De novo genome assembly of white clover (Trifolium repens L.) reveals the role of copy number variation in rapid environmental adaptation.

BACKGROUND: White clover (Trifolium repens) is a globally important perennial forage legume. This species also serves as an eco-evolutionary model system for studying within-species chemical defense variation; it features a well-studied polymorphism for cyanogenesis (HCN release following tissue damage), with higher frequencies of cyanogenic plants favored in warmer locations worldwide. Using a newly generated haplotype-resolved genome and two other long-read assemblies, we tested the hypothesis that copy number variants (CNVs) at cyanogenesis genes play a role in the ability of white clover to rapidly adapt to local environments. We also examined questions on subgenome evolution in this recently evolved allotetraploid species and on chromosomal rearrangements in the broader IRLC legume clade. RESULTS: Integration of PacBio HiFi, Omni-C, Illumina, and linkage map data yielded a completely de novo genome assembly for white clover (created without a priori sequence assignment to subgenomes). We find that white clover has undergone extensive transposon diversification since its origin but otherwise shows highly conserved genome organization and composition with its diploid progenitors. Unlike some other clover species, its chromosomal structure is conserved with other IRLC legumes. We further find extensive evidence of CNVs at the major cyanogenesis loci; these contribute to quantitative variation in the cyanogenic phenotype and to local adaptation across wild North American populations. CONCLUSIONS: This work provides a case study documenting the role of CNVs in local adaptation in a plant species, and it highlights the value of pan-genome data for identifying contributions of structural variants to adaptation in nature.

A Trifolium repens flavodoxin-like quinone reductase 1 (TrFQR1) improves plant adaptability to high temperature associated with oxidative homeostasis and lipids remodeling.

Maintenance of stable mitochondrial respiratory chains could enhance adaptability to high temperature, but the potential mechanism was not elucidated clearly in plants. In this study, we identified and isolated a TrFQR1 gene encoding the flavodoxin-like quinone reductase 1 (TrFQR1) located in mitochondria of leguminous white clover (Trifolium repens). Phylogenetic analysis indicated that amino acid sequences of FQR1 in various plant species showed a high degree of similarities. Ectopic expression of TrFQR1 protected yeast (Saccharomyces cerevisiae) from heat damage and toxic levels of benzoquinone, phenanthraquinone and hydroquinone. Transgenic Arabidopsis thaliana and white clover overexpressing TrFQR1 exhibited significantly lower oxidative damage and better photosynthetic capacity and growth than wild-type in response to high-temperature stress, whereas AtFQR1-RNAi A. thaliana showed more severe oxidative damage and growth retardation under heat stress. TrFQR1-transgenic white clover also maintained better respiratory electron transport chain than wild-type plants, as manifested by significantly higher mitochondrial complex II and III activities, alternative oxidase activity, NAD(P)H content, and coenzyme Q10 content in response to heat stress. In addition, overexpression of TrFQR1 enhanced the accumulation of lipids including phosphatidylglycerol, monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and cardiolipin as important compositions of bilayers involved in dynamic membrane assembly in mitochondria or chloroplasts positively associated with heat tolerance. TrFQR1-transgenic white clover also exhibited higher lipids saturation level and phosphatidylcholine:phosphatidylethanolamine ratio, which could be beneficial to membrane stability and integrity during a prolonged period of heat stress. The current study proves that TrFQR1 is essential for heat tolerance associated with mitochondrial respiratory chain, cellular reactive oxygen species homeostasis, and lipids remodeling in plants. TrFQR1 could be selected as a key candidate marker gene to screen heat-tolerant genotypes or develop heat-tolerant crops via molecular-based breeding.

Genome-wide identification and characterization of NAC transcription factor family members in Trifolium pratense and expression analysis under lead stress.

