Shifts in evolutionary lability underlie independent gains and losses of root-nodule symbiosis in a single clade of plants.

Root nodule symbiosis (RNS) is a complex trait that enables plants to access atmospheric nitrogen converted into usable forms through a mutualistic relationship with soil bacteria. Pinpointing the evolutionary origins of RNS is critical for understanding its genetic basis, but building this evolutionary context is complicated by data limitations and the intermittent presence of RNS in a single clade of ca. 30,000 species of flowering plants, i.e., the nitrogen-fixing clade (NFC). We developed the most extensive de novo phylogeny for the NFC and an RNS trait database to reconstruct the evolution of RNS. Our analysis identifies evolutionary rate heterogeneity associated with a two-step process: An ancestral precursor state transitioned to a more labile state from which RNS was rapidly gained at multiple points in the NFC. We illustrate how a two-step process could explain multiple independent gains and losses of RNS, contrary to recent hypotheses suggesting one gain and numerous losses, and suggest a broader phylogenetic and genetic scope may be required for genome-phenome mapping.

The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

Bringing CAM photosynthesis to the table: Paving the way for resilient and productive agricultural systems in a changing climate.

Modern agricultural systems are directly threatened by global climate change and the resulting freshwater crisis. A considerable challenge in the coming years will be to develop crops that can cope with the consequences of declining freshwater resources and changing temperatures. One approach to meeting this challenge may lie in our understanding of plant photosynthetic adaptations and water use efficiency. Plants from various taxa have evolved crassulacean acid metabolism (CAM), a water-conserving adaptation of photosynthetic carbon dioxide fixation that enables plants to thrive under semi-arid or seasonally drought-prone conditions. Although past research on CAM has led to a better understanding of the inner workings of plant resilience and adaptation to stress, successful introduction of this pathway into C3 or C4 plants has not been reported. The recent revolution in molecular, systems, and synthetic biology, as well as innovations in high-throughput data generation and mining, creates new opportunities to uncover the minimum genetic tool kit required to introduce CAM traits into drought-sensitive crops. Here, we propose four complementary research avenues to uncover this tool kit. First, genomes and computational methods should be used to improve understanding of the nature of variations that drive CAM evolution. Second, single-cell 'omics technologies offer the possibility for in-depth characterization of the mechanisms that trigger environmentally controlled CAM induction. Third, the rapid increase in new 'omics data enables a comprehensive, multimodal exploration of CAM. Finally, the expansion of functional genomics methods is paving the way for integration of CAM into farming systems.

Genetic architecture of terpene chemistry and growth traits and the impact of inbreeding on these traits in western redcedar (Thuja plicata).

Western redcedar (WRC; Thuja plicata) is a conifer of the Pacific Northwest of North America prized for its durable and rot-resistant wood. WRC has naturally low outcrossing rates and readily self-fertilizes in nature. Challenges faced in WRC breeding and propagation involve selecting trees for accelerated growth while also ensuring enhanced heartwood rot resistance and resistance to ungulate browsing, as well as mitigating potential effects of inbreeding depression. Terpenes, a large and diverse class of specialized metabolites, confer both rot and browse resistance in the wood and foliage of WRC, respectively. Using a Bayesian modelling approach, we isolated single nucleotide polymorphism (SNP) markers estimated to be associated with three different foliar terpene traits and four different heartwood terpene traits, as well as two growth traits. We found that all traits were complex, being associated with between 1700 and 3600 SNPs linked with putatively causal loci, with significant polygenic components. Growth traits tended to have a larger polygenic component while terpene traits had larger major gene components; SNPs with small or polygenic effect were spread across the genome, while larger-effect SNPs tended to be localized to specific linkage groups. To determine whether there was inbreeding depression for terpene chemistry or growth traits, we used mixed linear models for a genomic selection training population to estimate the effect of the inbreeding coefficient F on foliar terpenes, heartwood terpenes and several growth and dendrochronological traits. We did not find significant inbreeding depression for any assessed trait. We further assessed inbreeding depression across four generations of complete selfing and found that not only was inbreeding depression not significant but that selection for height growth was the only significant predictor for growth during selfing, suggesting that inbreeding depression due to selfing during operational breeding can be mitigated by increased selection intensity.

Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula.

BACKGROUND: Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. RESULTS: We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. CONCLUSIONS: Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

Single-nuclei transcriptome analysis of the shoot apex vascular system differentiation in Populus.

Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials - a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

Genomic selection reveals hidden relatedness and increased breeding efficiency in western redcedar polycross breeding.

