Nuclear Microsatellite Primers for the Endangered Relict Fir, Abies pinsapo (Pinaceae) and Cross-Amplification in Related Mediterranean Species (original) (raw)
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Plant Molecular Biology Reporter, 2017
The European black pine (Pinus nigra Arn.) is an ecologically and economically important conifer discontinuously and often patchily distributed across different ecological environments and climatic conditions around the Mediterranean Basin. Many studies aiming at understanding the evolutionary history of the species have been carried out, particularly in the hope of resolving the taxonomy of this collective species where many subspecies have been described. So far, neither morphology, enzyme, and metabolite polymorphism nor DNA marker studies have been able to converge on a single taxonomy, and the evolutionary history of the European black pine remains unclear. To date, nuclear microsatellites (nSSRs) are not available for P. nigra. In this work we designed two multiplexes of eight and six nSSRs each, both characterized by high amplification quality and polymorphism. Nine nSSRs were specifically developed for the European black pine starting from six transcriptomes sequenced using Next Generation Sequencing (NGS) techniques following the Illumina method, while five previously developed for other pines were successfully transferred to P. nigra. The mean number of alleles per locus was 5.9, and the observed heterozygosity ranged between 0.341 and 0.875. F ST was statistically significant for all loci, and Bayesian clustering approaches revealed a geographically meaningful population structure. We thus show this set of 14 nSSRs to be highly informative for population genetic studies and of significant interest to resolve the taxonomy of the European black pine.
PLOS ONE, 2018
Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive F IS values that were likely inflated by null alleles. Mantel's tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.
Genetic variation in Tertiary relics: The case of eastern-Mediterranean Abies (Pinaceae)
Ecology and evolution, 2017
The eastern-Mediterranean Abies taxa, which include both widely distributed species and taxa with minuscule ranges, represent a good model to study the impacts of range size and fragmentation on the levels of genetic diversity and differentiation. To assess the patterns of genetic diversity and phylogenetic relationships among eastern-Mediterranean Abies taxa, genetic variation was assessed by eight nuclear microsatellite loci in 52 populations of Abies taxa with a focus on those distributed in Turkey and the Caucasus. Both at the population and the taxon level, the subspecies or regional populations of Abies nordmanniana s.l. exhibited generally higher allelic richness, private allelic richness, and expected heterozygosity compared with Abies cilicica s.l. Results of both the structure analysis and distance-based approaches showed a strong differentiation of the two A. cilicica subspecies from the rest as well as from each other, whereas the subspecies of A. nordmanniana were disti...
Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra
Biologia, 2013
Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content.
Genetic structure of Pinus pinaster Ait. populations in Morocco revealed by nuclear microsatellites
Biochemical Systematics and Ecology, 2010
Pinus pinaster is one of the most popular conifers used for reforestation in Morocco and represents an economically and ecologically important species for the region. In this study, nuclear microsatellites (ncSSRs) are used to compare genetic structure and diversity estimates of natural populations of Moroccan maritime pine. Samples were collected among 10 natural populations distributed in three biogeographically different regions, the Rif Mountain, the Middle and the High Atlas. Forty-five nuclear alleles at seven variable loci were found with a mean of 6.4 alleles per locus. A number of private alleles (17.1%) were shown in populations from Rif and Middle Atlas. Moreover, in Morocco, P. pinaster showed a lower genetic diversity than in other parts of its geographic range. Significant departures from Hardy-Weinberg equilibrium with excess homozygosity are observed indicating a high level of mating inside populations. Genetic diversity was structured with high variability among populations (Fst ΒΌ 12%). Results show a correlation between genetic and geographic distances with an R-squared of 0.436. Two clusters were found using STRUCTURE, whereas three main clusters can be distinguished based on genetic distances of phylogenetic tree. Genetic relationships among maritime pine populations in Morocco appear to be related to historical, ecological as well as anthropogenic factors, suggesting the need for conservation strategies at the population level.
Forest Science, 2018
Predicting the coping responses of tree species to climate change is, in many cases, limited by a lack of fundamental genetic insights, such as detailed knowledge of their genomes. Relict tree species may serve as models to assess the general mechanisms and patterns of forest tree responses to the changing climate and to explore the adaptive capacity of trees. Here, we focus on Abies pinsapo, the southernmost European fir, whose marginal populations are prone to extinction. To discover new insights into the genetics of this species, Illumina de novo transcriptome sequencing was performed. We obtained a 24 Gb transcriptome dataset, and the assembly identified 97,768 unigenes with an average length of 1,100 nucleotides. A large number of molecular markers, 100,949 single nucleotide polymorphisms (SNPs), and 5,663 microsatellites (SSRs) were detected, and a gene-associated SNP validation pipeline was developed. The estimated accuracy of the SNP prediction was 55 percent. The novel reference transcriptome and the large number of molecular markers specific to Abies pinsapo reported in this study represent valuable resources for further genetic analyses and conservation guidelines for this endangered tree species and other Abies species.
Forest Ecology and Management, 2004
Pinus pinaster is a conifer native to western Europe and northern Africa. Following on-going breeding programmes, provenance and progeny trials were established in some of the countries of the species' range (France, Portugal and Spain) and quantitative traits were measured: growth, stem form, survival and pest and disease resistance, amongst others. Populations from the wide range of P. pinaster were recently screened with molecular markers in order to assess their genetic diversity. Data were obtained using allozymes, chloroplast (cpSSRs) and nuclear (nuSSRs) microsatellites and amplified fragment length polymorphisms (AFLPs). Based on mtDNA-RFLP analysis, three maternal lineages (named ''western'', ''eastern'' and ''Moroccan'') were identified and no population showed a mixed composition. In this study, the imprint that differentiation in putatively isolated glacial refugia (identified by the different maternal lineages) might have left on the nuclear genome was analysed using a wide range of molecular markers and adaptive traits. Multivariate ordination of populations based on nuclear molecular markers (allozymes and nuSSRs) showed a clear clustering of provenances sharing a given mtDNA lineage. However, that clustering was found to be less tight when only quantitative traits were investigated. In P. pinaster, the within-population estimates of gene diversity using different traits were not correlated. Therefore, caution is advisable when designing conservation strategies based on molecular marker studies or a limited number of populations. After these results, we recommend a conservation strategy characterised by gene flow consistent with the current population structure, careful seed transfer among maternal lineages (if any), selection of populations for conservation based on the originality of their allelic composition and definition of Management Units (MUs) based on adaptive traits. #