Effects of landscapes and range expansion on population structure and local adaptation.

Understanding the origin and distribution of genetic diversity across landscapes is critical for predicting the future of organisms in changing climates. This study investigated how adaptive and demographic forces have shaped diversity and population structure in Pinus densata, a keystone species on Qinghai-Tibetan Plateau (QTP). We examined the distribution of genomic diversity across the range of P. densata using exome capture sequencing. We applied spatially explicit tests to dissect the impacts of allele surfing, geographic isolation and environmental gradients on population differentiation and forecasted how this genetic legacy may limit the persistence of P. densata in future climates. We found that allele surfing from range expansion could explain the distribution of 39% of the c. 48 000 genotyped single nucleotide polymorphisms (SNPs). Uncorrected, these allele frequency clines severely confounded inferences of selection. After controlling for demographic processes, isolation-by-environment explained 9.2-19.5% of the genetic structure, with c. 4.0% of loci being affected by selection. Allele surfing and genotype-environment associations resulted in genomic mismatch under projected climate scenarios. We illustrate that significant local adaptation, when coupled with reduced diversity as a result of demographic history, constrains potential evolutionary response to climate change. The strong signal of genomic vulnerability in P. densata may be representative for other QTP endemics.

Late Pleistocene speciation of three closely related tree peonies endemic to the Qinling-Daba Mountains, a major glacial refugium in Central China.

Determining the factors promoting speciation is a major task in ecological and evolutionary research and can be aided by phylogeographic analysis. The Qinling-Daba Mountains (QDM) located in central China form an important geographic barrier between southern subtropical and northern temperate regions, and exhibit complex topography, climatic, and ecological diversity. Surprisingly, few phylogeographic analyses and studies of plant speciation in this region have been conducted. To address this issue, we investigated the genetic divergence and evolutionary histories of three closely related tree peony species (Paeonia qiui, P. jishanensis, and P. rockii) endemic to the QDM. Forty populations of the three tree peony species were genotyped using 22 nuclear simple sequence repeat markers (nSSRs) and three chloroplast DNA sequences to assess genetic structure and phylogenetic relationships, supplemented by morphological characterization and ecological niche modeling (ENM). Morphological and molecular genetic analyses showed the three species to be clearly differentiated from each other. In addition, coalescent analyses using DIYABC conducted on nSSR variation indicated that the species diverged from each other in the late Pleistocene, while ecological niche modeling (ENM) suggested they occupied a larger area during the Last Glacial Maximum (LGM) than at present. The combined genetic evidence from nuclear and chloroplast DNA and the results of ENM indicate that each species persisted through the late Pleistocene in multiple refugia in the Qinling, Daba, and Taihang Mountains with divergence favored by restricted gene flow caused by geographic isolation, ecological divergence, and limited pollen and seed dispersal. Our study contributes to a growing understanding of the origin and population structure of tree peonies and provides insights into the high level of plant endemism present in the Qinling-Daba Mountains of Central China.

Development of novel EST-SSR markers for Ephedra sinica (Ephedraceae) by transcriptome database mining.

PREMISE OF THE STUDY: Ephedra sinica (Ephedraceae) is a gymnosperm shrub with a wide distribution across Central and Eastern Asia. It is widely cultivated as a medicinal plant, but its wild populations are monitored to determine whether protection is needed. METHODS AND RESULTS: Thirty-six microsatellite markers, including 11 polymorphic markers, were developed from E. distachya RNA-Seq data deposited in the National Center for Biotechology Information dbEST database. Among 100 genotyped E. sinica individuals originating from five different population groups, the allele number ranged from three to 22 per locus. Levels of observed and expected heterozygosity ranged from 0 to 0.866 (average 0.176) and 0 to 0.876 (average 0.491), respectively. Allelic polymorphism information content ranged from 0.000 to 0.847 (average 0.333). Cross-species amplifications were successfully conducted with two related Ephedra species for all 11 di- or trinucleotide simple sequence repeats. CONCLUSIONS: This study provides the first set of microsatellite markers for genetic monitoring and surveying of this medicinal plant.

Genome sequence of Malania oleifera, a tree with great value for nervonic acid production.

BACKGROUND: Malania oleifera, a member of the Olacaceae family, is an IUCN red listed tree, endemic and restricted to the Karst region of southwest China. This tree's seed is valued for its high content of precious fatty acids (especially nervonic acid). However, studies on its genetic makeup and fatty acid biogenesis are severely hampered by a lack of molecular and genetic tools. FINDINGS: We generated 51 Gb and 135 Gb of raw DNA sequences, using Pacific Biosciences (PacBio) single-molecule real-time and 10× Genomics sequencing, respectively. A final genome assembly, with a scaffold N50 size of 4.65 Mb and a total length of 1.51 Gb, was obtained by primary assembly based on PacBio long reads plus scaffolding with 10× Genomics reads. Identified repeats constituted ∼82% of the genome, and 24,064 protein-coding genes were predicted with high support. The genome has low heterozygosity and shows no evidence for recent whole genome duplication. Metabolic pathway genes relating to the accumulation of long-chain fatty acid were identified and studied in detail. CONCLUSIONS: Here, we provide the first genome assembly and gene annotation for M. oleifera. The availability of these resources will be of great importance for conservation biology and for the functional genomics of nervonic acid biosynthesis.

