Plastome phylogenomics provide new perspective into the phylogeny and evolution of Betulaceae (Fagales).

BACKGROUND: Betulaceae is a relatively small but morphologically diverse family, with many species having important economic and ecological values. Although plastome structure of Betulaceae has been reported sporadically, a comprehensive exploration for plastome evolution is still lacking. Besides, previous phylogenies had been constructed based on limited gene fragments, generating unrobust phylogenetic framework and hindering further studies on divergence ages, biogeography and character evolution. Here, 109 plastomes (sixteen newly assembled and 93 previously published) were subject to comparative genomic and phylogenomic analyses to reconstruct a robust phylogeny and trace the diversification history of Betulaceae. RESULTS: All Betulaceae plastomes were highly conserved in genome size, gene order, and structure, although specific variations such as gene loss and IR boundary shifts were revealed. Ten divergent hotspots, including five coding regions (Pi > 0.02) and five noncoding regions (Pi > 0.035), were identified as candidate DNA barcodes for phylogenetic analysis and species delimitation. Phylogenomic analyses yielded high-resolution topology that supported reciprocal monophyly between Betula and Alnus within Betuloideae, and successive divergence of Corylus, Ostryopsis, and Carpinus-Ostrya within Coryloideae. Incomplete lineage sorting and hybridization may be responsible for the mutual paraphyly between Ostrya and Carpinus. Betulaceae ancestors originated from East Asia during the upper Cretaceous; dispersals and subsequent vicariance accompanied by historical environment changes contributed to its diversification and intercontinental disjunction. Ancestral state reconstruction indicated the acquisition of many taxonomic characters was actually the results of parallel or reversal evolution. CONCLUSIONS: Our research represents the most comprehensive taxon-sampled and plastome-level phylogenetic inference for Betulaceae to date. The results clearly document global patterns of plastome structural evolution, and established a well-supported phylogeny of Betulaceae. The robust phylogenetic framework not only provides new insights into the intergeneric relationships, but also contributes to a perspective on the diversification history and evolution of the family.

Species divergence and phylogeography of Corylus heterophylla Fisch complex (Betulaceae): Inferred from molecular, climatic and morphological data.

Historical geo-climatic changes have shaped the geographical distributions and genetic diversity of numerous plant taxa in East Asia, which promote species divergence and ultimately speciation. Here, we integrated multiple approaches, including molecular phylogeography, ecological niche modeling, and morphological traits to examine the nucleotide diversity and interspecific divergence within Corylus heterophylla complex (C. heterophylla, C. kweichowensis, and C. yunnanensis). These three sibling taxa harbored similar high levels of nucleotide diversity at the species level. The molecular data (SCNG and cpDNA) unanimously supported the division of C. heterophylla complex into two major clades, with C. yunnanensis diverged earlier from the complex, whereas C. heterophylla and C. kweichowensis could hardly be separated. The split between the two clades (c. 12.89 Ma) coincided with the formation of Sichuan Basin in the middle Miocene, while the divergence among and within the five subclades (YUN1-YUN3, HK1-HK2) occurred from the late Miocene to the Pleistocene. C. heterophylla of northern China experienced glacial contraction and interglacial expansion during the Quaternary, whereas C. kweichowensis and C. yunnanensis of southern China presented population expansion even during the last glacial maximum. Despite of high levels of genetic admixture between C. heterophylla and C. kweichowensis, significant ecological and morphological discrepancy as well as incomplete geographic isolation indicated that adaptive evolution triggered by divergent selection may have played important roles in incipient ecological speciation.

Multilocus data reveal deep phylogenetic relationships and intercontinental biogeography of the Eurasian-North American genus Corylus (Betulaceae).

