Genome-wide identification and characterization of the AP2/ERF gene family in loblolly pine (Pinus taeda L.).

The loblolly pine (Pinus taeda L.) is one of the most profitable forest species worldwide owing to its quick growth, high wood yields, and strong adaptability. The AP2/ERF gene family plays a widespread role in the physiological processes of plant defense responses and the biosynthesis of metabolites. Nevertheless, there are no reports on this gene family in loblolly pine (P. taeda). In this study, a total of 303 members of the AP2/ERF gene family were identified. Through multiple sequence alignment and phylogenetic analysis, they were classified into four subfamilies, including AP2 (34), RAV (17), ERF (251), and Soloist (1). An analysis of the conservation domains, conserved motifs, and gene structure revealed that every PtAP2/ERF transcription factor (TF) had at least one AP2 domain. While evolutionary conservation was displayed within the same subfamilies, the distribution of conserved domains, conserved motifs, and gene architectures varied between subfamilies. Cis-element analysis revealed abundant light-responsive elements, phytohormone-responsive elements, and stress-responsive elements in the promoter of the PtAP2/ERF genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of potential target genes showed that the AP2/ERF gene family might play a critical role in plant growth and development, the response to environmental stresses, and metabolite biosynthesis. Utilizing quantitative real-time PCR (qRT-PCR), we examined the expression patterns of 10 randomly selected genes from Group IX after 6 h of treatments with mechanical injury, ethephon (Eth), and methyl jasmonate (MeJA). The AP2/ERF gene family in the loblolly pine was systematically analyzed for the first time in this study, offering a theoretical basis for exploring the functions and applications of AP2/ERF genes.

The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability.

Salinity is an increasing problem in coastal areas affected by saltwater intrusion, with deleterious effects on tree health and forest growth. Ectomycorrhizal (ECM) fungi may improve the salinity tolerance of host trees, but the impact of external potassium (K+ ) availability on these effects is still unclear. Here, we performed several experiments with the ECM fungus Paxillus ammoniavirescens and loblolly pine (Pinus taeda L.) in axenic and symbiotic conditions at limited or sufficient K+ and increasing sodium (Na+ ) concentrations. Growth rate, biomass, nutrient content, and K+ transporter expression levels were recorded for the fungus, and the colonization rate, root development parameters, biomass, and shoot nutrient accumulation were determined for mycorrhizal and non-mycorrhizal plants. P. ammoniavirescens was tolerant to high salinity, although growth and nutrient concentrations varied with K+ availability and increasing Na+ exposure. While loblolly pine root growth and development decreased with increasing salinity, ECM colonization was unaffected by pine response to salinity. The mycorrhizal influence on loblolly pine salinity response was strongly dependent on external K+ availability. This study reveals that P. ammoniavirescens can reduce Na+ accumulation of salt-exposed loblolly pine, but this effect depends on external K+ availability.

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda).

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.

Spatial Distribution and Retention in Loblolly Pine Seedlings of Exogenous dsRNAs Applied through Roots.

Exogenously applied double-stranded RNA (dsRNA) can induce potent host specific gene knockdown and mortality in insects. The deployment of RNA-interference (RNAi) technologies for pest suppression is gaining traction in both agriculture and horticulture, but its implementation in forest systems is lagging. While numerous forest pests have demonstrated susceptibility to RNAi mediated gene silencing, including the southern pine beetle (SPB), Dendroctonus frontalis, multiple barriers stand between laboratory screening and real-world deployment. One such barrier is dsRNA delivery. One possible delivery method is through host plants, but an understanding of exogenous dsRNA movement through plant tissues is essential. Therefore, we sought to understand the translocation and persistence of dsRNAs designed for SPB throughout woody plant tissues after hydroponic exposure. Loblolly pine, Pinus taeda, seedlings were exposed to dsRNAs as a root soak, followed by destructive sampling. Total RNA was extracted from different tissue types including root, stem, crown, needle, and meristem, after which gel electrophoresis confirmed the recovery of the exogenous dsRNAs, which were further verified using Sanger sequencing. Both techniques confirmed the presence of the exogenously applied target dsRNAs in each tissue type after 1, 3, 5, and 7 d of dsRNA exposure. These findings suggest that root drench applications of exogenous dsRNAs could provide a viable delivery route for RNAi technology designed to combat tree feeding pests.

NLR diversity and candidate fusiform rust resistance genes in loblolly pine.