BACKGROUND: The NAC TF family is widely involved in plant responses to various types of stress. Red clover (Trifolium pratense) is a high-quality legume, and the study of NAC genes in red clover has not been comprehensive. The aim of this study was to analyze the NAC gene family of red clover at the whole-genome level and explore its potential role in the Pb stress response. RESULTS: In this study, 72 TpNAC genes were identified from red clover; collinearity analysis showed that there were 5 pairs of large fragment replicators of TpNAC genes, and red clover was found to be closely related to Medicago truncatula. Interestingly, the TpNAC genes have more homologs in Arabidopsis thaliana than in soybean (Glycine max). There are many elements in the TpNAC genes promoters that respond to stress. Gene expression analysis showed that all the TpNAC genes responded to Pb stress. qRT-PCR showed that the expression levels of TpNAC29 and TpNAC42 were significantly decreased after Pb stress. Protein interaction network analysis showed that 21 TpNACs and 23 other genes participated in the interaction. In addition, the TpNAC proteins had three possible 3D structures, and the secondary structure of these proteins were mainly of other types. These results indicated that most TpNAC members were involved in the regulation of Pb stress in red clover. CONCLUSION: These results suggest that most TpNAC members are involved in the regulation of Pb stress in red clover. TpNAC members play an important role in the response of red clover to Pb stress.

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress.

BACKGROUND: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. RESULTS: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. CONCLUSION: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Beyond cyanogenesis: Temperature gradients drive environmental adaptation in North American white clover (Trifolium repens L.).

Species that repeatedly evolve phenotypic clines across environmental gradients have been highlighted as ideal systems for characterizing the genomic basis of local environmental adaptation. However, few studies have assessed the importance of observed phenotypic clines for local adaptation: conspicuous traits that vary clinally may not necessarily be the most critical in determining local fitness. The present study was designed to fill this gap, using a plant species characterized by repeatedly evolved adaptive phenotypic clines. White clover is naturally polymorphic for its chemical defence cyanogenesis (HCN release with tissue damage); climate-associated cyanogenesis clines have evolved throughout its native and introduced range worldwide. We performed landscape genomic analyses on 415 wild genotypes from 43 locations spanning much of the North American species range to assess the relative importance of cyanogenesis loci vs. other genomic factors in local climatic adaptation. We find clear evidence of local adaptation, with temperature-related climatic variables best describing genome-wide differentiation between sampling locations. The same climatic variables are also strongly correlated with cyanogenesis frequencies and gene copy number variations (CNVs) at cyanogenesis loci. However, landscape genomic analyses indicate no significant contribution of cyanogenesis loci to local adaptation. Instead, several genomic regions containing promising candidate genes for plant response to seasonal cues are identified - some of which are shared with previously identified QTLs for locally adaptive fitness traits in North American white clover. Our findings suggest that local adaptation in white clover is likely determined primarily by genes controlling the timing of growth and flowering in response to local seasonal cues. More generally, this work suggests that caution is warranted when considering the importance of conspicuous phenotypic clines as primary determinants of local adaptation.

Starmerella kisarazuensis f.a., sp. nov., a novel yeast isolated from Trifolium pratense flowers.

Three yeast isolates, NBRC 115909T, NBRC 115910 and NBRC 116270, were isolated from Trifolium pratense (red clover) flowers collected from Kisarazu, Chiba, Japan. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions revealed that these isolates represent a single novel species within the genus Starmerella. Also, no ascospore formation was observed. The yeast isolates were closely related to Starmerella vitae UWOPS 00-107.2T and Starmerella bombi NRRL Y-17081T. They differed from S. vitae, the most closely related species with a validly published name, by ten nucleotide substitutions with two gaps in the D1/D2 domains and 20 nucleotide substitutions in the ITS region. Moreover, the three isolates exhibited distinct phenotypic characteristics from the closely related species. Therefore, we suggest that these three isolates represent a novel species, designated as Starmerella kisarazuensis f.a., sp. nov. The holotype is NBRC 115909T (isotype: CBS 18485T).

Lipid remodelling and the conversion of lipids into sugars associated with tolerance to cadmium toxicity during white clover seed germination.

