Based On The Transcriptome Analysed The Differences Between Female And Male And Explored Method Of Sex Discrimination Of Fraxinus Mandshurica Rupr

Fraxinus mandshurica Rupr belongs to the Oleaceae Fraxinus,it is dioecious woody plants.In order to analyse the difference between female and male,study the sex identification method preliminary,this experiment sequenced and obtained the transcriptome of Fraxinus mandshurica Rupr.We annotated difference gene function between female and male,got larger range pathway,obtained sex related difference genes between female and male.At the same time,based on the transcriptome data,detected SSR and SNP loci of Fraxinus mandshurica Rupr,got primers based on sites difference between female and male,revealed the difference between female and male,these established foundation for breeding and founding seed orchard.The main results are as follows:(1)We obtained the transcriptome database of Fraxinus mandshurica Rupr flowers,contained 90,532 sequences,all could be compared to known database.25,961 genes obtained GO annotation,10,879 genes obtained KOG annotation,and 9,084 genes obtained KEGG annotation.Which consisted 330 carbon metabolism related genes(3.62%),326 amino acid biosynthetic related genes(3.37%),280 plant hormones transduction related genes(3.08%),108 circadian related genes(1.19%),35 nitrogen metabolism related genes(0.39%),31 flavonoids metabolism related genes(0.34%).(2)We screened and obtained 2,930 differentially expressed genes between female and male flowers,included 1,441 superiority expression in female,and 1,489 superiority expression in male.Larger range pathways included biosynthesis of secondary metabolites,plant hormone signal transduction pathway,proteins in the endoplasmic reticulum,and glycolysis/gluconeogenesis pathway.The development of female and male and sex related pathways,also showed differences between female and male,included flavonoids biosynthesis,nitrogen metabolism,carbon metabolism and and light cycle.(3)Through analysis of RT-PCR,we obtained 10 genes which were related to sex differentiation and development,these genes played an important role in these process,included 4 superiority expression genes in female,6 superiority expression genes in male.In hormone signal transduction pathway,included AUX/IAA(2.02 times).A-A RR(-26.5 times)、EIN3(8.4 times)and ABF(1.2 times).In.Nitrogen metabolism pathway.In included GS(-1.6 times)and GDH(-9.1 times).In flavonoids synthesis pathway.included FLS(-6.2 times).In photoperiod pathway,included CaM(-8.7 times)、PKA(-13.1 times)and CRY(10.4 times).(4)Based on transcriptome sequencing of Fraxinus mandshurica Rupr,we searched SSR site,we also searched SSR in Fraxinus EST sequences from public database NCBI.We obtained 3,557 and 9,488 SSR sites,respectively.The frequency of SSR were 13.87 kb and 0.32 kb,respectively.The predominant type is dinucleotide by 58.05%and 72.87%,respectively.We designed 232 SSR primers,and 208(89.66%)pairs could be amplified effectively.After three rounds of PCR verification,FM56 and FM19 primers from the transcriptome got difference between female and male,the rates were 89.5%and 64.5%.(5)Based on the transcriptome,we got 214,367 SNP sites.included 5,104 female specific SNP sites and 6,532 male specific SNP sites.We designed 60 pairs of SNP primers,amplification rate was 100%.SNP test rate is 91.67%.The SNP site FM65697,the difference mutation rate was 74.21%between female and male.