Resistance to fusiform rust disease in loblolly pine (Pinus taeda) is a classic gene-for-gene system. Early resistance gene mapping in the P. taeda family 10-5 identified RAPD markers for a major fusiform rust resistance gene, Fr1. More recently, single nucleotide polymorphism (SNP) markers associated with resistance were mapped to a full-length gene model in the loblolly pine genome encoding for a nucleotide-binding site leucine-rich repeat (NLR) protein. NLR genes are one of the most abundant gene families in plant genomes and are involved in effector-triggered immunity. Inter- and intraspecies studies of NLR gene diversity and expression have resulted in improved disease resistance. To characterize NLR gene diversity and discover potential resistance genes, we assembled de novo transcriptomes from 92 loblolly genotypes from across the natural range of the species. In these transcriptomes, we identified novel NLR transcripts that are not present in the loblolly pine reference genome and found significant geographic diversity of NLR genes providing evidence of gene family evolution. We designed capture probes for these NLRs to identify and map SNPs that stably cosegregate with resistance to the SC20-21 isolate of Cronartium quercuum f.sp. fusiforme (Cqf) in half-sib progeny of the 10-5 family. We identified 10 SNPs and 2 quantitative trait loci associated with resistance to SC20-21 Cqf. The geographic diversity of NLR genes provides evidence of NLR gene family evolution in loblolly pine. The SNPs associated with rust resistance provide a resource to enhance breeding and deployment of resistant pine seedlings.

Prediction ability of genome-wide markers in Pinus taeda L. within and between population is affected by relatedness to the training population and trait genetic architecture.

Genomic prediction has the potential to significantly increase the rate of genetic gain in tree breeding programs. In this study, a clonally replicated population (n = 2063) was used to train a genomic prediction model. The model was validated both within the training population and in a separate population (n = 451). The prediction abilities from random (20% vs 80%) cross validation within the training population were 0.56 and 0.78 for height and stem form, respectively. Removal of all full-sib relatives within the training population resulted in ∼50% reduction in their genomic prediction ability for both traits. The average prediction ability for all 451 individual trees was 0.29 for height and 0.57 for stem form. The degree of genetic linkage (full-sib family, half sib family, unrelated) between the training and validation sets had a strong impact on prediction ability for stem form but not for height. A dominant dwarfing allele, the first to be reported in a conifer species, was discovered via genome-wide association studies on linkage Group 5 that conferred a 0.33-m mean height reduction. However, the QTL was family specific. The rapid decay of linkage disequilibrium, large genome size, and inconsistencies in marker-QTL linkage phase suggest that large, diverse training populations are needed for genomic selection in Pinus taeda L.

Genomic prediction for fusiform rust disease incidence in a large cloned population of Pinus taeda.

In this study, 723 Pinus taeda L. (loblolly pine) clonal varieties genotyped with 16920 SNP markers were used to evaluate genomic selection for fusiform rust disease caused by the fungus Cronartium quercuum f. sp. fusiforme. The 723 clonal varieties were from five full-sib families. They were a subset of a larger population (1831 clonal varieties), field-tested across 26 locations in the southeast US. Ridge regression, Bayes B, and Bayes Cπ models were implemented to study marker-trait associations and estimate predictive ability for selection. A cross-validation scenario based on a random sampling of 80% of the clonal varieties for the model building had higher (0.71-0.76) prediction accuracies of genomic estimated breeding values compared with family and within-family cross-validation scenarios. Random sampling within families for model training to predict genomic estimated breeding values of the remaining progenies within each family produced accuracies between 0.38 and 0.66. Using four families out of five for model training was not successful. The results showed the importance of genetic relatedness between the training and validation sets. Bayesian whole-genome regression models detected three QTL with large effects on the disease outcome, explaining 54% of the genetic variation in the trait. The significance of QTL was validated with GWAS while accounting for the population structure and polygenic effect. The odds of disease incidence for heterozygous AB genotypes were 10.7 and 12.1 times greater than the homozygous AA genotypes for SNP11965 and SNP6347 loci, respectively. Genomic selection for fusiform rust disease incidence could be effective in P. taeda breeding. Markers with large effects could be fit as fixed covariates to increase the prediction accuracies, provided that their effects are validated further.

Major QTL confer race-nonspecific resistance in the co-evolved Cronartium quercuum f. sp. fusiforme-Pinus taeda pathosystem.

