Application of High Resolution Melting analysis for haplotype detection in phylogeographic research and case studies of Arenaria ciliata, A. norvegica and Minuartia recurva (Caryophyllaceae)

• Main Author: Dang, Xiaodong
• Format: Thesis
• Language: English
• Published: 2012
• Subjects: Biology; Arctic
• Online Access: https://mural.maynoothuniversity.ie/4325/; https://mural.maynoothuniversity.ie/4325/1/Xiao-Dong_Dang_PhD_NUIM_2012.pdf

The present study includes two aspects in the field of phylogeography. First, the technology of High Resolution Melting (HRM) analysis based on real-time PCR is introduced as a tool for haplotype detection in phylogeographic research. Second, phylogeographic study of three arctic-alpine or alpine plant species in the family Caryophyllaceae is carried out based on the haplotype data obtained through established protocol of HRM analysis. In Chapter 2, experimental protocols of HRM analysis combined with posterior DNA sequencing as a complementary and confirmatory method are established for haplotype detection in the case study of three species, Arenaria ciliata, A. norvegica and Minuartia recurva. Non-coding chloroplast DNA loci, rps16 intron (c.750bp for Arenaria and c.690bp for Minuartia) and trnT-trnL (c.640bp for Arenaria and c.540bp for Minuartia) are used in HRM analysis, where they are split into smaller (<400bp) amplicons for each real-time PCR reaction. The protocol is able to reveal 19 out of 20 haplotypes of rps16 and all of the 24 haplotypes of trnT-trnL in the case of Arenaria species, and to reveal all of the four haplotypes of rps16 and three out of eight (or five if variation in SSRs is not considered) haplotypes of trnT-trnL in the case of M. recurva. Posterior DNA sequencing reveals only one more haplotype with rps16 in the case of Arenaria species, which indicates a high sensitivity of HRM analysis in both cases. In Chapter 3 and 4, phylogeographic studies are carried out for the Arenaria species and M. recurva. Based on the haplotype identities and their distribution within and among the sampled populations, a complete phylogeographic study becomes possible. Maximum-likelihood phylogenetic trees and statistical parsimony networks are constructed among the haplotypes, and the genealogical relationship among the haplotypes is combined with their geographic distribution to understand the migratory history of the populations, population genetic analysis is made to understand the genetic ...