Dataset Information

A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae).

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. • METHODS:Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compared interspecific variation between cpSSRs and nucleotide sequences of ycf1 and tested intraspecific variation for cpSSRs using 911 samples in the P. ponderosa species complex. • RESULTS:The cpSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions, and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. • DISCUSSION:The protocol allows efficient genotyping of many samples. The cpSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for cpSSR loci in a plastome alignment for the target group.

SUBMITTER: Wofford AM

PROVIDER: S-EPMC4103111 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae).

Wofford Austin M AM Finch Kristen K Bigott Adam A Willyard Ann A

Applications in plant sciences 20140430 5

<h4>Unlabelled</h4><h4>Premise of the study</h4>Recently released Pinus plastome sequences support characterization of 15 plastid simple sequence repeat (cpSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. •<h4>Methods</h4>Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We ...[more]

PMID: 25202625

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Project description:BACKGROUND AND AIMS:Pinus kwangtungensis is a five-needled pine, inhabiting isolated mountain tops, cliffs or slopes in the montane areas of southern China and northern Vietnam. Global warming and long-term deforestation in southern China threaten its existence and genetic integrity, and this species is listed as vulnerable in the China Species Red List. However, the level and distribution of genetic diversity in this vulnerable species are completely unknown. In this paper, the genetic diversity and structure are examined using paternally inherited plastid markers to shed light on its evolutionary history and to provide a genetic perspective for its conservation. METHODS:By means of direct sequencing, a new polymorphic fragment containing a minisatellite site was identified within the plastid genome of P. kwangtungensis. Using the minisatellite site along with five SNPs (one indel and four substitutions) within the same fragment, the population genetic structure and pollen flow were analysed in 17 populations of P. kwangtungensis in southern China. KEY RESULTS:Analysis of 227 individuals from 17 populations revealed ten haplotypes at the minisatellite site. The haplotype diversity at species level was relatively high (0.629). Genetic diversity of each population ranged from 0 to 0.779, and the western populations harboured more genetic variation than the eastern and Hainan populations, although the former appeared to have experienced a bottleneck in recent history. Population subdivision based on this site was high (F(ST) = 0.540 under IAM; R(ST) = 0.677 under SMM). Three major clusters (eastern, western and Hainan) were identified based on a neighbor-joining dendrogram generated from genetic distances among the populations. The genetic structures inferred from all the polymorphic sites and the SNPs were in concordance with that from the minisatellite site. CONCLUSIONS:The results suggest that there are at least three refugia for P. kwangtungensis and that populations in these refugia should be treated as separate evolutionarily significant units or conservation units. The high diversities in the western populations suggest that these were much larger in the past (e.g. glacial stages) and that the shrinking population size might have been caused by recent events (e.g. deforestation, global warming, etc.). The western populations should be given priority for conservation due to their higher genetic diversity and limited population sizes. It is concluded that the newly found minisatellite may serve as a novel and applicable molecular marker for unravelling evolutionary processes in P. kwangtungensis.

Project description:BackgroundThrough next-generation sequencing, the amount of sequence data potentially available for phylogenetic analyses has increased exponentially in recent years. Simultaneously, the risk of incorporating 'noisy' data with misleading phylogenetic signal has also increased, and may disproportionately influence the topology of weakly supported nodes and lineages featuring rapid radiations and/or elevated rates of evolution.ResultsWe investigated the influence of phylogenetic noise in large data sets by applying two fundamental strategies, variable site removal and long-branch exclusion, to the phylogenetic analysis of a full plastome alignment of 107 species of Pinus and six Pinaceae outgroups. While high overall phylogenetic resolution resulted from inclusion of all data, three historically recalcitrant nodes remained conflicted with previous analyses. Close investigation of these nodes revealed dramatically different responses to data removal. Whereas topological resolution and bootstrap support for two clades peaked with removal of highly variable sites, the third clade resolved most strongly when all sites were included. Similar trends were observed using long-branch exclusion, but patterns were neither as strong nor as clear. When compared to previous phylogenetic analyses of nuclear loci and morphological data, the most highly supported topologies seen in Pinus plastome analysis are congruent for the two clades gaining support from variable site removal and long-branch exclusion, but in conflict for the clade with highest support from the full data set.ConclusionsThese results suggest that removal of misleading signal in phylogenomic datasets can result not only in increased resolution for poorly supported nodes, but may serve as a tool for identifying erroneous yet highly supported topologies. For Pinus chloroplast genomes, removal of variable sites appears to be more effective than long-branch exclusion for clarifying phylogenetic hypotheses.

Dataset Information

A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae).

Publications

A set of plastid loci for use in multiplex fragment length genotyping for intraspecific variation in Pinus (Pinaceae).

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