SNPs Development And Genetic Diversity Of Stipa Breviflora Population

Biodiversity is the base of survival and development of human beings, the research and protection of biodiversity has been one of the most hot topics in the world. Biodiversity is not only the source of adaption and evolution, but the essential nature resource. Therefore, the study of biodiversity plays an indispensible role in the theoretical knowledge and practical application. Our experiment samples came from 8 Stipa breviflora populations (30 individuals of each population), which were collected from Inner Mongolian Plateau, Ordos Plateau, Alxa Plateau, Loess Plateau and Qinghai-Tibet Plateau. We sent 8 samples, coming from 8 different populations, to conduct transcriptome sequencing. By transcriptome sequencing, analyzing the results of sequencing, identifying SNP sites, designing the primers of SNP, utilizing the PCR to filter and amplify the primer,we developed SNPs. At last, we used these SNPs to illuminate the genetic diversity and genetic structure of the S. breviflora population and to explore the related influence factors. The main results of our study are as follows:1. We succeeded to construct relatively completed transcriptome data set of S. breviflora. There were 601116112 raw reads in total, which mean length was 150bp from 8 samples. After filtering the low quality reads, we got 551569834 clean reads. There were 82735475100 high quality basic groups, taking the 91.76 percents of the total sequencing data, which contained the number of GC reaching to 54.54%. At the same time, we got 178901 Unigenes that mean length was 1235bp, among which 128777 Unigenes acquired annotation results. Our research identified 493411 SNP sites in total. The number of transition was 321887 and the number of transversion was 171524, the number of transition was nearly as twice as transversion.2. Our research designed 26 pairs of SNP primers and established the best reaction system of SNP-PCR to S. breviflora (25μL).The reaction system was as follows: 30ng/μL template DNA 1μL, 10μM each primer 0.5μL, Premix TaqTM 12.5μL and ddH2O 10.5μL. PCR amplification program of better repeatability and stability were set up after a pre-degeneration 3 min at 94℃; degeneration 30s at 94℃; annealing and extension,35 cycles from degeneration to extension in total; then extension 10 min at 72℃, finally conserved at 4℃. Through conventional sequencing,7 pairs of SNP primers were with good amplification; through conventional sequencing, sequencing results were screened out, which including 56 SNPs used for genetic diversity analysis.3. The genetic diversity of S. breviflora population is medium level or slightly lower. On the species level, the expected heterozygosity was 0.1308, Nei’s genetic distance was 0.1306, and the 91.49% genetic variation came from individuals within populations. On the population level, the expected heterozygosity was 0.122, the gene flow was 2.504, which indicated that there existed frequent gene flow between the populations.4. There was significant positive correlation between geographic distances and genetic distances among pairwise populations. The 8 populations of S. breviflora could be divided into 4 groups, and the genetic variation among populations took relatively small proportion.This study laid foundation for the further analysis about genetic diversity, gengtic structure and the fluctuation of population dynamics of S. breviflora.