| Asparagus officinalis is a highly nutritious crop,and it is also a model plant for the study of sex-determining mechanisms.However,it is still not clear about the molecular mechanisms of sex determination,especially their sex differentiation in unisexual flower plants and developmental genes.In this study,based on the transcriptome high-throughput sequencing data,using bioinformatics,RT-PCR,real-time quantitative PCR,RACE and tissue in situ hybridization,we analyzed the male and female differentially expressed genes in asparagus.Three sex-related genes were cloned and their expression patterns were studied in flowers.Below are main research findings:1.By using High-throughput transcriptome sequencing with Illumina paird-end sequencing technique,a total of over 260 G high quality data were obtained of from flower buds of male and female asparagus.Totally,72,626 unigenes with an average length of 979 bp were assembled.In the transcriptome analysis of female and male / super-male flower buds,4876 differentially expressed genes(DEGs)were identified in the possible sex determination stage.Of these DEGs,a total of 433 gender-biased gene annotations were associated with flower development,including 285 male / super-male and 149 female-biased genes.Of the male / super-male genes,102 may be involved in anther development.In addition,43 DEGs were found to be involved in gender expression and reproduction;128 are transcription factors(TFs)belonging to various families.These results can provide a valuable basis for further investigation of the sex determination and differentiation regulatory networks in asparagus and to facilitate further genetic and genomic research.2.In order to verify the reliability of the sex-differentially expressed genes,RT-PCR and Q-PCR analysis of 30 sexually-differentially expressed genes were performed on roots,stems,flowers and leaves.The RT-PCR and Q-PCR results of 26 sex-differentiated genes were consistent with the sequencing results,including 20 males and 6 females,of which 8 genes were only expressed in male flowers.The other12 genes were specificall expressed in female individuls,with high expression in flowers and low expression in other tissues.Six genes were expressed in both male and female flowers,and the expression level in female flowers was much higher than male ones.3.In order to further obtain the full cDNA sequence of the genes related to sexual development,we used RACE technique to select 5 of 26 stably differentially expressed genes for full-length gene cloning.The results showed that three of the five genes were successfully cloned and named as AoM1,AoM2 and AoM10,respectively.AoM1,AoM2 and AoM10 encode 141,382 and 532 amino acids,respectively.BLAST analysis showed that AoM2 encodes type III polyketide synthase B-class and AoX10 encodes carbohydrate carrier C-class.Associated with the newly published Asparagus genomic sequence,the AoM2 gene was located on chromosome 4,consisted of 5 exons and 6 introns.The full-length gene was 382 bp.The AoM10 gene is located on chromosome 5 and includes 5 introns and 6 exons,with a total length of 532 bp.4.Using tissue in situ hybridization,the tissue expression patterns were analyzed on male and female asparagus flowers.The experimental results showed that the AoM1 gene had a hybridization signal only in the male flower primordium,and the signal was stronger in the pistil primordium and stamen primordium of the male flower.The AoM2 gene showed a significant hybridization signal between the pistil primordium and the stamen primordium of the male flowers,a signal was also found on the receptacle of the female flowers,and a weak signal was observed on the stamens and stamens’ primordium of female flowers.The signal intensity of AoM10 in male flowers is greater than that of female flowers,and their signals are focused on the pistil and stamen primordium of flowers. |
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