Genome-wide methylation landscape during somatic embryogenesis in Medicago truncatula reveals correlation between Tnt1 retrotransposition and hyperactive methylation regions.

Medicago truncatula is a model legume for fundamental research on legume biology and symbiotic nitrogen fixation. Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants in M. truncatula R108. Approximately 21 000 insertion lines have been generated and publicly available. Tnt1 retro-transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE of M. truncatula R108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2-kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site of Tnt1 insertions in M. truncatula R108 and stronger hypermethylation of genes correlated with higher number of Tnt1 insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and the Tnt1 retrotransposition correlates with the hyperactive methylation regions.

Transcript profiling of hairy vetch (Vicia villosa Roth) identified interesting genes for seed dormancy.

Hairy vetch, a diploid annual legume species, has a robust growth habit, high biomass yield, and winter hardy characteristics. Seed hardness is a major constraint for growing hairy vetch commercially. Hard seeded cultivars are valuable as forages, whereas soft seeded and shatter resistant cultivars have advantages for their use as a cover crop. Transcript analysis of hairy vetch was performed to understand the genetic mechanisms associated with important hairy vetch traits. RNA was extracted from leaves, flowers, immature pods, seed coats, and cotyledons of contrasting soft and hard seeded "AU Merit" plants. A range of 31.22-79.18 Gb RNA sequence data per tissue sample were generated with estimated coverage of 1040-2639×. RNA sequence assembly and mapping of the contigs against the Medicago truncatula (V4.0) genome identified 76,422 gene transcripts. A total of 24,254 transcripts were constitutively expressed in hairy vetch tissues. Key genes, such as KNOX4 (a class II KNOTTED-like homeobox KNOXII gene), qHs1 (endo-1,4-ß-glucanase), GmHs1-1 (calcineurin-like metallophosphoesterase), chitinase, shatterproof 1 and 2 (SHP1, SHP2), shatter resistant 1-5 (SHAT1-5)(NAC transcription factor), PDH1 (prephenate dehydrogenase 1), and pectin methylesterases with a potential role in seed hardness and pod shattering, were further explored based on genes involved in seed hardness from other species to query the hairy vetch transcriptome data. Identification of interesting candidate genes in hairy vetch can facilitate the development of improved cultivars with desirable seed characteristics for use as a forage and as a cover crop.

Elucidating the unknown transcriptional responses and PHR1-mediated biotic and abiotic stress tolerance during phosphorus limitation.

Phosphorus (P) limitation in the majority of world soils is a major constraint for plant growth and crop productivity. RNA sequencing was used to discover novel P-responsive gene transcripts (PRGTs) in leaves and roots of Arabidopsis. Hisat StringTie and the Cufflinks TopHat transcript assembler were used to analyze reads and identify 1074 PRGTs with a >5-fold altered abundance during P limitation. Interestingly, 60% of these transcripts were not previously reported. Among the novel PRGTs, 106 were from unannotated genes, and some were among the most P-responsive, including At2g36727 which encodes a novel miRNA. Annotated novel PRGTs encode transcription factors, miRNAs, small signaling peptides, long non-coding RNAs, defense-related proteins, and transporters, along with proteins involved in many biological processes. We identified several genes that undergo alternative splicing during P limitation, including a novel miR399-resistant splice variant of PHOSPHATE2 (PHO2.2). Several novel P-responsive genes were regulated by PHOSPHATE STARVATION RESPONSE1 (PHR1), PHR1-LIKE 1 (PHL1), and PHO2. We discovered that P-limited plants show increased resistance to pathogens and drought stress mediated by PHR1-PHL1. Identification of novel P-responsive transcripts and the discovery of the influence of P limitation on biotic and abiotic stress adds a significant component to our understanding of plant P signaling.

Comparative Genome Analyses of Plant Rust Pathogen Genomes Reveal a Confluence of Pathogenicity Factors to Quell Host Plant Defense Responses.

