Protocol: A high-throughput DNA extraction system suitable for conifers

<p>Abstract</p> <p>Background</p> <p>High throughput DNA isolation from plants is a major bottleneck for most studies requiring large sample sizes. A variety of protocols have been developed for DNA isolation from plants. However, many species, including conifers, have high contents of secondary metabolites that interfere with the extraction process or the subsequent analysis steps. Here, we describe a procedure for high-throughput DNA isolation from conifers.</p> <p>Results</p> <p>We have developed a high-throughput DNA extraction protocol for conifers using an automated liquid handler and modifying the Qiagen MagAttract Plant Kit protocol. The modifications involve change to the buffer system and improving the protocol so that it almost doubles the number of samples processed per kit, which significantly reduces the overall costs. We describe two versions of the protocol: one for medium-throughput (MTP) and another for high-throughput (HTP) DNA isolation. The HTP version works from start to end in the industry-standard 96-well format, while the MTP version provides higher DNA yields per sample processed. We have successfully used the protocol for DNA extraction and genotyping of thousands of individuals of several spruce and a pine species.</p> <p>Conclusion</p> <p>A high-throughput system for DNA extraction from conifer needles and seeds has been developed and validated. The quality of the isolated DNA was comparable with that obtained from two commonly used methods: the silica-spin column and the classic CTAB protocol. Our protocol provides a fully automatable and cost effective solution for processing large numbers of conifer samples.</p

Higher fine-scale genetic structure in peripheral than in core populations of a long-lived and mixed-mating conifer - eastern white cedar (Thuja occidentalis L.)

<p>Abstract</p> <p>Background</p> <p>Fine-scale or spatial genetic structure (SGS) is one of the key genetic characteristics of plant populations. Several evolutionary and ecological processes and population characteristics influence the level of SGS within plant populations. Higher fine-scale genetic structure may be expected in peripheral than core populations of long-lived forest trees, owing to the differences in the magnitude of operating evolutionary and ecological forces such as gene flow, genetic drift, effective population size and founder effects. We addressed this question using eastern white cedar (<it>Thuja occidentalis</it>) as a model species for declining to endangered long-lived tree species with mixed-mating system.</p> <p>Results</p> <p>We determined the SGS in two core and two peripheral populations of eastern white cedar from its Maritime Canadian eastern range using six nuclear microsatellite DNA markers. Significant SGS ranging from 15 m to 75 m distance classes was observed in the four studied populations. An analysis of combined four populations revealed significant positive SGS up to the 45 m distance class. The mean positive significant SGS observed in the peripheral populations was up to six times (up to 90 m) of that observed in the core populations (15 m). Spatial autocorrelation coefficients and correlograms of single and sub-sets of populations were statistically significant. The extent of within-population SGS was significantly negatively correlated with all genetic diversity parameters. Significant heterogeneity of within-population SGS was observed for 0-15 m and 61-90 m between core and peripheral populations. Average <it>Sp</it>, and gene flow distances were higher in peripheral (<it>Sp </it>= 0.023, σ_g= 135 m) than in core (<it>Sp </it>= 0.014, σ_g= 109 m) populations. However, the mean neighborhood size was higher in the core (<it>Nb </it>= 82) than in the peripheral (<it>Nb </it>= 48) populations.</p> <p>Conclusion</p> <p>Eastern white cedar populations have significant fine-scale genetic structure at short distances. Peripheral populations have several-folds higher within-population fine-scale genetic structure than core populations. Anthropogenic disturbances and population fragmentation presumably have significant effects on fine-scale genetic structure in eastern white cedar. Core populations have higher neighborhood size than peripheral populations, whereas gene flow distances are higher in peripheral than in core populations. The results of our study contribute to the knowledge of poorly-understood spatial genetic structure of core versus peripheral populations in plants. As well, the information is of significance for conservation of genetic resources of eastern white cedar and perhaps of other long-lived forest trees with mixed-mating system.</p

Near-saturated and complete genetic linkage map of black spruce (Picea mariana)