Fusiform rust disease, caused by the endemic fungus Cronartium quercuum f. sp. fusiforme, is the most damaging disease affecting economically important pine species in the southeast United States. Unlike the major epidemics of agricultural crops, the co-evolved pine-rust pathosystem is characterized by steady-state dynamics and high levels of genetic diversity within environments. This poses a unique challenge and opportunity for the deployment of large-effect resistance genes. We used trait dissection to study the genetic architecture of disease resistance in two P. taeda parents that showed high resistance across multiple environments. Two mapping populations (full-sib families), each with ~1000 progeny, were challenged with a complex inoculum consisting of 150 pathogen isolates. High-density linkage mapping revealed three major-effect QTL distributed on two linkage groups. All three QTL were validated using a population of 2057 cloned pine genotypes in a 6-year-old multi-environmental field trial. As a complement to the QTL mapping approach, bulked segregant RNAseq analysis revealed a small number of candidate nucleotide binding leucine-rich repeat genes harboring SNP associated with disease resistance. The results of this study show that in P. taeda, a small number of major QTL can provide effective resistance against genetically diverse mixtures of an endemic pathogen. These QTL vary in their impact on disease liability and exhibit additivity in combination.

Turgor regulation defect 1 proteins play a conserved role in pollen tube reproductive innovation of the angiosperms.

Sexual reproduction in angiosperms is siphonogamous, and the interaction between pollen tube and pistil is critical for successful fertilization. Our previous study demonstrated that mutation of the Arabidopsis turgor regulation defect 1 (TOD1) gene leads to reduced male fertility, a result of retarded pollen tube growth in the pistil. TOD1 encodes a Golgi-localized alkaline ceramidase, a key enzyme for the production of sphingosine-1-phosphate (S1P), which is involved in the regulation of turgor pressure in plant cells. However, whether TOD1s play a conserved role in the innovation of siphonogamy is largely unknown. In this study, we provide evidence that OsTOD1, which is similar to AtTOD1, is also preferentially expressed in rice pollen grains and pollen tubes. OsTOD1 knockout results in reduced pollen tube growth potential in rice pistil. Both the OsTOD1 genomic sequence with its own promoter and the coding sequence under the AtTOD1 promoter can partially rescue the attod1 mutant phenotype. Furthermore, TOD1s from other angiosperm species can partially rescue the attod1 mutant phenotype, while TOD1s from gymnosperm species are not able to complement the attod1 mutant phenotype. Our data suggest that TOD1 acts conservatively in angiosperms, and this opens up an opportunity to dissect the role of sphingolipids in pollen tube growth in angiosperms.

Involvement of VNS NAC-domain transcription factors in tracheid formation in Pinus taeda.

Vascular plants have two types of water-conducting cells, xylem vessel cells (in angiosperms) and tracheid cells (in ferns and gymnosperms). These cells are commonly characterized by secondary cell wall (SCW) formation and programmed cell death (PCD), which increase the efficiency of water conduction. The differentiation of xylem vessel cells is regulated by a set of NAC (NAM, ATAF1/2 and CUC2) transcription factors, called the VASCULAR-RELATED NAC-DOMAIN (VND) family, in Arabidopsis thaliana Linne. The VNDs regulate the transcriptional induction of genes required for SCW formation and PCD. However, information on the transcriptional regulation of tracheid cell differentiation is still limited. Here, we performed functional analysis of loblolly pine (Pinus taeda Linne) VND homologs (PtaVNS, for VND, NST/SND, SMB-related protein). We identified five PtaVNS genes in the loblolly pine genome, and four of these PtaVNS genes were highly expressed in tissues with tracheid cells, such as shoot apices and developing xylem. Transient overexpression of PtaVNS genes induced xylem vessel cell-like patterning of SCW deposition in tobacco (Nicotiana benthamiana Domin) leaves, and up-regulated the promoter activities of loblolly pine genes homologous to SCW-related MYB transcription factor genes and cellulose synthase genes, as well as to cysteine protease genes for PCD. Collectively, our data indicated that PtaVNS proteins possess transcriptional activity to induce the molecular programs required for tracheid formation, i.e., SCW formation and PCD. Moreover, these findings suggest that the VNS-MYB-based transcriptional network regulating water-conducting cell differentiation in angiosperm and moss plants is conserved in gymnosperms.

Predicting Adaptive Genetic Variation of Loblolly Pine (Pinus taeda L.) Populations Under Projected Future Climates Based on Multivariate Models.

