Structural Reorganization in Two Alfalfa Mitochondrial Genome Assemblies and Mitochondrial Evolution in Medicago Species.

Plant mitochondria are crucial for species evolution, phylogenetics, classification, and identification as maternal genetic material. However, the presence of numerous repetitive sequences, complex structures, and a low number of genes in the mitochondrial genome has hindered its complete assembly and related research endeavors. In this study, we assembled two mitochondrial genomes of alfalfa varieties of Zhongmu No.1 (299,123 bp) and Zhongmu No.4 (306,983 bp), based on a combination of PacBio, Illumina, and Hi-C sequences. The comparison of genome assemblies revealed that the same number of mitochondrial genes, including thirty-three protein-coding genes, sixteen tRNA genes, and three rRNA genes existed in the two varieties. Additionally, large fragments of repetitive sequences were found underlying frequent mitochondrial recombination events. We observed extensive transfer of mitochondrial fragments into the nuclear genome of Zhongmu No.4. Analysis of the cox1 and rrn18s genes in 35 Medicago accessions revealed the presence of population-level deletions and substitutions in the rrn18s gene. We propose that mitochondrial structural reorganizations may contribute to alfalfa evolution.

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.

Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.

Haplotype-Resolved, Chromosome-Level Assembly of White Clover (Trifolium repens L., Fabaceae).

White clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. Here, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively.

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.

Genome-wide analysis of the CDPK gene family and their important roles response to cold stress in white clover.

Calcium-dependent protein kinases (CDPKs) are an important class of calcium-sensitive response proteins that play an important regulatory role in response to abiotic stresses. To date, little is known about the CDPK genes in white clover. White clover is a high-quality forage grass with high protein content, but it is susceptible to cold stress. Therefore, we performed a genome-wide analysis of the CDPK gene family in white clover and identified 50 members of the CDPK genes. Phylogenetic analysis using CDPKs from the model plant Arabidopsis divided the TrCDPK genes into four groups based on their sequence similarities. Motif analysis showed that TrCDPKs within the same group had similar motif compositions. Gene duplication analysis revealed the evolution and expansion of TrCDPK genes in white clover. Meanwhile, a genetic regulatory network (GRN) containing TrCDPK genes was reconstructed, and gene ontology (GO) annotation analysis of these functional genes showed that they contribute to signal transduction, cellular response to stimuli, and biological regulation, all of which are important processes in response to abiotic stresses. To determine the function of TrCDPK genes, we analyzed the RNA-seq dataset and found that most TrCDPK genes were highly up-regulated under cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiments, implying that TrCDPK genes are involved in various gene regulatory pathways in response to cold stress. Our study may help to further investigate the function of TrCDPK genes and their role in response to cold stress, which is important for understanding the molecular mechanisms of cold tolerance in white clover and improving its cold tolerance.

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.

Molecular marker development and genetic diversity exploration in Medicago polymorpha.

Medicago polymorpha L. (bur clover), an invasive plant species of the genus Medicago, has been traditionally used in China as an edible vegetable crop because of its high nutritive value. However, few molecular markers for M. polymorpha have been identified. Using the recently published high-quality reference genome of M. polymorpha, we performed a specific-locus amplified fragment sequencing (SLAF-seq) analysis of 10 M. polymorpha accessions to identify molecular markers and explore genetic diversity. A total of 52,237 high-quality single nucleotide polymorphisms (SNPs) were developed. These SNPs were mostly distributed on pseudochromosome 3, least distributed on pseudochromosome 7, and relatively evenly distributed on five other pseudochromosomes of M. polymorpha. Phenotypic analysis showed that there was a great difference in phenotypic traits among different M. polymorpha accessions. Moreover, clustering all M. polymorpha accessions based on their phenotypic traits revealed three groups. Both phylogenetic analysis and principal component analysis (PCA) of all M. polymorpha accessions based on SNP markers consistently indicated that all M. polymorpha accessions could be divided into three distinct groups (I, II, and III). Subsequent genetic diversity analysis for the 10 M. polymorpha accessions validated the effectiveness of the M. polymorpha germplasm molecular markers in China. Additionally, SSR mining analysis was also performed to identify polymorphic SSR motifs, which could provide valuable candidate markers for the further breeding of M. polymorpha. Since M. polymorpha genetics have not been actively studied, the molecular markers generated from our research will be useful for further research on M. polymorpha resource utilization and marker-assisted breeding.

