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Microsatellite primers identified by 454 sequencing in the floodplain tree species Eucalyptus victrix (Myrtaceae).

ABSTRACT: UNLABELLED: PREMISE OF THE STUDY:Microsatellite primers were developed for Eucalyptus victrix (Myrtaceae) to evaluate the population and spatial genetic structure of this widespread northwestern Australian riparian tree species, which may be impacted by hydrological changes associated with mining activity. • METHODS AND RESULTS:454 GS-FLX shotgun sequencing was used to obtain 1895 sequences containing putative microsatellite motifs. Ten polymorphic microsatellite loci were identified and screened for variation in individuals from two populations in the Pilbara region. Observed heterozygosities ranged from 0.44 to 0.91 (mean: 0.66) and the number of alleles per locus ranged from five to 25 (average: 11). • CONCLUSIONS:These microsatellite loci will be useful in future studies of population and spatial genetic structure in E. victrix, and inform the development of seed sourcing strategies for the species.

SUBMITTER: Nevill PG

PROVIDER: S-EPMC4105038 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Microsatellite primers identified by 454 sequencing in the floodplain tree species Eucalyptus victrix (Myrtaceae).

Nevill Paul G PG Bradbury Donna D Jørgensen Tina T Krauss Siegfried S Samaraweera Sunil S Gardner Michael G MG

Applications in plant sciences 20130430 5

<h4>Unlabelled</h4><h4>Premise of the study</h4>Microsatellite primers were developed for Eucalyptus victrix (Myrtaceae) to evaluate the population and spatial genetic structure of this widespread northwestern Australian riparian tree species, which may be impacted by hydrological changes associated with mining activity. •<h4>Methods and results</h4>454 GS-FLX shotgun sequencing was used to obtain 1895 sequences containing putative microsatellite motifs. Ten polymorphic microsatellite loci were ...[more]

PMID: 25202545

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Project description:BACKGROUND AND AIMS:Few phylogeographic studies have been undertaken of species confined to narrow, linear coastal systems where past sea level and geomorphological changes may have had a profound effect on species population sizes and distributions. In this study, a phylogeographic analysis was conducted of Eucalyptus gomphocephala (tuart), a tree species restricted to a 400 × 10 km band of coastal sand-plain in south west Australia. Here, there is little known about the response of coastal vegetation to glacial/interglacial climate change, and a test was made as to whether this species was likely to have persisted widely through the Last Glacial Maximum (LGM), or conforms to a post-LGM dispersal model of recovery from few refugia. METHODS:The genetic structure over the entire range of tuart was assessed using seven nuclear (21 populations; n = 595) and four chloroplast (24 populations; n = 238) microsatellite markers designed for eucalypt species. Correlative palaeodistribution modelling was also conducted based on five climatic variables, within two LGM models. KEY RESULTS:The chloroplast markers generated six haplotypes, which were strongly geographically structured (GST = 0·86 and RST = 0·75). Nuclear microsatellite diversity was high (overall mean HE 0·75) and uniformly distributed (FST = 0·05), with a strong pattern of isolation by distance (r(2) = 0·362, P = 0·001). Distribution models of E. gomphocephala during the LGM showed a wide distribution that extended at least 30 km westward from the current distribution to the palaeo-coastline. CONCLUSIONS:The chloroplast and nuclear data suggest wide persistence of E. gomphocephala during the LGM. Palaeodistribution modelling supports the conclusions drawn from genetic data and indicates a widespread westward shift of E. gomphocephala onto the exposed continental shelf during the LGM. This study highlights the importance of the inclusion of complementary, non-genetic data (information on geomorphology and palaeoclimate) to interpret phylogeographic patterns.

Project description:BackgroundThe invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources.MethodologyWe use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis.ConclusionsWe demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics.

Dataset Information

Microsatellite primers identified by 454 sequencing in the floodplain tree species Eucalyptus victrix (Myrtaceae).

Publications

Microsatellite primers identified by 454 sequencing in the floodplain tree species Eucalyptus victrix (Myrtaceae).

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