The evolutionary history of the genus Corylus, a tertiary disjunct lineage consisting of approximately 15-20 taxa with New and Old World distribution, has not been fully studied using molecular tools. In this research, we reconstructed comprehensive phylogenies of this genus using multiple datasets (genome-wide SNPs; complete chloroplast genomes; and nuclear ribosomal ITS sequences) based on detailed sampling of 17 Corylus species currently recognized. Divergence times were estimated using a fossil calibrated relaxed clock model, and ancestral area reconstruction were inferred using Bayesian binary MCMC (BBM) method and the dispersal-extinction-cladogenesis (DEC) model. Phylogenetic incongruences were detected from datasets, with nuclear SNP and ITS phylogenies supporting four major clades that correspond well with morphological traits, while chloroplast phylogeny revealed geographic partitioning. Recombination and introgressive hybridization played important roles in Corylus diversification. Molecular dating and biogeographical analyses unambiguously revealed that Corylus originated in Southwest China during the middle Eocene. The westward migration of Phyllochlamys (Clade C) and Colurnae (Clade D) and the uplift of Qinghai-Tibet Plateau drove the formation of European taxa, whereas the transoccanic migration crossing the Bering Land Bridge of Siphonochlamys (Clade B) and Phyllochlamys (Clade C) led to the occurrence of North American taxa. The topographic heterogeneity and climatic oscillations from Miocene to Pleistocene made East Asia the diversity center for Corylus. This study offers important insights into the phylogenetic relationships and biogeography history of the genus Corylus.

Population genomics reveals demographic history and selection signatures of hazelnut (Corylus).

Hazelnut (Corylus spp.) is known as one of the four famous tree nuts in the world due to its pleasant taste and nutritional benefits. However, hazelnut promotion worldwide is increasingly challenged by global climate change, limiting its production to a few regions. Focusing on the eurytopic Section Phyllochlamys, we conducted whole-genome resequencing of 125 diverse accessions from five geo-ecological zones in Eurasia to elucidate the genomic basis of adaptation and improvement. Population structure inference outlined five distinct genetic lineages corresponding to climate conditions and breeding background, and highlighted the differentiation between European and Asian lineages. Demographic dynamics and ecological niche modeling revealed that Pleistocene climatic oscillations dominantly shaped the extant genetic patterns, and multiple environmental factors have contributed to the lineage divergence. Whole-genome scans identified 279, 111, and 164 selective sweeps that underlie local adaptation in Corylus heterophylla, Corylus kweichowensis, and Corylus yunnanensis, respectively. Relevant positively selected genes were mainly involved in regulating signaling pathways, growth and development, and stress resistance. The improvement signatures of hybrid hazelnut were concentrated in 312 and 316 selected genes, when compared to C. heterophylla and Corylus avellana, respectively, including those that regulate protein polymerization, photosynthesis, and response to water deprivation. Among these loci, 22 candidate genes were highly associated with the regulation of biological quality. Our study provides insights into evolutionary processes and the molecular basis of how sibling species adapt to contrasting environments, and offers valuable resources for future climate-resilient breeding.

[Methylenetetrahydrofolate reductase and methionine synthase reductase gene polymorphisms in ethnic Han women from Linyi].

OBJECTIVE: To explore the distribution of genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) 677C/T, 1298A/C and methionine synthase reductase (MTRR) 66A/G among ethnic Han females from Linyi, and to correlate it with serum level of homocysteine (Hcy). METHODS: A cross-sectional study was carried out. Oral epithelial cell samples were collected from 825 subjects. MTHFR and MTRR gene polymorphisms were determined with a Taqman-Minor Groove Binder (MGB) method. Distribution of gene polymorphisms was analyzed and compared with others regions of China including Weifang, Zhengzhou, Deyang and Hainan. A biochemical assay was also carried out to determine the total Hcy in plasma of 281 subjects. The reductase activity of MTHFR was classified into decreased and stable groups according to genetic polymorphism of MTHFR. Correlation between MTHFR groups and total Hcy level were also explored. RESULTS: (1) The frequencies of MTHFR677CC, CT and TT genotypes of the selected subjects were 16.7%, 48.3% and 35.0%, respectively. The frequencies of MTHFR 1298AA, AC and CC genotypes were 76.0%, 21.6% and 2.4%, respectively. And those of MTRR 66AA, AG and GG genotypes were 54.7%, 39.4% and 5.9%, respectively. For the selected subjects, their frequency of MTHFR 677TT genotype was higher than that of Deyang and Hainan (P< 0.01), whilst the frequency of MTHFR 1298CC genotype was lower than that of Deyang and Hainan (P < 0.01), and the frequency of MTRR 66 GG genotype was lower than that of Hainan (P< 0.01). (2) The Hcy level for those with decreased MTHFR activity was significantly higher than those with stable MTHFR activity (P< 0.05). CONCLUSION: MTHFR gene 677C/T, 1298A/C and MTRR 66A/G polymorphisms in ethnic Han women from Linyi have differed significantly from other regions of China. Decreased MTHFR activity caused by genetic polymorphisms is a risk factor for raised Hcy level.

