| Veratrilla baillonii Franch, a perennial herb belonging to Gentianaceae genus Veratrilla, is limited distributed in the highlands above 3600 m in the eastern Himalayas and Hengduan Mountains region. It is a good material to study the genetic differentiation of sky-island plants. In addition, V. baillonii is an important medical herb and is collected extensively in some regions, so understandings on the genetic structure of this species would be helpful for effective conservation. However, in recent years, most studies on V. baillonii are focused on the biological characteristics, medicinal value and effective chemical constituents, but the genetic diversity remains unclear. In this thesis, I conducted RNA-seq of V. baillonii, with the aim to find SSR primers with microsatellite searching software. Based on this result, genetic diversity was analyzed in 18 populations of V. bailloni.The main results are as follows:After filtering and splicing the raw reads, we got 133,019 contigs and the average length was 1263 bp. N50 was 2104 bp. By using the microsatellite searching software, 40,885 microsatellite sequences were identified. We designed and synthesized 151 pairs of SSRs primers and 74 of them could be amplified successfully. Besides, 14 pairs of those amplified SSRs primers were polymorphic. Finally, we selected 11 pairs of primers(HQJ19、HQJ34、HQJ37、HQJ40、HQJ45、HQJ56、HQJ63、HQJ79、HQJ99、HQJ103、HQJ115)to examine the genetic diversity of V. baillonii samples from 273 individuals of 18 populations.Among the 11 microsatellite primers, We detected 131 alleles, and the alleles(Na) ranged from 6 to 24 with a mean value of 11.9. Effective number of alleles(Ne) was from 1.313 to 3.053 and the mean value was 2.066. Observed heterozygosity(Ho) varied between 0.143 and 0.849, with a mean value of 0.397. Expected heterozygosity(He) changed from 0.182 to 0.614, which mean value was 0.397.At the population level, the average numbers of Na and Ne for each population were 3.202 and 2.066.(Ho) and(He) were 0.374 and 0.397, respectively. The highest genetic diversity was found in Jiulong county(JL population), Sichuan province(Na=4.723, Ne=2.406, H_O=0.437, H_E=0.500 and PPB=100%). In contrast, the lowest level of genetic diversity(Na=1.818, Ne=1.468, H_O=0.209, H_E=0.231 and PPB=100%) was found in Lixian county(LX population), Sichuan province. The average genetic differentiation value(Fst), and gene flow value(Nm) were 0.283 and 0.404, respectively. The results of AMOVA analysis showed that the major genetic variation of V. baillonii existed within population(58.89%) rather than among populations(41.11%).The UPGMA and STRUCTURE clustering analysis suggested that the closely distributed populations were not aggregated in a clade, and the results were corresponded with Mantel Test(r=0.197, p=0.106).Collectively, these results showed a relatively middle level of genetic diversity in V. baillonii, which migh result from genetic drift or bottleneck effect. The sky-island distribution pattern of V. baillonii could have incurred the high level of genetic differentiation within populations, and this might be facilitated by the breeding system of this species. |
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