Switchgrass rust caused by Puccinia novopanici (P. novopanici) has the ability to significantly affect the biomass yield of switchgrass, an important biofuel crop in the United States. A comparative genome analysis of P. novopanici with rust pathogen genomes infecting monocot cereal crops wheat, barley, oats, maize and sorghum revealed the presence of larger structural variations contributing to their genome sizes. A comparative alignment of the rust pathogen genomes resulted in the identification of collinear and syntenic relationships between P. novopanici and P. sorghi; P. graminis tritici 21-0 (Pgt 21) and P. graminis tritici Ug99 (Pgt Ug99) and between Pgt 21 and P. triticina (Pt). Repeat element analysis indicated a strong presence of retro elements among different Puccinia genomes, contributing to the genome size variation between ~1 and 3%. A comparative look at the enriched protein families of Puccinia spp. revealed a predominant role of restriction of telomere capping proteins (RTC), disulfide isomerases, polysaccharide deacetylases, glycoside hydrolases, superoxide dismutases and multi-copper oxidases (MCOs). All the proteomes of Puccinia spp. share in common a repertoire of 75 secretory and 24 effector proteins, including glycoside hydrolases cellobiohydrolases, peptidyl-propyl isomerases, polysaccharide deacetylases and protein disulfide-isomerases, that remain central to their pathogenicity. Comparison of the predicted effector proteins from Puccinia spp. genomes to the validated proteins from the Pathogen-Host Interactions database (PHI-base) resulted in the identification of validated effector proteins PgtSR1 (PGTG_09586) from P. graminis and Mlp124478 from Melampsora laricis across all the rust pathogen genomes.

Transcriptome of Endophyte-Positive and Endophyte-Free Tall Fescue Under Field Stresses.

Tall fescue is one of the primary sources of forage for livestock. It grows well in the marginal soils of the temperate zones. It hosts a fungal endophyte (Epichloë coenophiala), which helps the plants to tolerate abiotic and biotic stresses. The genomic and transcriptomic resources of tall fescue are very limited, due to a complex genetic background and outbreeding modes of pollination. The aim of this study was to identify differentially expressed genes (DEGs) in two tissues (pseudostem and leaf blade) between novel endophyte positive (E+) and endophyte-free (E-) Texoma MaxQ II tall fescue genotypes. Samples were collected at three diurnal time points: morning (7:40-9:00 am), afternoon (1:15-2:15 pm), and evening (4:45-5:45 pm) in the field environment. By exploring the transcriptional landscape via RNA-seq, for the first time, we generated 226,054 and 224,376 transcripts from E+ and E- tall fescue, respectively through de novo assembly. The upregulated transcripts were detected fewer than the downregulated ones in both tissues (S: 803 up and 878 down; L: 783 up and 846 down) under the freezing temperatures (-3.0-0.5°C) in the morning. Gene Ontology enrichment analysis identified 3 out of top 10 significant GO terms only in the morning samples. Metabolic pathway and biosynthesis of secondary metabolite genes showed lowest number of DEGs under morning freezing stress and highest number in evening cold condition. The 1,085 DEGs were only expressed under morning stress condition and, more importantly, the eight candidate orthologous genes of rice identified under morning freezing temperatures, including orthologs of rice phytochrome A, phytochrome C, and ethylene receptor genes, might be the possible route underlying cold tolerance in tall fescue.

Transcriptional Programs and Regulators Underlying Age-Dependent and Dark-Induced Senescence in Medicago truncatula.

In forage crops, age-dependent and stress-induced senescence reduces forage yield and quality. Therefore, delaying leaf senescence may be a way to improve forage yield and quality as well as plant resilience to stresses. Here, we used RNA-sequencing to determine the molecular bases of age-dependent and dark-induced leaf senescence in Medicago truncatula. We identified 6845 differentially expressed genes (DEGs) in M3 leaves associated with age-dependent leaf senescence. An even larger number (14219) of DEGs were associated with dark-induced senescence. Upregulated genes identified during age-dependent and dark-induced senescence were over-represented in oxidation-reduction processes and amino acid, carboxylic acid and chlorophyll catabolic processes. Dark-specific upregulated genes also over-represented autophagy, senescence and cell death. Mitochondrial functions were strongly inhibited by dark-treatment while these remained active during age-dependent senescence. Additionally, 391 DE transcription factors (TFs) belonging to various TF families were identified, including a core set of 74 TFs during age-dependent senescence while 759 DE TFs including a core set of 338 TFs were identified during dark-induced senescence. The heterologous expression of several senescence-induced TFs belonging to NAC, WKRY, bZIP, MYB and HD-zip TF families promoted senescence in tobacco leaves. This study revealed the dynamics of transcriptomic responses to age- and dark-induced senescence in M. truncatula and identified senescence-associated TFs that are attractive targets for future work to control senescence in forage legumes.

