High-throughput Sequencing Analysis And Development Of SSR Molecular Markers In The Transcriptome Of Tirpitzia Sinensis

Tirpitzia sinensis has very good ornamental value and medicinal value,but its relative lack of genomic information has limited its research on molecular biology and gene function.In order to obtain and mining Tirpitzia sinensis genetic data and functionality,this study first used a new generation of high-throughput sequencing technology platform Illumina Hi SeqTM2000 for Tirpitzia sinensis transcriptome sequencing,received 66755 Unigene after De novo assembly.27935 Unigene were annotated by a similarity search against seven dadabases.The results showed that 558 genes were assigned to second metabolic pathway.Among them,2 Unigenes were mapped to the betalain,18 to glucosinolate,44 to flavonoid,70 to monobactam,231 to phenylpropanoid biosynthesis pathways,These genes are likely to be involved in the biosynthesis of medicinal active substances in the Tirpitzia sinensis.The Unigene gene structure analysis was performed on the sequencing results.A total of 69,388 Unigenes obtained CDS predictions.Meanwhile,transcription factors prediction and analysis will provide preliminary insights into mechanisms of Tirpitzia sinensis for Karst environmental adaptation.To explore the SSR loci information in the Tirpitzia sinensis transcriptome,develop the molecular marker technology,SSR locus data mining for all Unigenes using bioinformatics analysis software MISA.Overall,22,542 SSRs distributed in 18,972 Unigenes were detected,accounting for 33.77% of all Unigenes.The average length of the transcriptomic SSRs was 15 bp,and the distribution distance 3.98 kb.The mononucleotide repeats?the trinucleotide repeats and the dinucleotide were the main types.accounting for 47.59%?27.95% and 23.45%.respectively,of all the SSRs,There were 161 kinds of repeat motifs in Tirpitzia sinensis transcriptome,with A/T?GA/TC?AG/CT?AT/AT?G/C and GAA/TTC as the most frequent motifs,The repeated sequence length was between 10~144bp,mostly smaller than 24 bp,and only 0.22%(48 SSR sites)were greater than 24 bp.In order to verify the feasibility of these SSR loci,50 pairs of primers were designed in this experiment,and 20 Tirpitzia sinensis materials from four regions of Dushan,Libo,Pingtang,Huishui,and Changshun were used.The leaf DNA was extracted and amplified by PCR.Eight pairs of primers with obvious polymorphic bands were screened and used for the genetic evolution analysis of different regions of Tirpitzia sinensis species.The acquisition of the transcriptome bioinformation of the Tirpitzia sinensis provided the basis for the research on the molecular level of the Tirpitzia sinensis.The results will provide a basis for the cloning of gene,gene expression and genetic diversity.