Western redcedar (WRC) is an ecologically and economically important forest tree species characterized by low genetic diversity with high self-compatibility and high heartwood durability. Using sequence capture genotyping of target genic and non-genic regions, we genotyped 44 parent trees and 1520 offspring trees representing 26 polycross (PX) families collected from three progeny test sites using 45,378 SNPs. Trees were phenotyped for eight traits related to growth, heartwood and foliar chemistry associated with wood durability and deer browse resistance. We used the genomic realized relationship matrix for paternity assignment, maternal pedigree correction, and to estimate genetic parameters. We compared genomics-based (GBLUP) and two pedigree-based (ABLUP: polycross and reconstructed full-sib [FS] pedigrees) models. Models were extended to estimate dominance genetic effects. Pedigree reconstruction revealed significant unequal male contribution and separated the 26 PX families into 438 FS families. Traditional maternal PX pedigree analysis resulted in up to 51% overestimation in genetic gain and 44% in diversity. Genomic analysis resulted in up to 22% improvement in offspring breeding value (BV) theoretical accuracy, 35% increase in expected genetic gain for forward selection, and doubled selection intensity for backward selection. Overall, all traits showed low to moderate heritability (0.09-0.28), moderate genotype by environment interaction (type-B genetic correlation: 0.51-0.80), low to high expected genetic gain (6.01%-55%), and no significant negative genetic correlation reflecting no large trade-offs for multi-trait selection. Only three traits showed a significant dominance effect. GBLUP resulted in smaller but more accurate heritability estimates for five traits, but larger estimates for the wood traits. Comparison between all, genic-coding, genic-non-coding and intergenic SNPs showed little difference in genetic estimates. In summary, we show that GBLUP overcomes the PX limitations, successfully captures expected historical and hidden relatedness as well as linkage disequilibrium (LD), and results in increased breeding efficiency in WRC.

The western redcedar genome reveals low genetic diversity in a self-compatible conifer.

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity.

Decoding exceptional plant traits by comparative single-cell genomics.

Some plants have acquired traits of remarkable adaptive value to thrive under stress. Transferring these traits to crops could improve agriculture, but uncovering the toolkit required has remained largely elusive. We propose that single-cell genomics offers a framework to compare species with contrasting developmental traits and to identify the regulators of evolutionary innovations.

An LCO-responsive homolog of NODULE INCEPTION positively regulates lateral root formation in Populus sp.

The transcription factor NODULE INCEPTION (NIN) has been studied extensively for its multiple roles in root nodule symbiosis within plants of the nitrogen-fixing clade (NFC) that associate with soil bacteria, such as rhizobia and Frankia. However, NIN homologs are present in plants outside the NFC, suggesting a role in other developmental processes. Here, we show that the biofuel crop Populus sp., which is not part of the NFC, contains eight copies of NIN with diversified protein sequence and expression patterns. Lipo-chitooligosaccharides (LCOs) are produced by rhizobia and a wide range of fungi, including mycorrhizal ones, and act as symbiotic signals that promote lateral root formation. RNAseq analysis of Populus sp. treated with purified LCO showed induction of the PtNIN2 subfamily. Moreover, the expression of PtNIN2b correlated with the formation of lateral roots and was suppressed by cytokinin treatment. Constitutive expression of PtNIN2b overcame the inhibition of lateral root development by cytokinin under high nitrate conditions. Lateral root induction in response to LCOs likely represents an ancestral function of NIN retained and repurposed in nodulating plants, as we demonstrate that the role of NIN in LCO-induced root branching is conserved in both Populus sp. and legumes. We further established a visual marker of LCO perception in Populus sp. roots, the putative sulfotransferase PtSS1 that can be used to study symbiotic interactions with the bacterial and fungal symbionts of Populus sp.

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade.

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root-nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

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.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Genomic prediction in family bulks using different traits and cross-validations in pine.

Genomic prediction integrates statistical, genomic, and computational tools to improve the estimation of breeding values and increase genetic gain. Due to the broad diversity in mating systems, breeding schemes, propagation methods, and unit of selection, no universal genomic prediction approach can be applied in all crops. In a genome-wide family prediction (GWFP) approach, the family is the basic unit of selection. We tested GWFP in two loblolly pine (Pinus taeda L.) datasets: a breeding population composed of 63 full-sib families (5-20 individuals per family), and a simulated population with the same pedigree structure. In both populations, phenotypic and genomic data was pooled at the family level in silico. Marker effects were estimated to compute genomic estimated breeding values (GEBV) at the individual and family (GWFP) levels. Less than six individuals per family produced inaccurate estimates of family phenotypic performance and allele frequency. Tested across different scenarios, GWFP predictive ability was higher than those for GEBV in both populations. Validation sets composed of families with similar phenotypic mean and variance as the training population yielded predictions consistently higher and more accurate than other validation sets. Results revealed potential for applying GWFP in breeding programs whose selection unit are family, and for systems where family can serve as training sets. The GWFP approach is well suited for crops that are routinely genotyped and phenotyped at the plot-level, but it can be extended to other breeding programs. Higher predictive ability obtained with GWFP would motivate the application of genomic prediction in these situations.

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.

Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues' secondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

A new, simple, highly scalable, and efficient protocol for genomic DNA extraction from diverse plant taxa.