Greenhouse gas emission and global warming are likely to cause rapid climate change within the natural range of loblolly pine over the next few decades, thus bringing uncertainty to their adaptation to the environment. Here, we studied adaptive genetic variation of loblolly pine and correlated genetic variation with bioclimatic variables using multivariate modeling methods-Redundancy Analysis, Generalized Dissimilarity Modeling, and Gradient Forests. Studied trees (N = 299) were originally sampled from their native range across eight states on the east side of the Mississippi River. Genetic variation was calculated using a total of 44,317 single-nucleotide polymorphisms acquired by exome target sequencing. The fitted models were used to predict the adaptive genetic variation on a large spatial and temporal scale. We observed east-to-west spatial genetic variation across the range, which presented evidence of isolation by distance. Different key factors drive adaptation of loblolly pine from different geographical regions. Trees residing near the northeastern edge of the range, spanning across Delaware and Maryland and mountainous areas of Virginia, North Carolina, South Carolina, and northern Georgia, were identified to be most likely impacted by climate change based on the large difference in genetic composition under current and future climate conditions. This study provides new perspectives on adaptive genetic variation of loblolly pine in response to different climate scenarios, and the results can be used to target particular populations while developing adaptive forest management guidelines.

Environmental Genome-Wide Association Reveals Climate Adaptation Is Shaped by Subtle to Moderate Allele Frequency Shifts in Loblolly Pine.

Understanding the genomic basis of local adaptation is crucial to determine the potential of long-lived woody species to withstand changes in their natural environment. In the past, efforts to dissect the genomic architecture in gymnosperms species have been limited due to the absence of reference genomes. Recently, the genomes of some commercially important conifers, such as loblolly pine, have become available, allowing whole-genome studies of these species. In this study, we test for associations between 87k SNPs, obtained from whole-genome resequencing of loblolly pine individuals, and 270 environmental variables and combinations of them. We determine the geographic location of significant loci and identify their genomic location using our newly constructed ultradense 26k SNP linkage map. We found that water availability is the main climatic variable shaping local adaptation of the species, and found 821 SNPs showing significant associations with climatic variables or combinations of them based on the consistent results of three different genotype-environment association methods. Our results suggest that adaptation to climate in the species might have occurred by many changes in the frequency of alleles with moderate to small effect sizes, and by the smaller contribution of large effect alleles in genes related to moisture deficit, temperature and precipitation. Genomic regions of low recombination and high population differentiation harbored SNPs associated with groups of environmental variables, suggesting climate adaptation might have evolved as a result of different selection pressures acting on groups of genes associated with an aspect of climate rather than on individual environmental variables.

Exome Resequencing Reveals Evolutionary History, Genomic Diversity, and Targets of Selection in the Conifers Pinus taeda and Pinus elliottii.

Loblolly pine (Pinus taeda) and slash pine (Pinus elliottii) are ecologically and economically important pine species that dominate many forest ecosystems in the southern United States, but like all conifers, the study of their genetic diversity and demographic history has been hampered by their large genome size. A small number of studies mainly based on candidate-gene sequencing have been reported for P. taeda to date, whereas none are available for P. elliottii. Targeted exome resequencing has recently enabled population genomics studies for conifers, approach used here to assess genomic diversity, signatures of selection, population structure, and demographic history of P. elliottii and P. taeda. Extensive similarities were revealed between these species: both species feature rapid linkage disequilibrium decay and high levels of genetic diversity. Moreover, genome-wide positive correlations for measures of genetic diversity between the species were also observed, likely due to shared structural genomic constraints. Also, positive selection appears to be targeting a common set of genes in both pines. Demographic history differs between both species, with only P. taeda being affected by a dramatic bottleneck during the last glacial period. The ability of P. taeda to recover from a dramatic reduction in population size while still retaining high levels of genetic diversity shows promise for other pines facing environmental stressors associated with climate change, indicating that these too may be able to adapt successfully to new future conditions even after a drastic population size contraction.

Association mapping of ectomycorrhizal traits in loblolly pine (Pinus taeda L.).

To understand how diverse mutualisms coevolve and how species adapt to complex environments, a description of the underlying genetic basis of the traits involved must be provided. For example, in diverse coevolving mutualisms, such as the interaction of host plants with a suite of symbiotic mycorrhizal fungi, a key question is whether host plants can coevolve independently with multiple species of symbionts, which depends on whether those interactions are governed independently by separate genes or pleiotropically by shared genes. To provide insight into this question, we employed an association mapping approach in a clonally replicated field experiment of loblolly pine (Pinus taeda L.) to identify genetic components of host traits governing ectomycorrhizal (EM) symbioses (mycorrhizal traits). The relative abundances of different EM fungi as well as the total number of root tips per cm root colonized by EM fungi were analyzed as separate mycorrhizal traits of loblolly pine. Single-nucleotide polymorphisms (SNPs) within candidate genes of loblolly pine were associated with loblolly pine mycorrhizal traits, mapped to the loblolly pine genome, and their putative protein function obtained when available. The results support the hypothesis that ectomycorrhiza formation is governed by host genes of large effect that apparently have independent influences on host interactions with different symbiont species.

