Comparative Transcriptome Analysis Of Male And Female Flowers And Identification Of Gender-related MiRNA Targets In Asparagus Officinalis L.

Sex determination is an important developmental process that leads to distinction and separation of male and female reproductive organs into different individuals in flowering plants. The modes of sex determination are usually unstable and genetically diverse. Plant sex chromosomes, carring with sex determination genes, play leading roles in the process of sex determination. The micro RNAs (miRNAs) can participate in plant sex determination through regulating the expression levels of sex determination genes. Apart from this, some external and internal factors including environmental conditions and plant hormones may also affect the sex expression in plants. However, researches on the gene regulatory network of plant sex expression are still lacking at present. Asparagus is an economically and nutritionally important vegetable crop cultivated worldwidely. It is also regarded as a typical dioecious plant with its sex chromosomes at the early evolution stage, making it very suitable for the study of sex chromosome evolution and gender origin. So asparagus is also regarded as one of the most primary model system for plant sex determination studies. In this study, the transcriptome profiles between asparagus male and female flowers at different developmental stages were compared using RNA-Seq on the basis of morphological and cytological analysis. Thus some important genes and miRNA targets relating to sex determination and flower differentiation in asparagus were discovered and identified. Furthermore, some plant hormones and sugars were verified to regulate sex determination in asparagus using molecular and physiological methods. The results provide important theoretical bases for revealing the molecular mechanism of plant sex determination system regulation, and it also has some practical significance for artificial control of sex expression, breeding of whole-male asparagus varieties, and improvement of asparagus yield and quality. The main results are summarized as follows.(1) Using asparagus variety "Grande", the development process of male and female flowers were compared by stereoscopic microscope and SEM to determine the transition stage from bisexual flower primordium to unisexual flower. It indicated that male and female flowers with the length of 2.0 mm could be distinguished by sexual organs. Furthermore, transcriptomes of male and female flowers at three representative stages (0.5 mm,2.0 mm and 4.0 mm) before, during and after the sex determination were obtained by RNA-seq. As a result,93,658 unigenes were firstly assembled into a asparagus cDNA database, among which 4,130 unigenes had significantly different expression levels between male and female flowers at one or more than one stage, and the differentially expressed genes were mainly distributed in the last two stages, when the male and female flowers showed a significant difference in morphology. Furthermore,17 gender-biased genes related to floral development were identified according to their expression patterns, among them the transcription factors related to the development of stamen and pistil were suggested to be involving in sex determination. Four genes with up regulated expression in male flower and down regulated expression in female flower were screened out by analyzing the changes of gene expression level along with the floral development, their opposite expression changes in male and female flowers suggested their vital roles for male organ development. Furthermore,390 SNPs were firstly identified between male and female flowers, which were distributed into 343 unigenes, they will provide a valuable resource for developing molecular markers linked to sex determination gene on benefit on the breeding of whole-male varieties, as well as construction of genetic map in asparagus.(2) Functional classification of differentially expressed genes (DEGs) between male and female flowers was performed by GO analysis and pathway analysis. As a result,64 DEGs were proved to be involved in the biosynthesis and degradation of plant hormones and sugars, suggesting that plant hormones and sugars may participate in the regulation on the sex determination and differentiation. Among them,29 unigenes coding for key enzymes involved in the biosynthesis and degradation of hormones and sugars were screened out, and their expression patterns between male and female flowers were characterised in more depth and verified by qRT-PCR, which indicated that the expression level of these genes related to glucose, sucrose and starch metabolism would change during the process of male and female floral development, and the synthetase genes of gibberellin, cytokinin, as well as BR showed male-biased, while synthetase genes of auxin and JA showed female-biased. In order to further verify the distribution patterns of endogenous hormones in asparagus male and female flowers, the content of GA3, IAA and JA were determined through HPLC-MS and ELISA method, which showed that the content of GA3 was higher in male flower, whereas the contents of IAA and JA were higher in female flower. It indicated that the expression changes of some important enzyme genes could affect the accumulation of plant endogenous hormone, thus regulate asparagus sex determination and differentiation.(3) Target genes of some miRNAs with differential expression level between male and female asparagus plants were predicted using bioinformatics methods in the unigene database constructed by RNA-seq. As a result,206 potential target genes of 27 miRNAs were obtained. Quite a number of the predicted targets were important transcription factors controlling plant growth and development, and 24 of the predicted targets showed differential expression levels between male and female flowers, which were suggested to involve in asparagus male and female flower differentiation. The predicted targets of four miRNAs were verified by performing the RLM-5’RACE experiment, which discovered one or two miRNA-mediated splice sites on the mRNA of target gene. In order to screen out some miRNAs related to sex differentiation in asparagus, the expression pattern between male and female flowers at different stages of the differentially expressed miRNAs between male and female asparagus plants, with their targets, were analyzed by qRT-PCR, which indicated that miR160d was up regulated in female flower and negatively regulated the expression of it’s target gene, ARFs, while miR396f was up regulated in the middle stage of male flower and negatively regulated it’s target gene GRFs, suggesting that these two miRNAs were probably involved in the regulation of the asparagus sex determination and differentiation, Combined with RNA-seq results, we can speculate that the miR160d could achieve its function by influencing the auxin signal transduction.