Stigmatic Transcriptome Analysis of Self-Incompatible and Compatible Pollination in Corylus heterophylla Fisch. × Corylus avellana L.

Self-incompatibility (SI) protects plants from inbreeding depression due to self-pollination and promotes the outcrossing process to maintain a high degree of heterozygosity during evolution. Corylus is an important woody oil and nut species that shows sporophytic SI (SSI). Yet the molecular mechanism of SI in Corylus remains largely unknown. Here we conducted self- ("Dawei" × "Dawei") and cross-pollination ("Dawei" × "Liaozhen No. 7") experiments and then performed an RNA-Seq analysis to investigate the mechanism of pollen-stigma interactions and identify those genes that may be responsible for SSI in Corylus. We uncovered 19,163 up- and 13,314 downregulated genes from the comparison of different pollination treatments. These differentially expressed genes (DEGs) were significantly enriched in plant-pathogen interaction, plant hormone signal transduction, and MAPK signaling pathway-plant. We found many notable genes potentially involved in pollen-stigma interactions and SSI mechanisms, including genes encoding receptor-like protein kinases (RLK), calcium-related genes, disease-resistance genes, and WRKY transcription factors. Four upregulated and five downregulated DEGs were consistently identified in those comparison groups involving self-incompatible pollination, suggesting they had important roles in pollen-pistil interactions. We further identified the S-locus region of the Corylus heterophylla genome based on molecular marker location. This predicted S-locus contains 38 genes, of which 8 share the same functional annotation as the S-locus genes of Corylus avellana: two PIX7 homologous genes (EVM0002129 and EVM0025536), three MIK2 homologous genes (EVM0002422, EVM0005666, and EVM0009820), one aldose 1-epimerase (EVM0002095), one 3-dehydroquinate synthase II (EVM0021283), and one At3g28850 homologous gene (EVM0016149). By characterizing the pistil process during the early postpollination phase via transcriptomic analysis, this study provides new knowledge and lays the foundation for subsequent analyses of pollen-pistil interactions.

Wound healing mechanism of antimicrobial peptide cathelicidin-DM.

Background and Purpose: Chronic wound infections and the development of antibiotic resistance are serious clinical problems that affect millions of people worldwide. Cathelicidin-DM, an antimicrobial peptide from Duttaphrynus melanostictus, has powerful antimicrobial activity and wound healing efficacy. So, it could be a potential candidate to address this problem. In this paper, we investigate the wound healing mechanism of cathelicidin-DM to establish a basis for preclinical studies of the drug. Experimental Approach: The effects of cathelicidin-DM on cell proliferation and migration, cytokines, and mitogen-activated protein kinase (MAPK) signaling pathways were examined. Then mice whole skin wound model was constructed to evaluate the wound healing activity of cathelicidin-DM, and further histological changes in the wounds were assessed by hematoxylin-eosin staining (H&E) and immunohistochemical assays. Key Results: Cathelicidin-DM promotes the proliferation of HaCaT, HSF, and HUVEC cells in a concentration-dependent manner and the migration of HSF, HUVEC, and RAW.264.7 cells. Moreover,cathelicidin-DM can involve in wound healing through activation of the MAPK signaling pathway by upregulating phosphorylation of ERK, JNK, and P38. However, cathelicidin-DM didn't affect the secretion of IL-6 and TNF-α. At the animal level, cathelicidin-DM accelerated skin wound healing and early debridement in mice as well as promoted re-epithelialization and granulation tissue formation, α-SMA expression, and collagen I deposition in mice. Conclusion and Implications: Our data suggest that cathelicidin-DM can be engaged in the healing of infected and non-infected wounds through multiple pathways, providing a new strategy for the treatment of infected chronic wounds.

Effect of pre-pregnancy body mass index on adverse pregnancy outcome in north of China.