<p>Abstract</p> <p>Background</p> <p>Genetic maps provide an important genomic resource for understanding genome organization and evolution, comparative genomics, mapping genes and quantitative trait loci, and associating genomic segments with phenotypic traits. Spruce (<it>Picea</it>) genomics work is quite challenging, mainly because of extremely large size and highly repetitive nature of its genome, unsequenced and poorly understood genome, and the general lack of advanced-generation pedigrees. Our goal was to construct a high-density genetic linkage map of black spruce (<it>Picea mariana</it>, 2n = 24), which is a predominant, transcontinental species of the North American boreal and temperate forests, with high ecological and economic importance.</p> <p>Results</p> <p>We have developed a near-saturated and complete genetic linkage map of black spruce using a three-generation outbred pedigree and amplified fragment length polymorphism (AFLP), selectively amplified microsatellite polymorphic loci (SAMPL), expressed sequence tag polymorphism (ESTP), and microsatellite (mostly cDNA based) markers. Maternal, paternal, and consensus genetic linkage maps were constructed. The maternal, paternal, and consensus maps in our study consistently coalesced into 12 linkage groups, corresponding to the haploid chromosome number (1n = 1x = 12) of 12 in the genus <it>Picea</it>. The maternal map had 816 and the paternal map 743 markers distributed over 12 linkage groups each. The consensus map consisted of 1,111 markers distributed over 12 linkage groups, and covered almost the entire (> 97%) black spruce genome. The mapped markers included 809 AFLPs, 255 SAMPL, 42 microsatellites, and 5 ESTPs. Total estimated length of the genetic map was 1,770 cM, with an average of one marker every 1.6 cM. The maternal, paternal and consensus genetic maps aligned almost perfectly.</p> <p>Conclusion</p> <p>We have constructed the first high density to near-saturated genetic linkage map of black spruce, with greater than 97% genome coverage. Also, this is the first genetic map based on a three-generation outbred pedigree in the genus <it>Picea</it>. The genome length in <it>P. mariana </it>is likely to be about 1,800 cM. The genetic maps developed in our study can serve as a reference map for various genomics studies and applications in <it>Picea a</it>nd Pinaceae.</p

Data from: Genetic divergence and signatures of natural selection in marginal populations of a keystone, long-lived conifer, eastern white pine (Pinus strobus) from northern Ontario

Marginal populations are expected to provide the frontiers for adaptation, evolution and range shifts of plant species under the anticipated climate change conditions. Marginal populations are predicted to show genetic divergence from central populations due to their isolation, and divergent natural selection and genetic drift operating therein. Marginal populations are also expected to have lower genetic diversity and effective population size (Ne) and higher genetic differentiation than central populations. We tested these hypotheses using eastern white pine (Pinus strobus) as a model for keystone, long-lived widely-distributed plants. All 614 eastern white pine trees, in a complete census of two populations each of marginal old-growth, central old-growth, and central second-growth, were genotyped at 11 microsatellite loci. The central populations had significantly higher allelic and genotypic diversity, latent genetic potential (LGP) and Ne than the marginal populations. However, heterozygosity and fixation index were similar between them. The marginal populations were genetically diverged from the central populations. Model testing suggested predominant north to south gene flow in the study area with curtailed gene flow to northern marginal populations. Signatures of natural selection were detected at three loci in the marginal populations; two showing divergent selection with directional change in allele frequencies, and one balancing selection. Contrary to the general belief, no significant differences were observed in genetic diversity, differentiation, LGP, and Ne between old-growth and second-growth populations. Our study provides information on the dynamics of migration, genetic drift and selection in central versus marginal populations of a keystone long-lived plant species and has broad evolutionary, conservation and adaptation significance

Data from: Effects of harvesting of increasing intensities on genetic diversity and population structure of white spruce

