Transcriptome Analysis And Development Of EST-SSR Markers In Rosa Roxburghii Tratt

Rosa roxburghii Tratt.(Rosaceae) is a perennial rosebush native to China. The fruits of this species are valued for their nutritional and medicinal characteristics, especially their high ascorbic acid(As A) levels. Now it is an important industry in Guizhou province. Genomic information is currently unavailable for R. roxburghii, the researches on genetic background and specific molecular markers development of R. roxburghii are still lack. All these lead to the progress of its researches on functional metabolism, resources and genetic breeding lagging behind that of many other important crops. In this study, we generated a normalized c DNA library prepared from R. roxburghii fruits and established a substantial EST dataset using high-throughput Illumina RNA sequencing. Using these data, we analyzed the R. roxburghii fruit transcriptome and developed a set of SSRs. Then the SSRs were used to analyze the genetic diversity within R. roxburghii germplasms and the genetic differentiation of natural populations of wild R. roxburghii from Guizhou province. The main results are as follows:1. The total c DNA library prepared from the fruit of R. roxburghii was sequenced using an Illumina Hi Seq 2000 platform, resulting in 53 535 304 clean reads. De novo assembly yielded 106 590 unigenes, with an average length of 343 bp. On the basis of sequence similarity to known proteins, 9 301 and 2 393 unigenes were classified into GO and COG categories, respectively. There were 7 480 unigenes assigned to 124 KEGG pathways. Among them, 20 Unigenes were mapped to ascorbate and aldarate metabolism pathway. The remaining unigenes with no homologs in Nr, Swiss-Prot, KEGG, and COG databases were analyzed using ESTScan, which identified 8 855 putative protein coding unigenes. 498 unique putative transcripts were identified as transcription factors(TFs) or regulators belonging to at least 16 different families in the R. roxburghii transcriptome data. Through transcriptome sequencing, we obtained a lot of functional genomic information for R. roxburghii for the first time.2. 109 590 unigenes were screened using MISA software. A total of 21 711 SSRs that occurred in 18 155 unigenes were identified,and the frequency of these SSRs was 20.37% and mean distance was 1.68 kb in the unigenes. Trinucleotide, tetranucleotide and dinucleotide were major types, accounting for 26.87%, 26.77% and 24.93%, respectively. AG/CT, AAG/CTT were respectively most frequent motifs in dinucleotide and trinucleotiderepeats, accounting for 17.80% and 11.55%, respectively. Using t he Primer 3.0, 102 primers were designed and synthesized based on different dominant motifs types, and verified with R. roxburghii germplasms for validity and Rosa germplasms for transferability. The results showed that the products of 71 primers are clear and effective, 71 pairs could be transferable to Rosa germplasms and 31 pairs were polymorphic among the 16 R. roxburghii germplasms. Large amounts of effective SSR markers for R. roxburghii were obtained for the first time. And the SSR markers also enriched the molecular markers for Rosa.3. 10 primers were used to analyze the genetic diversity within 256 R. roxburghii germplasms and 7 Rosa germplasms. Based on the UPMGA method, the accessions were classified into 4 groups with the distance index at 0.30. The range of distance index in 256 R. roxburghii germplasms was from 0 to 0.6916, suggesting that abundant genetic diversity existed. Construction of core collections showed that 8 materials of R. roxburghii core collections retained 92.5% alleles of 256 original collections and utmost genetic diversity of the original whole germplasm with 3.23% of the sampling ratio. Furthermore, genetic diversity and genetic structure of 12 wild R. roxburghii populations were investigated. The range of Shannon’s information index(I) was from 0.62 to 0.88, Nei’s gene diversity from 0.40 to 0.52. Total percentage of polymorphic loci(P) was 100% in these populations, except for Fuquan and Qinglong population. These indicated that there was a high level in genetic diversity among the 12 populations of R. roxburghii. The Chi-square test results argued that the 12 populations of R. roxburghii did not follow with the Hardy-Weinberg Equilibrium at most loci, and the Fit and Fis value was negative, all of which indicated that heterozygosity excess existed in those 12 populations. The mean Fst was 0.0403, which demonstrated that genetic differentiation mainly occurred inner-population. Futhermore, Mantel test revealed a significant correlation between genetic and geographic distance among populations. Characteristics, such as strong gene flow(Nem = 5.9484), high genetic identity(GI > 0.9068) and low genetic distance(GD < 0.0978) were discovered among 12 populations.