Overexpression of Arabidopsis nucleolar GTP-binding 1 (NOG1) proteins confers drought tolerance in rice.

As a major adverse environmental factor in most parts of the world, drought causes substantial crop yield losses. Rice (Oryza sativa) is one of the staple foods for more than one-half of the world's population. Rice plants are sensitive to even mild drought stress and need almost twice the amount of water compared to wheat (Triticum aestivum) or maize (Zea mays). Arabidopsis (Arabidopsis thaliana) small GTPase Nucleolar GTP-binding protein 1 (AtNOG1) plays a role in biotic stress tolerance. Here, we created transgenic rice lines constitutively overexpressing AtNOG1-1 or AtNOG1-2. We also developed rice RNA interference (RNAi) lines that show downregulation of OsNOG1. AtNOG1-1 and AtNOG1-2 overexpressors showed enhanced drought tolerance without compromising grain yield, whereas OsNOG1-RNAi was more susceptible to drought when compared to wild-type plants. Analysis of physiological parameters showed increased cell sap osmolality, relative water content, and abscisic acid (ABA) level, but decreased leaf water loss in AtNOG1-1 or AtNOG1-2 overexpressor lines compared to the control. We found upregulation of several genes involved in ABA and jasmonic acid (JA) signaling, stomata regulation, osmotic potential maintenance, stress protection, and disease resistance in AtNOG1-1 and AtNOG1-2 overexpressor lines compared to the control. We elucidated the role of NOG1-2 and NOG1-1 in regulation of silica body formation around stomata to prevent transpirational water loss. These results provide an avenue to confer drought tolerance in rice.

Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genotyping-by-sequencing and genomic selection applications in hexaploid triticale.

Triticale, a hybrid species between wheat and rye, is one of the newest additions to the plant kingdom with a very short history of improvement. It has very limited genomic resources because of its large and complex genome. Objectives of this study were to generate dense marker data, understand genetic diversity, population structure, linkage disequilibrium (LD), and estimate accuracies of commonly used genomic selection (GS) models on forage yield of triticale. Genotyping-by-sequencing (GBS), using PstI and MspI restriction enzymes for reducing genome complexity, was performed on a triticale diversity panel (n = 289). After filtering for biallelic loci with more than 70% genome coverage, and minor allele frequency (MAF) > 0.05, de novo variant calling identified 16,378 single nucleotide polymorphism (SNP) markers. Sequences of these variants were mapped to wheat and rye reference genomes to infer their homologous groups and chromosome positions. About 45% (7430), and 58% (9500) of the de novo identified SNPs were mapped to the wheat and rye reference genomes, respectively. Interestingly, 28.9% (2151) of the 7430 SNPs were mapped to the D genome of hexaploid wheat, indicating substantial substitution of the R genome with D genome in cultivated triticale. About 27% of marker pairs were in significant LD with an average r2 > 0.18 (P < 0.05). Genome-wide LD declined rapidly to r2 < 0.1 beyond 10 kb physical distance. The three sub-genomes (A, B, and R) showed comparable LD decay patterns. Genetic diversity and population structure analyses identified five distinct clusters. Genotype grouping did not follow prior winter vs spring-type classification. However, one of the clusters was largely dominated by winter triticale. GS accuracies were estimated for forage yield using three commonly used models with different training population sizes and marker densities. GS accuracy increased with increasing training population size while gain in accuracy tended to plateau with marker densities of 2000 SNPs or more. Average GS accuracy was about 0.52, indicating the potential of using GS in triticale forage yield improvement.

Drought Responsive Putative Marker-Trait Association in Tall Fescue as Influenced by the Presence of a Novel Endophyte.

