature,we concluded that the male sterility of NJCMS1A might be related to the disturbed functions and metabolism pathways of some key DEGs,such as DEGs involved in carbohydrate and energy metabolism,transcription factors,regulation of pollen development,elimination of reactive oxygen species(ROS),cellular signal transduction,and programmed cell death(PCD)etc.3.Study on differential proteomics between the male sterile line NJCMS1A and its maintainer NJCMS1B in soybeanThe differential proteomic analysis between flower buds of cytoplasmic-nuclear male sterile line NJCMS1A and its maintainer NJCMS1B in soybean was conducted using iTRAQ technology.As a result,180 differential expressed proteins(DEPs)between NJCMS1A and NJCMS1B were screened by "|Fold Change|?1.5 and p ? 0.05",as the threshold,among which,60 DEPs down-regulated and 120 DEPs up-regulated in NJCMS1A compared to in NJCMSIB.GO annotation showed that 167 DEPs(92.78%)were annotated to 41 functional categories.COG annotation showed that 106 DEPs(58.89%)were classified into 20 COG categories.KEGG enrichment analysis showed that 128 DEPs(71.11%)were assigned to 53 metabolic pathways.Multiple reaction monitoring(MRM)analysis and qRT-PCR analysis indicated that iTRAQ results was reliable.According to the differences proteome analysis results combined with reported literature,we concluded that the male sterility of NJCMS1A might be associated with the abnormal of carbohydrate and energy metabolism,unbalanced of protein synthesis and proteolysis,out of control regulation of pollen development,programmed cell death,material/secondary substance metabolism turbulence,abnormal of fertility restoration genes expressed and some known or uncharacterized DEPs differently expressed,and so on.4.Conjoint analysis of the differential transcriptomic and proteomic results between the male sterile line NJCMS1A and its maintainer NJCMS1B in soybeanThe conjoint analysis of the differential transcriptomic and proteomic results between the male sterile line NJCMS1A and its maintainer NJCMS1B in soybean was conducted.As a result,15 DEGs/DEPs with the same expression pattern were identified.GO annotation showed that 15 DEGs/DEPs were annotated to 31 functional categories.COG annotation showed that 11 DEGs/DEPs(73.33%)were classified into 7 COG categories.KEGG enrichment analysis showed that 13 DEGs/DEPs(86.67%)were assigned to 15 metabolic pathways.Cluster analysis indicated that the correlation between the differential transcriptomic and proteomic results was high.Finally,qRT-PCR analysis confirmed that the two kinds of "omics" results were reliable.According to the conjoint analysis combined with the reported literature,we concluded that the male sterility of NJCMS1A might be mainly related to the energy supply insufficient,material synthesis and metabolic turbulence,out of control respond to stress and programmed cell death(PCD),etc.