Genetic Variation Related to High Elevation Adaptation Revealed by Common Garden Experiments in Pinus yunnanensis.

Local adaptation, adaptation to specialized niches and environmental clines have been extensively reported for forest trees. Investigation of the adaptive genetic variation is crucial for forest resource management and breeding, especially in the context of global climate change. Here, we utilized a Pinus yunnanensis common garden experiments established at high and low elevation sites to assess the differences in growth and survival among populations and between the two common garden sites. The studied traits showed significant variation between the two test sites and among populations, suggesting adaptive divergence. To detect genetic variation related to environment, we captured 103,608 high quality SNPs based on RNA sequencing, and used them to assess the genetic diversity and population structure. We identified 321 outlier SNPs from 131 genes showing significant divergence in allelic frequency between survival populations of two sites. Functional categories associated with adaptation to high elevation were found to be related to flavonoid biosynthesis, response to UV, DNA repair, response to reactive oxygen species, and membrane lipid metabolic process. Further investigation of the outlier genes showed overrepresentation of the flavonoid biosynthesis pathway, suggesting that this pathway may play a key role in P. yunnanensis adaptation to high elevation environments. The outlier genes identified, and their variants, provide a basic reference for advanced investigations.

High-quality assembly of the reference genome for scarlet sage, Salvia splendens, an economically important ornamental plant.

Background: Salvia splendens Ker-Gawler, scarlet or tropical sage, is a tender herbaceous perennial widely introduced and seen in public gardens all over the world. With few molecular resources, breeding is still restricted to traditional phenotypic selection, and the genetic mechanisms underlying phenotypic variation remain unknown. Hence, a high-quality reference genome will be very valuable for marker-assisted breeding, genome editing, and molecular genetics. Findings: We generated 66 Gb and 37 Gb of raw DNA sequences, respectively, from whole-genome sequencing of a largely homozygous scarlet sage inbred line using Pacific Biosciences (PacBio) single-molecule real-time and Illumina HiSeq sequencing platforms. The PacBio de novo assembly yielded a final genome with a scaffold N50 size of 3.12 Mb and a total length of 808 Mb. The repetitive sequences identified accounted for 57.52% of the genome sequence, and 54,008 protein-coding genes were predicted collectively with ab initio and homology-based gene prediction from the masked genome. The divergence time between S. splendens and Salvia miltiorrhiza was estimated at 28.21 million years ago (Mya). Moreover, 3,797 species-specific genes and 1,187 expanded gene families were identified for the scarlet sage genome. Conclusions: We provide the first genome sequence and gene annotation for the scarlet sage. The availability of these resources will be of great importance for further breeding strategies, genome editing, and comparative genomics among related species.

Development of high transferability cpSSR markers for individual identification and genetic investigation in Cupressaceae species.

Given the low substitution rate in plastomes, the polymorphic and codominant nature of chloroplast SSRs (cpSSRs) makes them ideal markers, complementing their nuclear counterpart. In Cupressaceae, cpSSRs are mostly paternally inherited, thus, they are useful in mating systems and pollen flow studies. Using e-PCR, 92 SSR loci were identified across six Cupressaceae plastomes, and primers were designed for 26 loci with potential interspecific transferability. The 26 developed cpSSRs were polymorphic in four genera, Platycladus, Sabina, Juniperus, and Cupressus and are suitable for Cupressaceae molecular genetic studies and utilization. We genotyped 192 Platycladus orientalis samples from a core breeding population using 10 of the developed cpSSRs and 10 nuclear SSRs, and these individuals were identified with high confidence. The developed cpSSRs can be used in (1) a marker-assisted breeding scheme, specifically when paternity identification is required, (2) population genetics investigations, and (3) biogeography of Cupressaceae and unraveling the genetic relationships between related species.

De Novo Transcriptome Assembly and Characterization for the Widespread and Stress-Tolerant Conifer Platycladus orientalis.

Platycladus orientalis, of the family Cupressaceae, is a widespread conifer throughout China and is extensively used for ecological reforestation, horticulture, and in medicine. Transcriptome assemblies are required for this ecologically important conifer for understanding genes underpinning adaptation and complex traits for breeding programs. To enrich the species' genomic resources, a de novo transcriptome sequencing was performed using Illumina paired-end sequencing. In total, 104,073,506 high quality sequence reads (approximately 10.3 Gbp) were obtained, which were assembled into 228,948 transcripts and 148,867 unigenes that were longer than 200 nt. Quality assessment using CEGMA showed that the transcriptomes obtained were mostly complete for highly conserved core eukaryotic genes. Based on similarity searches with known proteins, 62,938 (42.28% of all unigenes), 42,158 (28.32%), and 23,179 (15.57%) had homologs in the Nr, GO, and KOG databases, 25,625 (17.21%) unigenes were mapped to 322 pathways by BLASTX comparison against the KEGG database and 1,941 unigenes involved in environmental signaling and stress response were identified. We also identified 43 putative terpene synthase (TPS) functional genes loci and compared them with TPSs from other species. Additionally, 5,296 simple sequence repeats (SSRs) were identified in 4,715 unigenes, which were assigned to 142 motif types. This is the first report of a complete transcriptome analysis of P. orientalis. These resources provide a foundation for further studies of adaptation mechanisms and molecular-based breeding programs.