Cadmium (Cd) is a leading environmental issue worldwide. The current study was conducted to investigate Cd tolerance of 10 commercial white clover (Trifolium repens) cultivars during seed germination and to further explore differences in lipid remodelling, glycometabolism, and the conversion of lipids into sugars contributing to Cd tolerance in the early phase of seedling establishment as well as the accumulation of Cd in seedlings and mature plants. The results show that Cd stress significantly reduced seed germination of 10 cultivars. Compared to Cd-sensitive Sulky, Cd-tolerant Pixie accelerated amylolysis to produce more glucose, fructose, and sucrose by maintaining higher amylase and sucrase activities under Cd stress. Pixie maintained higher contents of various lipids, higher DGDG/MGDG ratio, and lower unsaturation levels of lipids, which could be beneficial to membrane stability and integrity as well as signal transduction in cells after being subjected to Cd stress. In addition, Pixie upregulated expression levels of key genes (TrACX1, TrACX4, TrSDP6, and TrPCK1) involved in the conversion of lipids into sugars for early seedling establishment under Cd stress. These findings indicate that lipid remodelling, enhanced glycometabolism, and accelerated conversion of lipids into sugars are important adaptive strategies for white clover seed germination and subsequent seedling establishment under Cd stress. In addition, Pixie not only accumulated more Cd in seedlings and mature plants than Sulky but also had significantly better growth and phytoremediation efficiency under Cd stress. Pixie could be used as a suitable and critical germplasm for the rehabilitation and re-establishment of Cd-contaminated areas.

Physiological and comprehensive transcriptome analysis reveals distinct regulatory mechanisms for aluminum tolerance of Trifolium repens.

It is estimated that up to 50 % of arable lands worldwide are acidic, and most crops are severely inhibited due to the high active aluminum (Al). Trifolium repens is an excellent legume forage with a certain acid tolerance, although it is affected by Al toxicity in acidic soil. In this study, physiological and transcriptomic responses of different white clover varieties were analyzed when exposed to a high-level of Al stress. The results revealed that Trifolium repens had a high level of Al toxicity tolerance, and accumulated nearly 70 % of Al3+ in its roots. Al toxicity significantly inhibited the root length and root activity, decreased the chlorophyll (Chl) content and photosynthetic pigments, while significantly increased the intercellular CO2 concentration (Ci). The content of malondialdehyde (MDA), electrolyte leakage (EL), proline and reactive oxygen species (ROS) were significantly accumulated under Al stress. Furthermore, a total of 27,480 differentially expressed genes (DEGs) were identified after the treatment. Gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that most Al-responsive genes enriched to chloroplast thylakoid membrane, chloroplast stroma and photosynthesis in Haifa leaf while in MAG leaf highly enriched in response to regulation of defense response, which could induce the different tolerance of the two cultivars to Al stress. Besides, pectin methylesterase (PME), glycosyl transferases (GT1) and chalcone synthase genes associated with cell wall biosynthesis may improve the Al accumulation and enhance tolerance of Al toxicity. The results established here would help to understand the morphological structure, physiological and biochemical response, and molecular mechanism of white clover under Al tolerance.

The White Clover TrMYB33-TrSAMS1 Module Contributes to Drought Tolerance by Modulation of Spermidine Biosynthesis via an ABA-Dependent Pathway.

Spermidine is well known to accumulate in plants exposed to drought, but the regulatory network associated with its biosynthesis and accumulation and the underlying molecular mechanisms remain unclear. Here, we demonstrated that the Trifolium repens TrMYB33 relayed the ABA signal to modulate drought-induced spermidine production by directly regulating the expression of TrSAMS1, which encodes an S-adenosylmethionine synthase. This gene was identified by transcriptome and expression analysis in T. repens. TrSAMS1 overexpression and its pTRV-VIGS-mediated silencing demonstrated that TrSAMS1 is a positive regulator of spermidine synthesis and drought tolerance. TrMYB33 was identified as an interacting candidate through yeast one-hybrid library screening with the TrSAMS1 promoter region as the bait. TrMYB33 was confirmed to bind directly to the predicted TAACCACTAACCA (the TAACCA MYB binding site is repeated twice in tandem) within the TrSAMS1 promoter and to act as a transcriptional activator. Additionally, TrMYB33 contributed to drought tolerance by regulating TrSAMS1 expression and modulating spermidine synthesis. Additionally, we found that spermidine accumulation under drought stress depended on ABA and that TrMYB33 coordinated ABA-mediated upregulation of TrSAMS1 and spermidine accumulation. This study elucidated the role of a T. repens MYB33 homolog in modulating spermidine biosynthesis. The further exploitation and functional characterization of the TrMYB33-TrSAMS1 regulatory module can enhance our understanding of the molecular mechanisms responsible for spermidine accumulation during drought stress.