Tall fescue (Festuca arundinacea Schreb.) is one of the most important cool-season perennial obligatory outcrossing forage grasses in the United States. The production and persistence of tall fescue is significantly affected by drought in the south-central United States. Shoot-specific endophyte (Epichloë coenophiala)-infected tall fescue showed superior performance under both biotic and abiotic stress conditions. We performed a genome-wide association analysis using clonal pairs of novel endophyte AR584-positive (EP) and endophyte-free (EF) tall fescue populations consisting of 205 genotypes to identify marker-trait associations (MTAs) that contribute to drought tolerance. The experiment was performed through November 2014 to June 2018 in the field, and phenotypic data were taken on plant height, plant spread, plant vigor, and dry biomass weight under natural summer conditions of sporadic drought. Genotyping-by-sequencing of the population generated 3,597 high quality single nucleotide polymorphisms (SNPs) for further analysis. We identified 26 putative drought responsive MTAs (17 specific to EP, eight specific to EF, and one in both EP and EF populations) and nine of them (i.e., V.ep_10, S.ef_12, V.ep_27, HSV.ef_31, S.ep_30, SV.ef_32, V.ep_68, V.ef_56, and H.ef_57) were identified within 0.5 Mb region in the tall fescue genome (44.5-44.7, 75.3-75.8, 77.5-77.9 and 143.7-144.2 Mb). Using 26 MTAs, 11 tall fescue genotypes were selected for subsequent study to develop EP and EF drought tolerant tall fescue populations. Ten orthologous genes (six for EP and four for EF population) were identified in Brachypodium genome as potential candidates for drought tolerance in tall fescue, which were also earlier reported for their involvement in abiotic stress tolerance. The MTAs and candidate genes identified in this study will be useful for marker-assisted selection in improving drought tolerance of tall fescue as well opening avenue for further drought study in tall fescue.

Serendipita Fungi Modulate the Switchgrass Root Transcriptome to Circumvent Host Defenses and Establish a Symbiotic Relationship.

The fungal family Serendipitaceae encompasses root-associated lineages with endophytic, ericoid, orchid, and ectomycorrhizal lifestyles. Switchgrass is an important bioenergy crop for cellulosic ethanol production owing to high biomass production on marginal soils otherwise unfit for food crop cultivation. The aim of this study was to investigate the host plant responses to Serendipita spp. colonization by characterizing the switchgrass root transcriptome during different stages of symbiosis in vitro. For this, we included a native switchgrass strain, Serendipita bescii, and a related strain, S. vermifera, isolated from Australian orchids. Serendipita colonization progresses from thin hyphae that grow between root cells to, finally, the production of large, bulbous hyphae that fill root cells during the later stages of colonization. We report that switchgrass seems to perceive both fungi prior to physical contact, leading to the activation of chemical and structural defense responses and putative host disease resistance genes. Subsequently, the host defense system appears to be quenched and carbohydrate metabolism adjusted, potentially to accommodate the fungal symbiont. In addition, prior to contact, switchgrass exhibited significant increases in root hair density and root surface area. Furthermore, genes involved in phytohormone metabolism such as gibberellin, jasmonic acid, and salicylic acid were activated during different stages of colonization. Both fungal strains induced plant gene expression in a similar manner, indicating a conserved plant response to members of this fungal order. Understanding plant responsiveness to Serendipita spp. will inform our efforts to integrate them into forages and row crops for optimal plant-microbe functioning, thus facilitating low-input, sustainable agricultural practices.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Four chromosome scale genomes and a pan-genome annotation to accelerate pecan tree breeding.

Genome-enabled biotechnologies have the potential to accelerate breeding efforts in long-lived perennial crop species. Despite the transformative potential of molecular tools in pecan and other outcrossing tree species, highly heterozygous genomes, significant presence-absence gene content variation, and histories of interspecific hybridization have constrained breeding efforts. To overcome these challenges, here, we present diploid genome assemblies and annotations of four outbred pecan genotypes, including a PacBio HiFi chromosome-scale assembly of both haplotypes of the 'Pawnee' cultivar. Comparative analysis and pan-genome integration reveal substantial and likely adaptive interspecific genomic introgressions, including an over-retained haplotype introgressed from bitternut hickory into pecan breeding pedigrees. Further, by leveraging our pan-genome presence-absence and functional annotation database among genomes and within the two outbred haplotypes of the 'Lakota' genome, we identify candidate genes for pest and pathogen resistance. Combined, these analyses and resources highlight significant progress towards functional and quantitative genomics in highly diverse and outbred crops.

Three Common Symbiotic ABC Subfamily B Transporters in Medicago truncatula Are Regulated by a NIN-Independent Branch of the Symbiosis Signaling Pathway.

