Transcriptomic Analysis And EST-SSR Molecular Markers Development Of Rhododendron Rex Subsp.Rex,An Endemic Plant In Southwest Of China

Rhododendron rex subsp.rex is a perennial woody species from the Ericaceae family and subgenus Hymenanthes.This plant is endangered and endemic to Southwestern China with high ornamental value(Fu 1992).It is mainly distributed in the southwest of Sichuan and northeastern Yunnan in China.The populations are becoming fragmented and declined due to long-term anthropogenic disturbance,and the wild populations have been severely damaged.To date,there are few research on the basic biology characterisits of R.rex subsp.rex reported.Transcriptomic and molecular marker development of R.rex subsp.rex is still very limited,which severly hindering its research on germplasm resources and genetic diversity studies of this species.Thus,based on the investigation of the population distribution of R.rex subsp.rex,transcriptome analysis,environmental adaptability and gene mining of chemical substance synthesis,EST-SSR and SNPs molecular markers development of R.rex subsp.rex were studied in this thesis.The purpose is to provide a scientific basis for the protection and exploitation of germplasm resources of this species.The major results and conclusions are as follows: 1.Transcriptome sequencing analysis in R.rex subsp.rexBased on the next-generation sequencing(NGS)technologies,fresh leaf samples from R.rex subsp.rex was sequenced using Illumina HiSeq4000 sequencing platform.The highquality reads were obtained and deposited into the U.S.National Center for Biotechnology Information(NCBI)sequence read archive(SRA)database(Raw Illumina reads: NCBI SRP108011).Blast algorithm(E < 1E-5)were used to compare with Nr,String,Swissprot,and KEGG database to obtain similarity sequence and the corresponding annotation information.Of the 164,242 unigenes,115,089 unigenes were successfully annotated in the databases,and 97,839(59.57%)unigenes were aligned to the Nr database,60600(36.9%),62018(37.76%),17247(10.5%),63183(38.47%)unigenes were assigned to theSwiss-Prot,GO,COG,KEGG databases,respectively.A total of 15314 potential SSRs were detected using the program MISA version 1.0,and 29,654 high-quality SNPs were detected by using Samtools.2.Gene mining related to cold stress and terpene,phenol,flavonoid biosynthesisThe assembly data of the R.rex subsp.rex transcriptome were compared with the angiosperm data in KEGG Automatic Annotation Server(HUhttp://www.genome.jp/kegg/kaas/UH)database,and we got the KO number of the unigene sequence.Then,the sequences that may be involved in specific biological pathways and rucial genes involved in the regulation of these pathways were also obtained.Finally,we analysised these gene information.A total of 147 cold-stress related potential genes from 2293 unigene sequences were found.57 candidate genes synthesized with terpenoids were obtained.17 candidate genes related to phenols and 17 candidate genes related to flavonoid synthesis were also determined in the present study.3.Development and Validation of New EST-SSR and SNPs MarkersA total of 15,314 potential EST-SSRs were identified from 12,188 unigenes,100 primer pairs were randomly selected and synthesized,45 produced the expected size of the amplified products.Using 20 R.rex subsp.rex individuals from four populations as PCR templates,the results showed that 36 primer pairs were polymorphic.A total of 36 pairs of primers were evaluated for transferability in its related species of the genus Rhododendron,the transferability ratios ranged from 58.33 to 83.33%.Furthermore,50 SNPs were randomly selected from 29,654 high-quality SNPs and 14 SNP loci showed bi-allelic polymorphism in 12 sampled individuals of R.rex subsp.rex.In summary,the R.rex subsp.rex transcriptome data was obtained using the Illumina HiSeq4000 sequencing platform.Novel gene resources related to cold stress and terpene,phenol,flavonoid biosynthesis of R.rex subsp.rex were determined in the presest study.In addition,the polymorphism EST-SSR and SNPs markers were development and validation,The results will provide an important scientific basis for the future studies on genetic diversity,genetic structure,environmental adaptation,protection and utilization of germplasm resources of R.rex subsp.rex.