Innovation Of Male Sterile Germplasm And Its Identification Of Male Sterile Related Genes In Cotton

Cotton is very important economic crop.Heterosis utilization is a chief topic of cotton breeding for a long time,the discovery and creation of male sterile mutants supply available materials for heterosis.The Identification of the male sterility related genes has important application value in cotton male sterility mechanism research,molecular marker assisted breeding,heterosis utilization and germplasm innovation.In this study,13 strains and 3 cultivated varieties of upland cotton and 6 strains of sea-island cotton were used as the wild type receptor,the kenaf disulfide bond isomerase gene(Hcpdil5-2a),rice pollenless gene(pollenless3),gDNA of "DONG A" sterile plants and gDNA of kenaf cytoplasmic sterile line P3A were transformed through the method of pollen tube.Male sterile mutants were investigated including morphological characteristics,fertility,cell morphology,genetic analysis and gene expression.At the same time,RNA sequencing of sterile anther and fertile anther in the early microspore mononuclear of"DONG A" were completed.The following results were obtained:1.This study established a system to create,preserve and breeding male sterile material in cotton,which based on transformation of gene Hcpdil5-2a.2.Observation of morphology and fertility performance of male sterile mutants indicate that all the 13 male sterile mutants in upland cotton(S2-S13)have little flowers,long stigma and indehiscent anther compared with wild type controls,but there are no difference in agronomic traits between mutants and wild types.Besides all the differences in flower organs above,male sterile mutant 276s in the sea island cotton has many branches and a compact plant,but has no significant change in plant height.Pollen abortion rate of all above mutations are 100%.3.Genetic analysis indicated that the BC,of 13 male sterile mutants in upland cotton were completely fertile.After BC,self-bred nextly,the segregation ratio of fertile plants and sterile plants matched 3:1,showed that 13 male sterile mutations were a single recessive nuclear sterility.The BC,and BC2 of male sterile mutant 276s were completely sterile,BC1 of 276s backcrossed with 10 upland cotton strains were still infertility,which showed that 276s with a inheritance characteristics of cytoplasmic male sterility(CMS).4.The cytological study of microspore abortion of S4,S5,S6 and 276s were carried on.The results show that the abortion of S4,S5 and S6 occurred in microspore meiosis period,all have the characteristics of retardation in the dyad period,and most serious abortion appeared in the microspore mononuclear stage.The abortion of 276s was associated with the dysplasia of microspore mother cell and occurred from the dyad period.5.The full length of PDIL(protein disulphide isomerase)gene in cotton was cloned through e-clone.Sequence analysis showed that introns were absent in the gene.It contains a,293bp open reading frame,encodes a deduced protein of 430 aminoacid residues,and shows 97%homology to Hcpdil5-2a which regulates fertility in kenaf.This gene was tentatively designated as Ghpdil5-2a,and have the same expression pattern in "DONG A"sterility line and fertile line,even have no differences in other varneties.6.Cell morphological observation showed that early microspore mononuclear pernod was the most abortion stage of upland male sterility line "Dong A".The relationship between periods of anther development and bud diameter in "Dong A" were confirmed:during meiosis bud diameter d=1-2mm,at the beginning of tetrads formed d=2-3mm,in tetrads d=3-4mm,d=4-5mm in dikaryophase,d=5mm pollen grains start to take shape,and d=6mm-in pollen maturation stage.7.In our study,cDNA libraries founded from equal amount of RNA taken from male sterile anthers and fertile anthers were sequenced using Illumina/Solexa platform.More than 107,805,664 high-quality reads were found.Based on sequence similarity search,a total of 70,067 genes were identified(E-value<10-5).Differential expression analysis showed that 1208 genes were different expression in all annotated 66 535 unigenes,and 70 specific expression genes were selected as the candidates most likely to be involved in male sterility.At the same time 51 unigenes related to hormone differently expressed,including 23 genes up-regulated and 28 genes down-regulated in sterile anthers.It is the first time to analyze the different expression of hormone related genes though transcriptome sequencing,and identify two of these genes.It provides direct insight into male sterility mechanism and catching sight of key genes in upland cotton "DONG A".