Genomic architecture of complex traits in loblolly pine.

Dissecting the genetic and genomic architecture of complex traits is essential to understand the forces maintaining the variation in phenotypic traits of ecological and economical importance. Whole-genome resequencing data were used to generate high-resolution polymorphic single nucleotide polymorphism (SNP) markers and genotype individuals from common gardens across the loblolly pine (Pinus taeda) natural range. Genome-wide associations were tested with a large phenotypic dataset comprising 409 variables including morphological traits (height, diameter, carbon isotope discrimination, pitch canker resistance), and molecular traits such as metabolites and expression of xylem development genes. Our study identified 2335 new SNP × trait associations for the species, with many SNPs located in physical clusters in the genome of the species; and the genomic location of hotspots for metabolic × genotype associations. We found a highly polygenic basis of quantitative inheritance, with significant differences in number, effects size, genomic location and frequency of alleles contributing to variation in phenotypes in the different traits. While mutation-selection balance might be shaping the genetic variation in metabolic traits, balancing selection is more likely to shape the variation in expression of xylem development genes. Our work contributes to the study of complex traits in nonmodel plant species by identifying associations at a whole-genome level.

Exploring the genetic basis of gene transcript abundance and metabolite levels in loblolly pine (Pinus taeda L.) using association mapping and network construction.

BACKGROUND: Identifying genetic variations that shape important complex traits is fundamental to the genetic improvement of important forest tree species, such as loblolly pine (Pinus taeda L.), which is one of the most commonly planted forest tree species in the southern U.S. Gene transcripts and metabolites are important regulatory intermediates that link genetic variations to higher-order complex traits such as wood development and drought response. A few prior studies have associated intermediate phenotypes including mRNA expression and metabolite levels with a limited number of molecular markers, but the identification of genetic variations that regulate intermediate phenotypes needs further investigation. RESULTS: We identified 1841 single nucleotide polymorphisms (SNPs) associated with 191 gene expression mRNA phenotypes and 524 SNPs associated with 53 metabolite level phenotypes using 2.8 million exome-derived SNPs. The identified SNPs reside in genes with a wide variety of functions. We further integrated the identified SNPs and the associated expressed genes and metabolites into networks. We described the SNP-SNP interactions that significantly impacted the gene transcript abundance and metabolite level in the networks. Key loci and genes in the wood development and drought response networks were identified and analyzed. CONCLUSIONS: This work provides new candidate genes for research on the genetic basis of gene expression and metabolism linked to wood development and drought response in loblolly pine and highlights the efficiency of using association-mapping-based networks to discover candidate genes with important roles in complex biological processes.

Exploring the loblolly pine (Pinus taeda L.) genome by BAC sequencing and Cot analysis.

Loblolly pine (LP; Pinus taeda L.) is an economically and ecologically important tree in the southeastern U.S. To advance understanding of the loblolly pine (LP; Pinus taeda L.) genome, we sequenced and analyzed 100 BAC clones and performed a Cot analysis. The Cot analysis indicates that the genome is composed of 57, 24, and 10% highly-repetitive, moderately-repetitive, and single/low-copy sequences, respectively (the remaining 9% of the genome is a combination of fold back and damaged DNA). Although single/low-copy DNA only accounts for 10% of the LP genome, the amount of single/low-copy DNA in LP is still 14 times the size of the Arabidopsis genome. Since gene numbers in LP are similar to those in Arabidopsis, much of the single/low-copy DNA of LP would appear to be composed of DNA that is both gene- and repeat-poor. Macroarrays prepared from a LP bacterial artificial chromosome (BAC) library were hybridized with probes designed from cell wall synthesis/wood development cDNAs, and 50 of the "targeted" clones were selected for further analysis. An additional 25 clones were selected because they contained few repeats, while 25 more clones were selected at random. The 100 BAC clones were Sanger sequenced and assembled. Of the targeted BACs, 80% contained all or part of the cDNA used to target them. One targeted BAC was found to contain fungal DNA and was eliminated from further analysis. Combinations of similarity-based and ab initio gene prediction approaches were utilized to identify and characterize potential coding regions in the 99 BACs containing LP DNA. From this analysis, we identified 154 gene models (GMs) representing both putative protein-coding genes and likely pseudogenes. Ten of the GMs (all of which were specifically targeted) had enough support to be classified as intact genes. Interestingly, the 154 GMs had statistically indistinguishable (α = 0.05) distributions in the targeted and random BAC clones (15.18 and 12.61 GM/Mb, respectively), whereas the low-repeat BACs contained significantly fewer GMs (7.08 GM/Mb). However, when GM length was considered, the targeted BACs had a significantly greater percentage of their length in GMs (3.26%) when compared to random (1.63%) and low-repeat (0.62%) BACs. The results of our study provide insight into LP evolution and inform ongoing efforts to produce a reference genome sequence for LP, while characterization of genes involved in cell wall production highlights carbon metabolism pathways that can be leveraged for increasing wood production.