High-quality chromosome-level de novo assembly of the Trifolium repens.

BACKGROUND: White clover (Trifolium repens L.), an excellent perennial legume forage, is an allotetraploid native to southeastern Europe and southern Asia. It has high nutritional, ecological, genetic breeding, and medicinal values and exhibits excellent resistance to cold, drought, trample, and weed infestation. Thus, white clover is widely planted in Europe, America, and China; however, the lack of reference genome limits its breeding and cultivation. This study generated a white clover de novo genome assembly at the chromosomal level and annotated its components. RESULTS: The PacBio third-generation Hi-Fi assembly and sequencing methods generated a 1096 Mb genome size of T. repens, with contigs of N50 = 14 Mb, scaffolds of N50 = 65 Mb, and BUSCO value of 98.5%. The newly assembled genome has better continuity and integrity than the previously reported white clover reference genome; thus provides important resources for the molecular breeding and evolution of white clover and other forage. Additionally, we annotated 90,128 high-confidence gene models from the genome. White clover was closely related to Trifolium pratense and Trifolium medium but distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum. The expansion, contraction, and GO functional enrichment analysis of the gene families showed that T. repens gene families were associated with biological processes, molecular function, cellular components, and environmental resistance, which explained its excellent agronomic traits. CONCLUSIONS: This study reports a high-quality de novo assembly of white clover genome obtained at the chromosomal level using PacBio Hi-Fi sequencing, a third-generation sequencing. The generated high-quality genome assembly of white clover provides a key basis for accelerating the research and molecular breeding of this important forage crop. The genome is also valuable for future studies on legume forage biology, evolution, and genome-wide mapping of quantitative trait loci associated with the relevant agronomic traits.

Rhizobium nodule diversity and composition are influenced by clover host selection and local growth conditions.

While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.

Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens).

PREMISE: ß-Cyanoalanine synthase (ß-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of ß-CAS and Aox genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species. METHODS: We used a natural polymorphism for cyanogenesis in white clover to examine ß-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure. We identified all ß-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a, and Aox2d subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue. RESULTS: ß-CAS and Aox2a expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression. Aox2d, in contrast, appeared to be upregulated as a generalized wounding response. CONCLUSIONS: These findings suggest a heightened constitutive role for HCN detoxification (via elevated ß-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a expression) in plants that are capable of cyanogenesis. As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.

Biochemical changes after cold acclimation in Nordic red clover (Trifolium pratense L.) accessions with contrasting levels of freezing tolerance.

The ability to tolerate low freezing temperatures is an important component of winter survival and persistence of red clover. Cold acclimation (CA) allows plants to acquire higher levels of freezing tolerance. However, the biochemical responses to cold and the importance of such changes for the plant to acquire adequate freezing tolerance have not been investigated in red clover of Nordic origin, which has a distinct genetic background. To shed light on this, we selected five freezing tolerant (FT) and five freezing susceptible (FS) accessions and studied the effect of CA on the contents of carbohydrates, amino acids, and phenolic compounds in the crowns. Among those compounds which increased during CA, FT accessions had higher contents of raffinose, pinitol, arginine, serine, alanine, valine, phenylalanine, and one phenolic compound (a pinocembrin hexoside derivative) than FS accessions, suggesting a role for these compounds in the freezing tolerance in the selected accessions. These findings, together with a description of the phenolic profile of red clover crowns, significantly add to the current knowledge of the biochemical changes during CA and their role in freezing tolerance in Nordic red clover.

Red clover root-associated microbiota is shaped by geographic location and choice of farming system.