Several ATP-binding cassette (ABC) transporters involved in the arbuscular mycorrhizal symbiosis and nodulation have been identified. We describe three previously unreported ABC subfamily B transporters, named AMN1, AMN2, and AMN3 (ABCB for mycorrhization and nodulation), that are expressed early during infection by rhizobia and arbuscular mycorrhizal fungi. These ABCB transporters are strongly expressed in symbiotically infected tissues, including in root-hair cells with rhizobial infection threads and arbusculated cells. During nodulation, the expression of these genes is highly induced by rhizobia and purified Nod factors and is dependent on DMI3 but is not dependent on other known major regulators of infection, such as NIN, NSP1, or NSP2. During mycorrhization their expression is dependent on DMI3 and RAM1 but not on NSP1 and NSP2. Therefore, they may be commonly regulated through a distinct branch of the common symbiotic pathway. Mutants with exonic Tnt1-transposon insertions were isolated for all three genes. None of the single or double mutants showed any differences in colonization by either rhizobia or mycorrhizal fungi, but the triple amn1 amn2 amn3 mutant showed an increase in nodule number. Further studies are needed to identify potential substrates of these transporters and understand their roles in these beneficial symbioses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The First Genomic Resources for Phymatotrichopsis omnivora, a Soilborne Pezizomycete Pathogen with a Broad Host Range.

Phymatotrichopsis omnivora is a destructive plant pathogen causing root rot disease of alfalfa, cotton, pecan, grape, and many other important dicotyledonous species. A member of the family Rhizinaceae, in the class Pezizomycetes, P. omnivora is a soilborne ascomycete fungus that is difficult to maintain in culture, currently genetically intractable, and for which there are no publicly available genomic resources. We have generated draft genome sequences of four P. omnivora isolates obtained from cotton and alfalfa, growing in Texas and Oklahoma. These genome sequences will provide new insights into the biology of the fungus, including the factors responsible for its broad host range and pathogenicity.

Distinguishing HapMap Accessions Through Recursive Set Partitioning in Hierarchical Decision Trees.

The HapMap (haplotype map) projects have produced valuable genetic resources in life science research communities, allowing researchers to investigate sequence variations and conduct genome-wide association study (GWAS) analyses. A typical HapMap project may require sequencing hundreds, even thousands, of individual lines or accessions within a species. Due to limitations in current sequencing technology, the genotype values for some accessions cannot be clearly called. Additionally, allelic heterozygosity can be very high in some lines, causing genetic and sometimes phenotypic segregation in their descendants. Genetic and phenotypic segregation degrades the original accession's specificity and makes it difficult to distinguish one accession from another. Therefore, it is vitally important to determine and validate HapMap accessions before one conducts a GWAS analysis. However, to the best of our knowledge, there are no prior methodologies or tools that can readily distinguish or validate multiple accessions in a HapMap population. We devised a bioinformatics approach to distinguish multiple HapMap accessions using only a minimum number of genetic markers. First, we assign each candidate marker with a distinguishing score (DS), which measures its capability in distinguishing accessions. The DS score prioritizes those markers with higher percentages of homozygous genotypes (allele combinations), as they can be stably passed on to offspring. Next, we apply the "set-partitioning" concept to select optimal markers by recursively partitioning accession sets. Subsequently, we build a hierarchical decision tree in which a specific path represents the selected markers and the homogenous genotypes that can be used to distinguish one accession from others in the HapMap population. Based on these algorithms, we developed a web tool named MAD-HiDTree (Multiple Accession Distinguishment-Hierarchical Decision Tree), designed to analyze a user-input genotype matrix and construct a hierarchical decision tree. Using genetic marker data extracted from the Medicago truncatula HapMap population, we successfully constructed hierarchical decision trees by which the original 262 M. truncatula accessions could be efficiently distinguished. PCR experiments verified our proposed method, confirming that MAD-HiDTree can be used for the identification of a specific accession. MAD-HiDTree was developed in C/C++ in Linux. Both the source code and test data are publicly available at https://bioinfo.noble.org/MAD-HiDTree/.

Genetic regulation of flowering time and inflorescence architecture by MtFDa and MtFTa1 in Medicago truncatula.

