Dang, Xiaodong (2012) Application of High Resolution Melting analysis for haplotype detection in phylogeographic research and case studies of Arenaria ciliata, A. norvegica and Minuartia recurva (Caryophyllaceae). PhD thesis, National University of Ireland Maynooth.
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Abstract
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 diversity and structure of the populations
and mismatch analysis is performed to understand demographic history of
the populations. In Chapter 3, deeply diverged clades are revealed which
are not in accordance of either subspecies or geographic localities. The
data indicate a much older establishment of the populations of A. ciliata
on Ben Bulben in northwest Ireland than thought before, possibly as early
as 150-250 thousand years ago. Thus these Irish populations may have
survived the last ice age in situ, rather than having immigrated after the
end of Pleistocene (c. 12, 000 years ago). However, the result shows that
the Irish populations are more closely related to the Iberian populations
than they are related to the Alps populations. In Chapter 4, much lower
level of genetic polymorphisms is revealed in the M. recurva populations,
although they cover a European range comparable to that in the Arenaria
case. The Balkans region is suggested as the refugium for the species,
while little variation is found across the species distribution from the Alps,
the Pyrenees, the north side of Spain and Ireland, which indicates a recent
dispersal of the species in west Europe. Also a close relationship is
suggested between the Irish populations and the Iberian populations.
In Chapter 5, a further theoretical assessment of possibility of missed
detection in HRM analysis is carried out via in silico HRM simulation.
Based on an amplicon of rps16I in A. ciliata, random mutations are made
and HRM sensitivity is evaluated to different classes of single nucleotide
substitutions. Class I and II class I (A/G or C/T) and II (A/C or G/T)
substitutions are demonstrated easier to be detected than class III (C/G)
and IV (A/T) substitutions. Further analyses suggest that between 50 and
650bp, amplicons of greater sizes are more likely to yield multiple
melting peaks, which is favourable for higher sensitivity in HRM analysis.
Between 100 and 550bp when all the amplicons render double melting
peaks, amplicons of greater sizes tend to provide lower sensitivity in
HRM analysis. Amplicons smaller than 350bp with double melting peaks
are considered to generate an acceptable rate of missed detection
(<10-20%). In addition, in silico HRM analysis is tested with available
DNA sequences of mitochondrial and chloroplast loci from published
phylogeographic studies, and is demonstrated to help distinguish most of
the extant haplotypes, although with a proportion of haplotypes missed
(typically 10-20%). The results provide information for possible
improvements of HRM analysis to be widely applied for haplotype
detection in phylogeographic research.
Item Type: | Thesis (PhD) |
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Keywords: | High Resolution Melting analysis; haplotype detection; phylogeographic research; Arenaria ciliata; A. norvegica; Minuartia recurva; Caryophyllaceae; |
Academic Unit: | Faculty of Science and Engineering > Biology |
Item ID: | 4325 |
Depositing User: | IR eTheses |
Date Deposited: | 19 Apr 2013 11:29 |
URI: | https://mural.maynoothuniversity.ie/id/eprint/4325 |
Use Licence: | This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here |
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