Targeted Capture Sequencing in Whitebark Pine Reveals Range-Wide Demographic and Adaptive Patterns Despite Challenges of a Large, Repetitive Genome.

Whitebark pine (Pinus albicaulis) inhabits an expansive range in western North America, and it is a keystone species of subalpine environments. Whitebark is susceptible to multiple threats - climate change, white pine blister rust, mountain pine beetle, and fire exclusion - and it is suffering significant mortality range-wide, prompting the tree to be listed as 'globally endangered' by the International Union for Conservation of Nature and 'endangered' by the Canadian government. Conservation collections (in situ and ex situ) are being initiated to preserve the genetic legacy of the species. Reliable, transferrable, and highly variable genetic markers are essential for quantifying the genetic profiles of seed collections relative to natural stands, and ensuring the completeness of conservation collections. We evaluated the use of hybridization-based target capture to enrich specific genomic regions from the 27 GB genome of whitebark pine, and to evaluate genetic variation across loci, trees, and geography. Probes were designed to capture 7,849 distinct genes, and screening was performed on 48 trees. Despite the inclusion of repetitive elements in the probe pool, the resulting dataset provided information on 4,452 genes and 32% of targeted positions (528,873 bp), and we were able to identify 12,390 segregating sites from 47 trees. Variations reveal strong geographic trends in heterozygosity and allelic richness, with trees from the southern Cascade and Sierra Range showing the greatest distinctiveness and differentiation. Our results show that even under non-optimal conditions (low enrichment efficiency; inclusion of repetitive elements in baits), targeted enrichment produces high quality, codominant genotypes from large genomes. The resulting data can be readily integrated into management and gene conservation activities for whitebark pine, and have the potential to be applied to other members of 5-needle pine group (Pinus subsect. Quinquefolia) due to their limited genetic divergence.

Multilocus analyses reveal little evidence for lineage-wide adaptive evolution within major clades of soft pines (Pinus subgenus Strobus).

Estimates from molecular data for the fraction of new nonsynonymous mutations that are adaptive vary strongly across plant species. Much of this variation is due to differences in life history strategies as they influence the effective population size (Ne ). Ample variation for these estimates, however, remains even when comparisons are made across species with similar values of Ne . An open question thus remains as to why the large disparity for estimates of adaptive evolution exists among plant species. Here, we have estimated the distribution of deleterious fitness effects (DFE) and the fraction of adaptive nonsynonymous substitutions (α) for 11 species of soft pines (subgenus Strobus) using DNA sequence data from 167 orthologous nuclear gene fragments. Most newly arising nonsynonymous mutations were inferred to be so strongly deleterious that they would rarely become fixed. Little evidence for long-term adaptive evolution was detected, as all 11 estimates for α were not significantly different from zero. Nucleotide diversity at synonymous sites, moreover, was strongly correlated with attributes of the DFE across species, thus illustrating a strong consistency with the expectations from the Nearly Neutral Theory of molecular evolution. Application of these patterns to genome-wide expectations for these species, however, was difficult as the loci chosen for the analysis were a biased set of conserved loci, which greatly influenced the estimates of the DFE and α. This implies that genome-wide parameter estimates will need truly genome-wide data, so that many of the existing patterns documented previously for forest trees (e.g. little evidence for signature of selection) may need revision.

Targeted enrichment strategies for next-generation plant biology.

PREMISE OF THE STUDY: The dramatic advances offered by modern DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome size, extensive variation in the proportion of organellar DNA in total DNA, polyploidy, and gene number/redundancy contribute to these challenges, and they demand flexible targeted enrichment strategies to achieve the desired goals. METHODS: In this article, we summarize the many available targeted enrichment strategies that can be used to target partial-to-complete organellar genomes, as well as known and anonymous nuclear targets. These methods fall under four categories: PCR-based enrichment, hybridization-based enrichment, restriction enzyme-based enrichment, and enrichment of expressed gene sequences. KEY RESULTS: Examples of plant-specific applications exist for nearly all methods described. While some methods are well established (e.g., transcriptome sequencing), other promising methods are in their infancy (hybridization enrichment). A direct comparison of methods shows that PCR-based enrichment may be a reasonable strategy for accessing small genomic targets (e.g., ≤50 kbp), but that hybridization and transcriptome sequencing scale more efficiently if larger targets are desired. CONCLUSIONS: While the benefits of targeted sequencing are greatest in plants with large genomes, nearly all comparative projects can benefit from the improved throughput offered by targeted multiplex DNA sequencing, particularly as the amount of data produced from a single instrument approaches a trillion bases per run.

Interspecific phylogenetic analysis enhances intraspecific phylogeographical inference: a case study in Pinus lambertiana.

Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 1600-km latitudinal range extending from Oregon, USA, to northern Baja California, Mexico. Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast DNA (cpDNA) survey of Pinus subsect. Strobus with comprehensive geographical sampling of P. lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographical differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an additional 72 individuals demarcated a narrow 150-km contact zone in northeastern California. In the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene resistance, previously determined for 41 seed zones, differ significantly among seed zones that are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that includes Pinus albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). These results are consistent with a Pleistocene migration of whitebark pine into North America and subsequent chloroplast introgression from whitebark pine to sugar pine. This study demonstrates the importance of placing phylogeographical results in a broader phylogenetic context.