PREMISE: Commonly used molecular techniques such as next-generation sequencing require reliable methods to extract DNA quickly and efficiently. Secondary compounds within plant tissues make this requirement all the more challenging, often forcing researchers to test different extraction methods tailored to their particular species of interest in order to obtain large amounts of high-quality genomic DNA. The opportunities provided by high-throughput, next-generation sequencing only exacerbate these problems, especially when trying to extract DNA from multiple species at the same time. Several methods have attempted to resolve the challenges of obtaining suitable DNA from plants; however, a rapid, high-yield, high-quality, and highly scalable DNA extraction method is still needed. METHODS AND RESULTS: We present a rapid DNA extraction protocol that utilizes a buffer with relatively large amounts of cetyltrimethylammonium bromide (CTAB) and sodium chloride, combined with a silica maxi-column cleanup of the extracted DNA. The new method is easy to implement using standard equipment and inexpensive reagents. The entire procedure (from grinding to the final elution) can be completed in less than two hours for a single sample and can be easily scaled to meet desired research goals. It works on diverse green plants with highly varied secondary chemistries (e.g., ferns, gymnosperms, and phylogenetically divergent angiosperms). CONCLUSIONS: Application of the protocol to various plant species yielded DNA of high quality in less than two hours and can be adjusted to extract DNA at large (maxi-preps) or small (96-well minipreps) scales. We anticipate that our method will be of wide utility for rapidly isolating large quantities of quality genomic DNA from diverse plant species and will have broad applications in phylogenetic studies utilizing PCR and short-read DNA sequencing.

Accelerating forest tree breeding by integrating genomic selection and greenhouse phenotyping.

Breeding forest species can be a costly and slow process because of the extensive areas needed for field trials and the long periods (e.g., five years) that are required to measure economically and environmentally relevant phenotypes (e.g., adult plant biomass or plant height). Genomic selection (GS) and indirect selection using early phenotypes (e.g., phenotypes collected in greenhouse conditions) are two ways by which tree breeding can be accelerated. These approaches can both reduce the costs of field-testing and the time required to make selection decisions. Moreover, these approaches can be highly synergistic. Therefore, in this study, we used a data set comprising DNA genotypes and longitudinal measurements of growth collected from a population of Populus deltoides W. Bartram ex Marshall (eastern cottonwood) in the greenhouse and the field, to evaluate the potential impact of integrating large-scale greenhouse phenotyping with conventional GS. We found that the integration of greenhouse phenotyping and GS can deliver very early selection decisions that are moderately accurate. Therefore, we conclude that the adoption of these approaches, in conjunction with reproductive techniques that shorten the generation interval, can lead to an unprecedented acceleration of selection gains in P. deltoides and, potentially, other commercially planted tree species.

Xylem systems genetics analysis reveals a key regulator of lignin biosynthesis in Populus deltoides.

Despite the growing resources and tools for high-throughput characterization and analysis of genomic information, the discovery of the genetic elements that regulate complex traits remains a challenge. Systems genetics is an emerging field that aims to understand the flow of biological information that underlies complex traits from genotype to phenotype. In this study, we used a systems genetics approach to identify and evaluate regulators of the lignin biosynthesis pathway in Populus deltoides by combining genome, transcriptome, and phenotype data from a population of 268 unrelated individuals of P. deltoides The discovery of lignin regulators began with the quantitative genetic analysis of the xylem transcriptome and resulted in the detection of 6706 and 4628 significant local- and distant-eQTL associations, respectively. Among the locally regulated genes, we identified the R2R3-MYB transcription factor MYB125 (Potri.003G114100) as a putative trans-regulator of the majority of genes in the lignin biosynthesis pathway. The expression of MYB125 in a diverse population positively correlated with lignin content. Furthermore, overexpression of MYB125 in transgenic poplar resulted in increased lignin content, as well as altered expression of genes in the lignin biosynthesis pathway. Altogether, our findings indicate that MYB125 is involved in the control of a transcriptional coexpression network of lignin biosynthesis genes during secondary cell wall formation in P. deltoides.

The Genetic Regulation of Alternative Splicing in Populus deltoides.

Alternative splicing (AS) is a mechanism of regulation of the proteome via enabling the production of multiple mRNAs from a single gene. To date, the dynamics of AS and its effects on the protein sequences of individuals in a large and genetically unrelated population of trees have not been investigated. Here we describe the diversity of AS events within a previously genotyped population of 268 individuals of Populus deltoides and their putative downstream functional effects. Using a robust bioinformatics pipeline, the AS events and resulting transcript isoforms were discovered and quantified for each individual in the population. Analysis of the AS revealed that, as expected, most AS isoforms are conserved. However, we also identified a substantial collection of new, unannotated splice junctions and transcript isoforms. Heritability estimates for the expression of transcript isoforms showed that approximately half of the isoforms are heritable. The genetic regulators of these AS isoforms and splice junction usage were then identified using a genome-wide association analysis. The expression of AS isoforms was predominately cis regulated while splice junction usage was generally regulated in trans. Additionally, we identified 696 genes encoding alternatively spliced isoforms that changed putative protein domains relative to the longest protein coding isoform of the gene, and 859 genes exhibiting this same phenomenon relative to the most highly expressed isoform. Finally, we found that 748 genes gained or lost micro-RNA binding sites relative to the longest protein coding isoform of a given gene, while 940 gained or lost micro-RNA binding sites relative to the most highly expressed isoform. These results indicate that a significant fraction of AS events are genetically regulated and that this isoform usage can result in protein domain architecture changes.