AIMS: This study evaluated the red clover (Trifolium pratense) root-associated microbiota to clarify the presence of pathogenic and beneficial microorganisms in 89 Swedish field sites. METHODS AND RESULTS: 16S rRNA and ITS amplicon sequencing analysis were performed on DNA extracted from the red clover root samples collected to determine the composition of the prokaryotic and eukaryotic root-associated microbe communities. Alpha and beta diversities were calculated and relative abundance of various microbial taxa and their co-occurrence were analyzed. Rhizobium was the most prevalent bacterial genus, followed by Sphingomonas, Mucilaginibacter, Flavobacterium, and the unclassified Chloroflexi group KD4-96. The Leptodontidium, Cladosporium, Clonostachys, and Tetracladium fungal genera known for endophytic, saprotrophic, and mycoparasitic lifestyles were also frequently observed in all samples. Sixty-two potential pathogenic fungi were identified with a bias toward grass pathogens and a higher abundance in samples from conventional farms. CONCLUSIONS: We showed that the microbial community was mainly shaped by geographic location and management procedures. Co-occurrence networks revealed that the Rhizobiumleguminosarum bv. trifolii was negatively associated with all fungal pathogenic taxa recognized in this study.

Optimization of Protocols for the Induction of Callus and Plant Regeneration in White Clover (Trifolium repens L.).

White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L-1 AgNO3 and 25 mg L-1 VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L-1 6-BA, 1 mg L-1 KT and 0.5 mg L-1 NAA. In addition, the transformation system for Agrobacterium tumefaciens-mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background.

Identification and Evolutionary Analysis of the Auxin Response Factor (ARF) Family Based on Transcriptome Data from Caucasian Clover and Analysis of Expression Responses to Hormones.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1-6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real-time RT-PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in Caucasian clover.

A de novo assembled high-quality chromosome-scale Trifolium pratense genome and fine-scale phylogenetic analysis.

BACKGROUND: Red clover (Trifolium pratense L.) is a diploid perennial temperate legume with 14 chromosomes (2n = 14) native to Europe and West Asia, with high nutritional and economic value. It is a very important forage grass and is widely grown in marine climates, such as the United States and Sweden. Genetic research and molecular breeding are limited by the lack of high-quality reference genomes. In this study, we used Illumina, PacBio HiFi, and Hi-C to obtain a high-quality chromosome-scale red clover genome and used genome annotation results to analyze evolutionary relationships among related species. RESULTS: The red clover genome obtained by PacBio HiFi assembly sequencing was 423 M. The assembly quality was the highest among legume genome assemblies published to date. The contig N50 was 13 Mb, scaffold N50 was 55 Mb, and BUSCO completeness was 97.9%, accounting for 92.8% of the predicted genome. Genome annotation revealed 44,588 gene models with high confidence and 52.81% repetitive elements in red clover genome. Based on a comparison of genome annotation results, red clover was closely related to Trifolium medium and distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum among legumes. Analyses of gene family expansions and contractions and forward gene selection revealed gene families and genes related to environmental stress resistance and energy metabolism. CONCLUSIONS: We report a high-quality de novo genome assembly for the red clover at the chromosome level, with a substantial improvement in assembly quality over those of previously published red clover genomes. These annotated gene models can provide an important resource for molecular genetic breeding and legume evolution studies. Furthermore, we analyzed the evolutionary relationships among red clover and closely related species, providing a basis for evolutionary studies of clover leaf and legumes, genomics analyses of forage grass, the improvement of agronomic traits.

Asynapsis and unreduced gamete formation in a Trifolium interspecific hybrid.