PURPOSE: To investigate the effect of pre-pregnancy BMI on adverse maternal and neonatal outcomes in pregnancy women of northern China. METHODS: We conducted a retrospective population-based cohort study of 5,047 singleton nulliparous pregnancies. The subjects were categorized into four groups by BMI-underweight (BMI < 18.5 kg/m(2)), normal (BMI 18.5-24 kg/m(2)), overweight (BMI 24-28 kg/m(2)) and obese (BMI ≥ 28 kg/m(2)). Logistic regression was used to adjust the potential confounder. Maternal and neonatal outcomes were evaluated with relative risks and 95% confidence intervals. RESULTS: 11.5, 63.4, 18.3, and 6.8% of the subjects were underweight, normal BMI, overweight and obese, respectively. Compared with women of normal BMI, the risk of the following outcomes was significantly increased in overweight and obese women and expressed as [adjusted RR (95% confidence interval)] respectively: pre-eclampsia [2.99 (2.21-4.06), 5.68 (3.97-8.11)]; gestational diabetes [2.49 (1.82-3.39), 4.35 (3.00-6.31)]; premature rupture of the membranes [1.64 (1.20-2.23), 1.73 (1.11-2.72)]; abruption placentae [1.84 (1.19-2.87), 2.79 (1.60-4.83)]; cesarian section [1.47 (1.27-1.70), 2.51 (1.97-3.20)]; postpartum hemorrhage [2.31 (1.51-3.54), 3.73 (2.37-5.04)]; perineal rupture [2.89 (1.44-5.81), 3.36 (1.55-7.30)]; large-for-gestational age [1.46 (1.02-2.08), 1.91 (1.17-3.10)]. However, anemia (2.54, 1.15-5.63), small-for-gestational age (1.67, 1.07-2.61) were significantly more common in the underweight group. CONCLUSIONS: The prevalence of overweight and obesity in women of northern China is much lower than in the Caucasian population or Chinese in Hong Kong. The increased maternal BMI is associated with many adverse pregnancy outcomes and its risk increases with the degree of obesity. Maternal underweight has a protective effect although increases the risk of having small-for-gestational age baby and anemia.

Seasonal Changes in Soil Microbial Community and Co-Occurrence Network of Species of the Genus Corylus.

Hazelnut is one of the four major nuts in the world and has high nutritional and economic value. This study employed Illumina sequencing of ITS rDNA and 16S rRNA genes to identify the seasonal changes in soil microbial community, the predominant environmental factors driving microbial community composition, and the differences in soil microbial composition among different species of the genus Corylus. We found that the soil microbial community composition of species of Corylus changed significantly with the change in seasons. Corylus heterophylla and Corylus kweichowensis had more ectomycorrhiza in their soil compared to Corylus avellane. The main factor influencing fungal community composition in soil was the available potassium, while that of bacteria was the total phosphorus content. Co-occurrence network analysis revealed that the ratio of positive interaction to negative interaction in soil of C. heterophylla and Ping'ou (C. heterophylla × C. avellane) was higher, while the negative interaction of soil community structure in C. avellane was greater. The bacterial community was more stable than the fungal community according to microbial diversity and co-occurrence network analyses. The findings of this research may facilitate improvements to the production and soil system management in hazel planting processes.

Characteristics of the Fungal Communities and Co-occurrence Networks in Hazelnut Tree Root Endospheres and Rhizosphere Soil.

Hazelnut has gained economic value in China in recent years, but its large-scale planting and research started later than other countries. Conducting basic research on hazelnut trees requires studying their related microorganisms. Here, we used high-throughput DNA sequencing to quantify the fungal communities in the root endospheres and rhizosphere soil of four hazelnut species. Fungal diversity in the rhizosphere soil was significantly higher than that in the root endospheres. Rhizosphere soil had more Mortierellomycota, and the fungal community compositions differed among the four hazelnut species. The root endospheres, especially those of the Ping'ou (Corylus heterophylla × Corylus avellana) trees, contained more ectomycorrhizal fungi. The co-occurrence networks in the rhizosphere soil were more sophisticated and stable than those in the root endospheres, even when the root endospheres had higher modularity, because the structural differentiation of the root endospheres differed from that of the rhizosphere soil. Two-factor correlation network analysis and linear regression analysis showed that the total organic carbon was the main environmental factor affecting the fungal communities. Our study revealed the community compositions, functional predictions, and co-occurrence network structural characteristics of fungi in hazelnut root endospheres and rhizosphere soil. We also examined the potential keystone taxa, and analyzed the environmental factors of the dominant fungal community compositions. This study provides guidance for the growth of hazelnut and the management of hazelnut garden, and provides an insight for future development of fungal inoculants to be used in hazelnut root.

Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis in Corylus heterophylla Fisch. × Corylus avellana L.

(1) Background: the species of Corylus have sporophytic type of self-incompatibility. Several genes related to recognition reaction between pollen and stigma have been identified in hazelnuts. To better understand the self-incompatibility (SI) response, we screened the suitable reference genes by using quantitative real-time reverse transcription PCR (qRT-PCR) analysis in hazelnut for the first time. (2) Methods: the major cultivar "Dawei" was used as material. A total of 12 candidate genes were identified and their expression profiles were compared among different tissues and in response to various treatments (different times after self- and cross-pollination) by RT-qPCR. The expression stability of these 12 candidate reference genes was evaluated using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder programs. (3) Results: the comprehensive ranking of RefFinder indicated that ChaActin, VvActin, ChaUBQ14, and ChaEF1-α were the most suitable reference genes. According to the stability analysis of 12 candidate reference genes for each sample group based on four software packages, ChaActin and ChaEF1-α were most stable in different times after self-pollination and 4 h after self- and cross-pollination, respectively. To further validate the suitability of the reference genes identified in this study, CavPrx, which the expression profiles in Corylus have been reported, was quantified by using ChaActin and ChaEF1-α as reference genes. (4) Conclusions: our study of reference genes selection in hazelnut shows that the two reference genes, ChaActin and ChaEF1-α, are suitable for the evaluation of gene expression, and can be used for the analysis of pollen-pistil interaction in Corylus. The results supply a reliable foundation for accurate gene quantifications in Corylus species, which will facilitate the studies related to the reproductive biology in Corylus.

Effects of Living Cover on the Soil Microbial Communities and Ecosystem Functions of Hazelnut Orchards.

Living cover is an important management measure for orchards in China, and has certain influences on soil properties, microorganisms, and the micro-ecological environment. However, there are few studies on the effects of living cover on the soil changes in hazelnut orchards. In this study, we compared the soils of living cover treatments with Vulpia myuros and the soils of no cover treatments, and analyzed the observed changes in soil properties, microorganisms, and microbial functions by using high-throughput ITS rDNA and 16S rRNA gene Illumina sequencing. The results demonstrated that the total organic carbon content in the 20-40 cm deep soils under the living cover treatments increased by 32.87 and 14.82% in May and July, respectively, compared with those under the no cover treatments. The living cover treatment with V. myuros also significantly increased the contents of total phosphorus (TP), total nitrogen (TN), available phosphorus (AP), and available potassium (AK) in the soil samples. Moreover, the influence of seasons was not as significant as that of soil depth. The living cover treatment also significantly improved the soil enzyme activity levels. The results demonstrated that Ascomycota, Mortierellomycota and Basidiomycota were the dominant fungal phyla in all samples, while Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, and Chloroflexi were the dominant bacterial phyla, but the different treatments impacted the compositions of fungal and bacterial communities. Principal component analysis (PCA) showed that living cover with V. myuros significantly changed the soil fungal community structures whereas the bacterial community structures may be more sensitive to seasonal changes. At the microbial functional level, the living cover treatment increased the fungal operational taxonomic units (OTUs) of symbiotrophs and decreased that of pathotrophs. According to this study, we believe that the application of a living cover with V. myuros has a favorable regulating influence on soil properties, microbial communities and microbial function. This treatment can also reduce the use of herbicides, reduce the cost of orchard management, and store more carbon underground to achieve sustainable intensification of production in hazelnut orchards, so it can be considered as a management measure for hazelnut orchards.

A chromosome-level reference genome of the hazelnut, Corylus heterophylla Fisch.

