| Heterosis is a common phenomenon in the biology and an important way to improve crops and greatly increase yields.Solanum lycopersicum is a self-pollination crop,and the heterosis is very obvious.Hybrids not only has the advantage of increasing yield,but also has the advantages of strong growth potential,strong adaptability,high degree of uniformity and strong resistance.At present,there are still some difficulties in the use of male sterile lines in tomato,and the hybrids widely promoted in production still rely on artificial castration.The high cost of seed production has become one of the main limiting factors for tomato heterosis utilization.A few genes that control the development of anthers and pollen in tomato have been cloned,but the molecular mechanism is still not clear.Through screening and mapping,we obtained a male sterility gene ms-10,and applied VIGS cloning and molecular biology methods,cloned the gene and preliminary elucidated its molecular mechanism regulating tomato fertility.The main research results are as follows:(1)The male sterility line of tomato with green stalk at the seedling stage and the wild type of fertile and inbred line with purple stalk at the seedling stage were used as materials.By constructing BC1 and F,segregating populations,the phenotype of the male sterile line and the segregation population were observed under field conditions.The results showed that the sterility performance was stable.The floral organs were degraded,although its lighter.To some degree,the stamen is abnormal.rare pollen in the anther,and shape of the anther is incomplete,being dry and dented.The style was slightly longer than the anther cylinder.The rate of fruit set in the field was 5.25%,of which the normal fruit rate was only 8.62%.The seedling green stalk gene and male sterility gene are both single recessive genes,and the two genes are closely linked,the exchange rate of the two genes is 1.1%.This research results show that the green stalk gene in seedling stage can be used to produce male sterile hybrid and trans-breeding male sterile gene.(2)As in the above case,the genetic male sterile was used as the female parent and the wild type was used as the male parent to construct the F2 segregating population.A total of 30 individual plants of male sterile and 30 individual plants of fertile,which DNA pools were established by the BSA method.The polymorphic SRAP markers were screened between the two DNA pools.In the meantime,Specific-locus amplified fragment sequencing(SLAF-seq)was used to detect sufficient amounts of SLAF.Results showing four pairs of polymorphic primer combinations were obtained from 544 pairs of SRAP primers.The four pairs of primer combinations contained 12 polymorphic sites between the two DNA pools.Using these polymorphic loci markers,60 F2 populations were identified by genetic analysis and linkage analysis.A genetic map of male sterile genes was finally obtained,and a total of five specific loci linked to the male sterile gene were identified.Then a total of 20.27 M Reads and 4.90 Gb data were obtained through SLAF-seq.A total of 103250 SLAF markers were obtained.The overall average depth of the markers reached 160.39 X.A polymorphism analysis of SLAF markers between two DNA pools was completed,and acquiring 6892 polymorphism markers.There are eight candidate regions of related to the target male sterile gene were obtained by association analysis between two DNA pools,the average size of eight candidate regions were 0.29 Mb.There were 27 polymorphism markers and 146 candidate genes in the candidate regions.And classify the candidate regions according to the strength of the correlation;perform functional annotation and enrichment analysis of the genes in the associated regions.To compare the two mapping results between the SLAF-seq and the SRAP markers,the overlay region be deemed to as the candidate region.The physical location of the candidate region was confirmed by blast the reference genome of tomato.At last,the male sterile gene was mapped on second chromosome,which candidate region size was 0.18 Mb(44063750-44244438 bp),and which containing 30 candidate genes.(3)Based on the tomato reference sequence,RT-PCR primers were designed for 30 candidate genes,distinguishing the differential expression of these 30 candidate genes between male sterile and fertile plants during the flower development.Furthermore,genes which differential expression during the flower development were selected as candidate genes.Primers for the candidate genes silence fragment(300-600 bp)were designed,and the silence fragment of each candidate gene was amplified by RT-PCR.A TRV-silence vector of each candidate gene was constructed by restriction enzyme digestion-ligation or Gateway recombination technology.Based on the reverse genetics,the agrobacterium containing each silence vector of candidate gene was used to infect a wild type fertile plant during the flower development.Once appear the male-sterile phenotype,and the target gene of male sterility would be identified.The results showed there are 18 genes of 30 candidate genes different expression between the two lines at different development stages of flower organs,and there are no different expression of the 9 genes between the two lines at different development stages of the floral organs,and there are no expression of the 3 genes between the two lines at different development stages of the floral organs.When the candidate gene Soly02079810 was silenced among the 18 genes with the trait of different expression,the flower organ showed an sterile phenotype,when silence the other 17 candidate genes,there are no apparent phenotype in the flower organ.Therefore,the candidate gene Soly02079810 was identified as the male sterility gene ms-10;Then,cloned the sterility gene ms-10 and its allele Ms-10 by silico cloning,which found that the length of DNA sequence of ms-10 was 1002 bp,with 4 exonic regions and 3 intron regions,mRNA sequence of 627 nucleotides,encoding 209 amino acids.Sequencing analysis revealed there are no mutations in the coding region,only 1 SNP mutation in intron region.However,there is a 398 bp fragment insertion of ms-10 gene at the position of-151 bp in the promoter region.And the ms-10 was demonstrated its to be a typical bHLH transcription factors by bioinformatics,and its belongs to the StbHLH subfamily of bHLH transcription factors by cluster analysis.(4)The same ms-10 sterile line and wild-type fertile line were used as materials.The regulation network of sterile lines and fertile lines in the whole reproductive metabolism was studied at the protein level by the iTRAQ.The total number of identified proteins was 8734 and 8272 of them can be quantified.Frequency of identified polypeptides were 251435 and the number of identified polypeptides was 64481.The significantly up-regulated and down-regulated differentially expressed proteins in the bud and flower phases of male fertile line were 214 and 476,respectively;in the bud and flower phases of male sterile line were 91 and 360,respectively;in the bud phase between male sterile line and fertile line were 22 and 178,respectively;in the flower phase between male sterile line and fertile line were 53 and 205,respectively;in the two phases of bud and flower being all significantly up-regulated and down-regulated differentially expressed proteins between male sterile line and fertile line were 127 and 440,respectively;in the two line of male sterile and fertile line being all significantly up-regulated and down-regulated differentially expressed proteins between bud and flower phase were 27 and 131,respectively.GO analysis showed that the tomato male fertile line,which the differential proteins between bud and flower stage were mainly related to the molecular function processes such as oxidoreductase activity and catalytic activity.At the bud stage between the male sterile and fertile lines,the differential protein were mainly related to oxidoreductase activity and inositol biosynthesis process.The differential proteins between buds and flowers in male sterile line were mainly related to cell composition.The differential proteins between male sterile and fertile line at flower stages were mainly related to the functions of carboxylic acid metabolism and oxidoreductase activity.The KEGG pathway analysis showed that the enrichment degree of the major pathway was higher than that of the following,secondary metabolite biosynthesis,biosynthesis of tropic alkanes,piperidine and pyridine alkaloids,alpha-linolenic acid metabolism and nitrogen metabolism.The interactions between ms-10 protein and other proteins in tomato were analyzed on-line,and there are 9 protein genes were revealed that interact with ms-10 protein. |
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