BACKGROUND: Unreduced gametes, a driving force in the widespread polyploidization and speciation of flowering plants, occur relatively frequently in interspecific or intergeneric hybrids. Studies of the mechanisms leading to 2n gamete formation, mainly in the wheat tribe Triticeae have shown that unreductional meiosis is often associated with chromosome asynapsis during the first meiotic division. The present study explored the mechanisms of meiotic nonreduction leading to functional unreduced gametes in an interspecific Trifolium (clover) hybrid with three sub-genomes from T. ambiguum and one sub-genome from T. occidentale. RESULTS: Unreductional meiosis leading to 2n gametes occurred when there was a high frequency of asynapsis during the first meiotic division. In this hybrid, approximately 39% of chromosomes were unpaired at metaphase I. Within the same cell at anaphase I, sister chromatids of univalents underwent precocious separation and formed laggard chromatids whereas paired chromosomes segregated without separation of sister chromatids as in normal meiosis. This asynchrony was frequently accompanied by incomplete or no movement of chromosomes toward the poles and restitution leading to unreduced chromosome constitutions. Reductional meiosis was restored in progeny where asynapsis frequencies were low. Two progeny plants with approximately 5 and 7% of unpaired chromosomes at metaphase I showed full restoration of reductional meiosis. CONCLUSIONS: The study revealed that formation of 2n gametes occurred when asynapsis (univalent) frequency at meiosis I was high, and that normal gamete production was restored in the next generation when asynapsis frequencies were low. Asynapsis-dependent 2n gamete formation, previously supported by evidence largely from wheat and its relatives and grasshopper, is also applicable to hybrids from the dicotyledonous plant genus Trifolium. The present results align well with those from these widely divergent organisms and strongly suggest common molecular mechanisms involved in unreduced gamete formation.

Identification of loci controlling timing of stem elongation in red clover using genotyping by sequencing of pooled phenotypic extremes.

MAIN CONCLUSION: Through selective genotyping of pooled phenotypic extremes, we identified a number of loci and candidate genes putatively controlling timing of stem elongation in red clover. We have identified candidate genes controlling the timing of stem elongation prior to flowering in red clover (Trifolium pratense L.). This trait is of ecological and agronomic significance, as it affects fitness, competitivity, climate adaptation, forage and seed yield, and forage quality. We genotyped replicate pools of phenotypically extreme individuals (early and late-elongating) within cultivar Lea using genotyping-by-sequencing in pools (pool-GBS). After calling and filtering SNPs and GBS locus haplotype polymorphisms, we estimated allele frequencies and searched for markers with significantly different allele frequencies in the two phenotypic groups using BayeScan, an FST-based test utilizing replicate pools, and a test based on error variance of replicate pools. Of the three methods, BayeScan was the least stringent, and the error variance-based test the most stringent. Fifteen significant markers were identified in common by all three tests. The candidate genes flanking the markers include genes with potential roles in the vernalization, autonomous, and photoperiod regulation of floral transition, hormonal regulation of stem elongation, and cell growth. These results provide a first insight into the potential genes and mechanisms controlling transition to stem elongation in a perennial legume, which lays a foundation for further functional studies of the genetic determinants regulating this important trait.

Genetic trade-offs underlie divergent life history strategies for local adaptation in white clover.

Local adaptation is common in plants, yet characterization of its underlying genetic basis is rare in herbaceous perennials. Moreover, while many plant species exhibit intraspecific chemical defence polymorphisms, their importance for local adaptation remains poorly understood. We examined the genetic architecture of local adaptation in a perennial, obligately-outcrossing herbaceous legume, white clover (Trifolium repens). This widespread species displays a well-studied chemical defence polymorphism for cyanogenesis (HCN release following tissue damage) and has evolved climate-associated cyanogenesis clines throughout its range. Two biparental F2  mapping populations, derived from three parents collected in environments spanning the U.S. latitudinal species range (Duluth, MN, St. Louis, MO and Gainesville, FL), were grown in triplicate for two years in reciprocal common garden experiments in the parental environments (6,012 total plants). Vegetative growth and reproductive fitness traits displayed trade-offs across reciprocal environments, indicating local adaptation. Genetic mapping of fitness traits revealed a genetic architecture characterized by allelic trade-offs between environments, with 100% and 80% of fitness QTL in the two mapping populations showing significant QTL×E interactions, consistent with antagonistic pleiotropy. Across the genome there were three hotspots of QTL colocalization. Unexpectedly, we found little evidence that the cyanogenesis polymorphism contributes to local adaptation. Instead, divergent life history strategies in reciprocal environments were major fitness determinants: selection favoured early investment in flowering at the cost of multiyear survival in the southernmost site versus delayed flowering and multiyear persistence in the northern environments. Our findings demonstrate that multilocus genetic trade-offs contribute to contrasting life history characteristics that allow for local adaptation in this outcrossing herbaceous perennial.