BACKGROUND: Corylus heterophylla Fisch. is a species of the Betulaceae family native to China. As an economically and ecologically important nut tree, C. heterophylla can survive in extremely low temperatures (-30 to -40 °C). To deepen our knowledge of the Betulaceae species and facilitate the use of C. heterophylla for breeding and its genetic improvement, we have sequenced the whole genome of C. heterophylla. FINDINGS: Based on >64.99 Gb (∼175.30×) of Nanopore long reads, we assembled a 370.75-Mb C. heterophylla genome with contig N50 and scaffold N50 sizes of 2.07 and 31.33 Mb, respectively, accounting for 99.23% of the estimated genome size (373.61 Mb). Furthermore, 361.90 Mb contigs were anchored to 11 chromosomes using Hi-C link data, representing 97.61% of the assembled genome sequences. Transcriptomes representing 4 different tissues were sequenced to assist protein-coding gene prediction. A total of 27,591 protein-coding genes were identified, of which 92.02% (25,389) were functionally annotated. The phylogenetic analysis showed that C. heterophylla is close to Ostrya japonica, and they diverged from their common ancestor ∼52.79 million years ago. CONCLUSIONS: We generated a high-quality chromosome-level genome of C. heterophylla. This genome resource will promote research on the molecular mechanisms of how the hazelnut responds to environmental stresses and serves as an important resource for genome-assisted improvement in cold and drought resistance of the Corylus genus.

Highly sensitive and spatially resolved polyvinyl alcohol/acrylamide photopolymer for real-time holographic applications.

By employing low molecular-weight polyvinyl alcohol (PVA) as binder, the spatial resolution of a red-sensitive PVA/acrylamide based photopolymer are improved from 1000 lines/mm to 3000 lines/mm. By increasing the ambient temperature during the holographic recording, the photosensitivity of photopolymer is also increased about 5 times. The optimized photopolymer system has high capacity such as high photosensitivity (8 mJ/cm(2)), high spatial resolution (over 3000 lines/mm) and high diffraction efficiency (over 94%). To our knowledge, its holographic recording performance is the best of ever reported PVA/acrylamide based photopolymer systems. It has good application prospects in real-time holographic interferometry, holographic storage and holographic display.

Molecular cloning and expression analysis of hybrid hazelnut (Corylus heterophylla × Corylus avellana) ChaSRK1/2 genes and their homologs from other cultivars and species.

The self-incompatibility system of Corylus is a sporophytic type that is phenotypically similar to that of Brassica. While the molecular mechanism of sporophytic self-incompatibility (SSI) has been investigated extensively in Brassica (Brassicaceae), little is known about the corresponding mechanism in Corylus (Betulaceae). Here, we discuss the SSI mechanism with respect to S-locus receptor kinase (SRK) gene homologs. To obtain two SRK candidate unigenes, we compared all of the unigenes in a transcriptional protein database from our previous study with BnSRK-1 (AB270767) using BLASTX with a cutoff e-value of 10-5. We then cloned the full-length cDNA of ChaSRK1 and ChaSRK2 genes from Ping'ou hybrid hazelnut (Corylus heterophylla × Corylus avellana) using RACE techniques. Bioinformatics approaches were used to analyze the cDNA sequences, protein sequences, and domains of the encoded proteins. The full-length ChaSRK1 cDNA was 2883 base pairs (bp) with a coding sequence (CDS) of 2,547 bp encoding 849 amino acid residues. The full-length ChaSRK2 cDNA was 2,693 bp, with a CDS of 2,433 bp encoding 811 amino acids. The ChaSRK1/2 proteins contained an S-domain (extracellular domain), a transmembrane domain, a Ser/Thr protein kinase active site (kinase domain), and DUF3660 and/or DUF3403 domains. The lengths of 18 partial SRK homologs ranged from 1347 to 1451 bp, and they contained the same structural domains as ChaSRK1 and ChaSRK2. Phylogenetic analysis revealed that all SRK homologs could be divided into two categories that were similar to the classification of SRKs in Brassica. The expression patterns of ChaSRK1 and ChaSRK2 differed: ChaSRK2 was predominantly expressed in mature stigmatic styles, while ChaSRK1 was expressed in other tissues with the highest in the root tips of Corylus. Using dual-color fluorescence in situ hybridization, ChaSRK1/2 expression was found to be localized in papillar cells. Collectively, these results revealed that SRKs from Corylus had similar characteristics to SRKs from Brassica. We therefore speculated that the SSI mechanism in Corylus might be more similar to the Brassica mechanism than to other SSI types.

The complete chloroplast genomes of three Betulaceae species: implications for molecular phylogeny and historical biogeography.