Forest harvesting of increasing intensities is expected to have intensifying impacts on the genetic diversity and population structure of postharvest naturally regenerated stands by affecting the magnitude of evolutionary processes, such as genetic drift, gene flow, mating system, and selection. We have tested this hypothesis for the first time by employing widely distributed boreal white spruce (Picea glauca) as a model and controlled, replicated experimental harvesting and regeneration experiment at the EMEND project site (http://www.emendproject.org). We used two approaches. First, genetic diversity and population structure of postharvest natural regeneration after five harvesting treatments (green tree retention of 75%, 50%, 20%, and 10%, and clearcut) were assessed and compared with those of the unharvested control (pristine preharvest old-growth) in two replicates each of conifer-dominated (CD) and mixed-wood (MW) forest, using 10 (six EST (expressed sequence tag) and four genomic) microsatellite markers. Second, genetic diversity and population structure of preharvest old-growth were compared with those of postharvest natural regeneration after five harvesting treatments in the same treatment blocks in one replicate each of CD and MW forests. Contrary to our expectations, genetic diversity, inbreeding levels, and population genetic structure were similar between unharvested control or preharvest old-growth and postharvest natural regeneration after five harvesting treatments, with clearcut showing no negative genetic impacts. The potential effects of genetic drift and inbreeding resulting from harvesting bottlenecks were counterbalanced by predominantly outcrossing mating system and high gene flow from the residual and/or surrounding white spruce. CD and MW forests responded similarly to harvesting of increasing intensities. Simulated data for 10, 50, and 100 microsatellite markers showed the same results as obtained empirically from 10 microsatellite markers. Similar patterns of genetic diversity and population structure were observed for EST and genomic microsatellites. In conclusion, harvesting of increasing intensities did not show any significant negative impact on genetic diversity, population structure, and evolutionary potential of white spruce in CD and MW forests. Our first of its kind of study addresses the broad central forest management question how forest harvesting and regeneration practices can best maintain genetic biodiversity and ecosystem integrity

Genetic divergence and signatures of natural selection in marginal populations of a keystone, long-lived conifer, Eastern White Pine (Pinus strobus) from Northern Ontario.

Marginal populations are expected to provide the frontiers for adaptation, evolution and range shifts of plant species under the anticipated climate change conditions. Marginal populations are predicted to show genetic divergence from central populations due to their isolation, and divergent natural selection and genetic drift operating therein. Marginal populations are also expected to have lower genetic diversity and effective population size (Ne) and higher genetic differentiation than central populations. We tested these hypotheses using eastern white pine (Pinus strobus) as a model for keystone, long-lived widely-distributed plants. All 614 eastern white pine trees, in a complete census of two populations each of marginal old-growth, central old-growth, and central second-growth, were genotyped at 11 microsatellite loci. The central populations had significantly higher allelic and genotypic diversity, latent genetic potential (LGP) and Ne than the marginal populations. However, heterozygosity and fixation index were similar between them. The marginal populations were genetically diverged from the central populations. Model testing suggested predominant north to south gene flow in the study area with curtailed gene flow to northern marginal populations. Signatures of natural selection were detected at three loci in the marginal populations; two showing divergent selection with directional change in allele frequencies, and one balancing selection. Contrary to the general belief, no significant differences were observed in genetic diversity, differentiation, LGP, and Ne between old-growth and second-growth populations. Our study provides information on the dynamics of migration, genetic drift and selection in central versus marginal populations of a keystone long-lived plant species and has broad evolutionary, conservation and adaptation significance

A simple method for estimating genetic diversity in large populations from finite sample sizes

<p>Abstract</p> <p>Background</p> <p>Sample size is one of the critical factors affecting the accuracy of the estimation of population genetic diversity parameters. Small sample sizes often lead to significant errors in determining the allelic richness, which is one of the most important and commonly used estimators of genetic diversity in populations. Correct estimation of allelic richness in natural populations is challenging since they often do not conform to model assumptions. Here, we introduce a simple and robust approach to estimate the genetic diversity in large natural populations based on the empirical data for finite sample sizes.</p> <p>Results</p> <p>We developed a non-linear regression model to infer genetic diversity estimates in large natural populations from finite sample sizes. The allelic richness values predicted by our model were in good agreement with those observed in the simulated data sets and the true allelic richness observed in the source populations. The model has been validated using simulated population genetic data sets with different evolutionary scenarios implied in the simulated populations, as well as large microsatellite and allozyme experimental data sets for four conifer species with contrasting patterns of inherent genetic diversity and mating systems. Our model was a better predictor for allelic richness in natural populations than the widely-used Ewens sampling formula, coalescent approach, and rarefaction algorithm.</p> <p>Conclusions</p> <p>Our regression model was capable of accurately estimating allelic richness in natural populations regardless of the species and marker system. This regression modeling approach is free from assumptions and can be widely used for population genetic and conservation applications.</p