Research On Transcriptome Sequencing And Mining Of SSR Markers Of Osmanthus Serrulatus Rehd.

Osmanthus serrulatus, a member of genus Osmanthus, is one of the rare fragrant trees blooming in April and occupies a narrow distribution in China, which bears a broad develpoment prospect and an enormous conservation value. In this study, RNA-seq was performed for flowers and leave buds of Osmanthus serrulatus using Illumina paired-end sequencing. Abundant transcriptome resources were achieved and comprehensively analyzed in bioinformatics ways. SSRs mining and SSR primer pairs develpoment were also performed. The main results are as followed:(1) Transcriptomes from two tissues were sequenced based on Illumina HISeq 2000 platform through paired-end sequencing. By using Trinity, the cleand raw reads were assembled into 189,993 transcripts and 92,798 unigenes with an average length of 1,169 and 697 bp, N50 2,017 and 1,200 bp, respectively, which indicated a good outcome by RNA-seq and de novo assembly.(2) All unigenes were assigned to 7 public databases, namely Nr, Nt, Swiss-Prot, Pfam, GO, KOG, KO and it turned out that 3,783(4.07%) unigenes were successfully annotated in all 7 databases. The unigenes compared to GO were divided into 3 categories including 46 biological function terms; 26 groups were involved in KOG, in which General function prediction only constituted the biggest share; 5 branches including 262 pathways were orgnized into KEGG, and 337 unigenes were searched out associcating pathways of pigment and frgrance biosythensis.(3) 64,576 CDSs were obtained altogether, 31,925 from comparing to the protain databases and 32,651 by Estscan software.(4) In total, 4,306 SSRs distributed in 4,189 SSR-containing sequences were identified throughout 92,798 unigenes in the transcriptome, with the frequency of 4.64% or 1/15.02 kb. Dinucleotide SSRs were the most abundant repeat type, and SSR abundance showed negative correlation with the size of repeat motif excluding hexanucleotide repeats; AG/CT(41.94%) was the most dominant within 61 kind of repeat motifs followed by AC/GT(14.40%), AT/TA(9.71%), AAG/CTT(9.24%), AAT/ATT(5.99%), and AG/CT, AAG/CTT were the most frequent repeat motif in dinucleotide and trinucleotide repeats, respectively. The length of 4,306 SSRs ranged from 12 to 120 bp with an average length of 16 bp, negative relation also existed between the frequency and the size of all SSRs; Furthermore, SSRs in shorter repeat motifs with longer length were predicted with higher polymorphism.(5) 2,366 SSR primer pairs were designed based on 4,189 SSR-containing unigenes using primer 3, and the success rate was 56.48%.(6) 50 primer pairs were randomly selected to validate the amplifications and to determine the degree of polymorphism in the genomic DNA pools. Results revealed that 28 primer pairs were successfully amplified with the precentage of 56%, in which 19 generated PCR amplicons at the expected size, 7 longer than expected and 2 shorter than expected. And 10 PCR products presented polymorphism, taking up 20% of all selected primer pairs.