BACKGROUND: Previous phylogenetic conclusions on the family Betulaceae were based on either morphological characters or traditional single loci, which may indicate some limitations. The chloroplast genome contains rich polymorphism information, which is very suitable for phylogenetic studies. Thus, we sequenced the chloroplast genome sequences of three Betulaceae species and performed multiple analyses to investigate the genome variation, resolve the phylogenetic relationships, and clarify the divergence history. METHODS: Chloroplast genomes were sequenced using the high-throughput sequencing. A comparative genomic analysis was conducted to examine the global genome variation and screen the hotspots. Three chloroplast partitions were used to reconstruct the phylogenetic relationships using Maximum Likelihood and Bayesian Inference approaches. Then, molecular dating and biogeographic inferences were conducted based on the whole chloroplast genome data. RESULTS: Betulaceae chloroplast genomes consisted of a small single-copy region and a large single copy region, and two copies of inverted repeat regions. Nine hotspots can be used as potential DNA barcodes for species delimitation. Phylogenies strongly supported the division of Betulaceae into two subfamilies: Coryloideae and Betuloideae. The phylogenetic position of Ostryopsis davidiana was controversial among different datasets. The divergence time between subfamily Coryloideae and Betuloideae was about 70.49 Mya, and all six extant genera were inferred to have diverged fully by the middle Oligocene. Betulaceae ancestors were probably originated from the ancient Laurasia. DISCUSSIONS: This research elucidates the potential of chloroplast genome sequences in the application of developing molecular markers, studying evolutionary relationships and historical dynamic of Betulaceae.It also reveals the advantages of using chloroplast genome data to illuminate those phylogenies that have not been well solved yet by traditional approaches in other plants.

Comparative Genomics and Phylogenetic Analysis Revealed the Chloroplast Genome Variation and Interspecific Relationships of Corylus (Betulaceae) Species.

Corylus L. is an economically and phylogenetically important genus in the family Betulaceae. Taxonomic and phylogenetic relationships of Corylus species have long been controversial for lack of effective molecular markers. In this study, the complete chloroplast (cp) genomes of six Corylus species were assembled and characterized using next-generation sequencing. We compared the genome features, repeat sequences, sequence divergence, and constructed the phylogenetic relationships of the six Corylus species. The results indicated that Corylus cp genomes were typical of the standard double-stranded DNA molecule, ranging from 160,445 base pairs (bp) (C. ferox var. thibetca) to 161,621 bp (C. yunnanensis) in length. Each genome contained a pair of inverted repeats (IRs), a large single-copy (LSC) region and a small single-copy (SSC) region. Each of the six cp genomes possessed 113 unique genes arranged in the same order, including 80 protein-coding, 29 tRNA, and 4 rRNA genes. C. yunnanensis contained the highest number of repeat sequences, and the richest SSRs in six cp genomes were A/T mononucleotides. Comparative analyses of six Corylus cp genomes revealed four hotspot regions (trnH-psbA, rpoB-trnC, trnF-ndhJ, and rpl32-trnL) that could be used as potential molecular markers. Phylogenetic analyses of the complete chloroplast genomes and 80 protein-coding genes exhibited nearly identical topologies that strongly supported the monophyly of Corylus and simultaneously revealed the generic relationships among Betulaceae. The availability of these genomes can offer valuable genetic information for further taxonomy, phylogeny, and species delimitation in Corylus or even Betulaceae plants.

Resolving the Speciation Patterns and Evolutionary History of the Intercontinental Disjunct Genus Corylus (Betulaceae) Using Genome-Wide SNPs.

Understanding the underlying mechanisms of species origin, divergence, and distribution patterns of the intercontinental disjunct taxa has long fascinated botanists. Based on 4,894 genome-wide single-nucleotide polymorphism dataset, we present a molecular phylogenetic reconstruction of genus Corylus (Betulaceae), which have a disjunct distribution between Eurasia and North America (NA). The aim is to explore the speciation patterns and evolutionary relationships of Corylus species by establishing a general phylogenetic framework with extensive sampling. Both the molecular phylogeny inferred from recombination-free dataset and structure analysis support the division of Corylus into four major clades (A-D). Recombination tests and hybridization detection reveal extensive recombination and hybridization events among different clades, which have potentially influenced the speciation process of Corylus. Divergence time estimation indicates that recent common ancestor (MRCA) of Corylus occurred in late Eocene (∼36.38 Ma) and subsequent rapid diversification began during Miocene. Ancestral area reconstruction shows that Corylus originated from southwest China. The arrival of two clades (Clades B and C) to NA was well supported by the long distance dispersal crossing the Bering land bridge. The Himalayas, European-Mediterranean area, and other distribution regions are primarily the recipients of dispersal taxa. Vicariance after dispersal plays an important role in speciation.