Regulation of floral transition and inflorescence development is crucial for plant reproductive success. FLOWERING LOCUS T (FT) is one of the central players in the flowering genetic regulatory network, whereas FLOWERING LOCUS D (FD), an interactor of FT and TERMINAL FLOWER 1 (TFL1), plays significant roles in both floral transition and inflorescence development. Here we show the genetic regulatory networks of floral transition and inflorescence development in Medicago truncatula by characterizing MtFTa1 and MtFDa and their genetic interactions with key inflorescence meristem (IM) regulators. Both MtFTa1 and MtFDa promote flowering; the double mutant mtfda mtfta1 does not proceed to floral transition. RNAseq analysis reveals that a broad range of genes involved in flowering regulation and flower development are up- or downregulated by MtFTa1 and/or MtFDa mutations. Furthermore, mutation of MtFDa also affects the inflorescence architecture. Genetic analyses of MtFDa, MtFTa1, MtTFL1, and MtFULc show that MtFDa is epistatic to MtFULc and MtTFL1 in controlling IM identity. Our results demonstrate that MtFTa1 and MtFDa are major flowering regulators in M. truncatula, and MtFDa is essential both in floral transition and secondary inflorescence development. The study will advance our understanding of the genetic regulation of flowering time and inflorescence development in legumes.

Serendipita bescii promotes winter wheat growth and modulates the host root transcriptome under phosphorus and nitrogen starvation.

Serendipita vermifera ssp. bescii, hereafter referred to as S. bescii, is a root-associated fungus that promotes plant growth in both its native switchgrass host and a variety of monocots and dicots. Winter wheat (Triticum aestivum L.), a dual-purpose crop, used for both forage and grain production, significantly contributes to the agricultural economies of the Southern Great Plains, USA. In this study, we investigated the influence of S. bescii on growth and transcriptome regulation of nitrogen (N) and phosphorus (P) metabolism in winter wheat. Serendipita bescii significantly improved lateral root growth and forage biomass under a limited N or P regime. Further, S. bescii activated sets of host genes regulating N and P starvation responses. These genes include, root-specific auxin transport, strigolactone and gibberellin biosynthesis, degradation of phospholipids and biosynthesis of glycerolipid, downregulation of ammonium transport and nitrate assimilation, restriction of protein degradation by autophagy and subsequent N remobilization. All these genes are hypothesized to regulate acquisition, assimilation and remobilization of N and P. Based on transcriptional level gene regulation and physiological responses to N or P limitation, we suggest S. bescii plays a critical role in modulating stress imposed by limitation of these two critical nutrients in winter wheat.

Dissection of physiological, transcriptional, and metabolic traits in two tall fescue genotypes with contrasting drought tolerance.

Tall fescue (Festuca arundinacea) is an important cool-season perennial forage grass that forms mutualistic symbioses with fungal endophytes. Physiological, biochemical and transcriptional comparisons were made between two tall fescue genotypes with contrasting drought tolerance (tolerant, T400, and sensitive, S279), either with or without endophyte (Epichloë coenophiala). Drought stress was applied by withholding watering until plants reached mild, moderate and severe stresses. Physiological characterization showed that T400 had narrower, thicker leaves, and lower leaf conductance under well-watered conditions, compared to S279. After severe drought and recovery, endophytic T400 had greater shoot and root biomass than other plant types. Under drought, leaf osmotic pressure increased much more in T400 than S279, consistent with accumulation of metabolites/osmolytes, especially proline. Gene Ontology enrichment analysis indicated that T400 had more active organic acid metabolism than S279 under drought, and implicated the role of endophyte in stimulating protein metabolism in both genotypes. Overall T400 and S279 responded to endophyte differently in aspects of physiology, gene transcription and metabolites, indicating plant genotype-specific reactions to endophyte infection.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element.

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.

Chromosome-Level Reference Genome of Venturia effusa, Causative Agent of Pecan Scab.

Pecan scab, caused by Venturia effusa, is a devastating disease of pecan (Carya illinoinensis), which results in economic losses on susceptible cultivars throughout the southeastern United States. To enhance our understanding of pathogenicity in V. effusa, we have generated a complete telomere-to-telomere reference genome of V. effusa isolate FRT5LL7-Albino. By combining Illumina MiSeq and Oxford Nanopore MinION data, we assembled a 45.2-Mb genome represented by 20 chromosomes and containing 10,820 putative genes, of which 7,619 have at least one functional annotation. The likely causative mutation of the albino phenotype was identified as a single base insertion and a resulting frameshift in the gene encoding the polyketide synthase ALM1. This genome represents the first full chromosome-level assembly of any Venturia sp.