Complete chloroplast genome sequence and comparative analysis of loblolly pine (Pinus taeda L.) with related species.

Pinaceae, the largest family of conifers, has a diversified organization of chloroplast (cp) genomes with two typical highly reduced inverted repeats (IRs). In the current study, we determined the complete sequence of the cp genome of an economically and ecologically important conifer tree, the loblolly pine (Pinus taeda L.), using Illumina paired-end sequencing and compared the sequence with those of other pine species. The results revealed a genome size of 121,531 base pairs (bp) containing a pair of 830-bp IR regions, distinguished by a small single copy (42,258 bp) and large single copy (77,614 bp) region. The chloroplast genome of P. taeda encodes 120 genes, comprising 81 protein-coding genes, four ribosomal RNA genes, and 35 tRNA genes, with 151 randomly distributed microsatellites. Approximately 6 palindromic, 34 forward, and 22 tandem repeats were found in the P. taeda cp genome. Whole cp genome comparison with those of other Pinus species exhibited an overall high degree of sequence similarity, with some divergence in intergenic spacers. Higher and lower numbers of indels and single-nucleotide polymorphism substitutions were observed relative to P. contorta and P. monophylla, respectively. Phylogenomic analyses based on the complete genome sequence revealed that 60 shared genes generated trees with the same topologies, and P. taeda was closely related to P. contorta in the subgenus Pinus. Thus, the complete P. taeda genome provided valuable resources for population and evolutionary studies of gymnosperms and can be used to identify related species.

An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly.

Genes Sufficient for Synthesizing Peptidoglycan are Retained in Gymnosperm Genomes, and MurE from Larix gmelinii can Rescue the Albino Phenotype of Arabidopsis MurE Mutation.

The endosymbiotic theory states that plastids are derived from a single cyanobacterial ancestor that possessed a cell wall. Peptidoglycan (PG), the main component of the bacteria cell wall, gradually degraded during plastid evolution. PG-synthesizing Mur genes have been found to be retained in the genomes of basal streptophyte plants, although many of them have been lost from the genomes of angiosperms. The enzyme encoded by bacterial MurE genes catalyzes the formation of the UDP-N-acetylmuramic acid (UDP-MurNAc) tripeptide in bacterial PG biosynthesis. Knockout of the MurE gene in the moss Physcomitrella patens resulted in defects of chloroplast division, whereas T-DNA-tagged mutants of Arabidopsis thaliana for MurE revealed inhibition of chloroplast development but not of plastid division, suggesting that AtMurE is functionally divergent from the bacterial and moss MurE proteins. Here, we could identify 10 homologs of bacterial Mur genes, including MurE, in the recently sequenced genomes of Picea abies and Pinus taeda, suggesting the retention of the plastid PG system in gymnosperms. To investigate the function of gymnosperm MurE, we isolated an ortholog of MurE from the larch, Larix gmelinii (LgMurE) and confirmed its presence as a single copy per genome, as well as its abundant expression in the leaves of larch seedlings. Analysis with a fusion protein combining green fluorescent protein and LgMurE suggested that it localizes in chloroplasts. Cross-species complementation assay with MurE mutants of A. thaliana and P. patens showed that the expression of LgMurE cDNA completely rescued the albefaction defects in A. thaliana but did not rescue the macrochloroplast phenotype in P. patens. The evolution of plastid PG and the mechanism behind the functional divergence of MurE genes are discussed in the context of information about plant genomes at different evolutionary stages.