Assessment of Genetic Diversity and Population Genetic Structure of Corylus mandshurica in China Using SSR Markers.

Corylus mandshurica, also known as pilose hazelnut, is an economically and ecologically important species in China. In this study, ten polymorphic simple sequence repeat (SSR) markers were applied to evaluate the genetic diversity and population structure of 348 C. mandshurica individuals among 12 populations in China. The SSR markers expressed a relatively high level of genetic diversity (Na = 15.3, Ne = 5.6604, I = 1.8853, Ho = 0.6668, and He = 0.7777). According to the coefficient of genetic differentiation (Fst = 0.1215), genetic variation within the populations (87.85%) were remarkably higher than among populations (12.15%). The average gene flow (Nm = 1.8080) significantly impacts the genetic structure of C. mandshurica populations. The relatively high gene flow (Nm = 1.8080) among wild C. mandshurica may be caused by wind-pollinated flowers, highly nutritious seeds and self-incompatible mating system. The UPGMA (unweighted pair group method of arithmetic averages) dendrogram was divided into two main clusters. Moreover, the results of STRUCTURE analysis suggested that C. mandshurica populations fell into two main clusters. Comparison of the UPGMA dendrogram and the Bayesian STRUCTURE analysis showed general agreement between the population subdivisions and the genetic relationships among populations of C. mandshurica. Group I accessions were located in Northeast China, while Group II accessions were in North China. It is worth noting that a number of genetically similar populations were located in the same geographic region. The results further showed that there was obvious genetic differentiation among populations from Northeast China to North China. Results from the Mantel test showed a weak but still significant positive correlation between Nei's genetic distance and geographic distance (km) among populations (r = 0.419, P = 0.005), suggesting that genetic differentiation in the 12 C. mandshurica populations might be related to geographic distance. These data provide comprehensive information for the development of conservation strategies of these valuable hazelnut resources.

Expression and Functional Analysis of WRKY Transcription Factors in Chinese Wild Hazel, Corylus heterophylla Fisch.

Plant WRKY transcription factors are known to regulate various biotic and abiotic stress responses. In this study we identified a total of 30 putative WRKY unigenes in a transcriptome dataset of the Chinese wild Hazel, Corylus heterophylla, a species that is noted for its cold tolerance. Thirteen full-length of these ChWRKY genes were cloned and found to encode complete protein sequences, and they were divided into three groups, based on the number of WRKY domains and the pattern of zinc finger structures. Representatives of each of the groups, Unigene25835 (group I), Unigene37641 (group II) and Unigene20441 (group III), were transiently expressed as fusion proteins with yellow fluorescent fusion protein in Nicotiana benthamiana, where they were observed to accumulate in the nucleus, in accordance with their predicted roles as transcriptional activators. An analysis of the expression patterns of all 30 WRKY genes revealed differences in transcript abundance profiles following exposure to cold, drought and high salinity conditions. Among the stress-inducible genes, 23 were up-regulated by all three abiotic stresses and the WRKY genes collectively exhibited four different patterns of expression in flower buds during the overwintering period from November to April. The organ/tissue related expression analysis showed that 18 WRKY genes were highly expressed in stem but only 2 (Unigene9262 and Unigene43101) were greatest in male anthotaxies. The expression of Unigene37641, a member of the group II WRKY genes, was substantially up-regulated by cold, drought and salinity treatments, and its overexpression in Arabidopsis thaliana resulted in better seedling growth, compared with wild type plants, under cold treatment conditions. The transgenic lines also had exhibited higher soluble protein content, superoxide dismutase and peroxidase activiety and lower levels of malondialdehyde, which collectively suggets that Unigene37641 expression promotes cold tolerance.