Leaf transcriptome analysis of Medicago ruthenica revealed its response and adaptive strategy to drought and drought recovery.

BACKGROUND: Drought is one of the main causes of losses in forage crop yield and animal production. Medicago ruthenica (L.) cv. Zhilixing is a high-yielding alfalfa cultivar also known for its high tolerance to drought. We analyzed the transcriptome profile of this cultivar throughout drought stress and recovery and we were able to describe its phased response through the expression profiles of overlapping gene networks and drought-specific genes. RESULTS: The ABA and auxin signal transduction pathways are overlapping pathways in response to drought and drought recovery in forage crops. Medicago ruthenica (L.) cv. Zhilixing adopts different strategies at different degrees of drought stress. On the 9th day of drought, transcriptional regulations related to osmoregulation are enhanced mainly through increased activities of carbohydrate and amino acid metabolism, while photosynthetic activities were reduced to slow down growth. With drought prolonging, on the 12th day of drought, the synthesis of proline and other stored organic substances was suppressed in general. After recovery, Medicago ruthenica synthesizes flavonoids through the flavonoid biosynthesis pathway to remove accumulated ROS and repair the oxidative damage from water stress. In addition, the regulation of circadian rhythm seems to accelerate the drought recovery process. CONCLUSIONS: Medicago ruthenica adapts to drought by regulating the osmoregulatory system and photosynthesis, which appears to involve the ABA and auxin signaling pathways as key regulators. Furthermore, the synthesis of flavonoids and the regulation of the circadian rhythm can accelerate the recovery process. These results enriched our knowledge of molecular responses to drought and drought recovery in Medicago ruthenica and provide useful information for the development of new legume forage grass varieties with improved adaptability to drought stress.

Phenotypic variation and quantitative trait loci for resistance to southern anthracnose and clover rot in red clover.

KEY MESSAGE: High variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding. Red clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces, and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses. A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover.

Mechanism of pod shattering in the forage legume Medicago ruthenica.

Pod shattering is a seed dispersal strategy and an important agronomical trait in domesticated crops. The relationship between pod shattering and pod morphology in the genus Medicago is well known; however, the detailed mechanism underlying pod dehiscence in Medicago ruthenica, a perennial legume used for forage production, is unknown. Here, the pod ventral sutures of shatter-resistant and shatter-susceptible M. ruthenica genotypes were examined at 8, 12, 16, and 20 d after flowering. The mechanism of pod shattering was analyzed through microscopic observations, polygalacturonase (PG) and cellulase (CE) activity analyses, and RNA-sequencing (RNA-Seq), and the results were verified via reverse transcriptase-quantitative polymerase chain reaction. Pod shattering at the ventral suture in M. ruthenica occurs via a combination of two mechanisms: degradation of the middle lamella at the abscission layers (ALs) and detachment of lignified cells on either side of the ALs triggered by physical forces. Increased PG and CE activities in the pod ventral suture are essential for AL cell-autolysis in the shatter-susceptible genotype. RNA-Seq revealed that 11 genes encoding PG and CE were highly expressed in the ventral sutures of the shatter-susceptible genotype. The expression levels of auxin biosynthesis-related genes decreased in the AL cells and they were negatively associated with pod dehiscence. These results enhance our understanding of the pod shattering mechanism not only in M. ruthenica but also in other leguminous plants.

Physiological and transcriptome analyses highlight multiple pathways involved in drought stress in Medicago falcata.

Medicago falcata is one of the leguminous forage crops, which grows well in arid and semiarid region. To fully investigate the mechanism of drought resistance response in M. falcata, we challenged the M. falcata plants with 30% PEG-6000, and performed physiological and transcriptome analyses. It was found that, the activities of antioxidant enzymes (eg. SOD, POD, and CAT) and soluble sugar content were all increased in the PEG-treated group, as compared to the control group. Transcriptome results showed that a total of 706 genes were differentially expressed in the PEG-treated plants in comparison with the control. Gene enrichment analyses on differentially expressed genes revealed that a number of genes in various pathway were significantly enriched, including the phenylpropanoid biosynthesis (ko00940) and glycolysis/gluconeogenesis (ko00010), indicating the involvement of these key pathways in drought response. Furthermore, the expression levels of seven differentially expressed genes were verified to be involved in drought response in M. falcata by qPCR. Taken together, these results will provide valuable information related to drought response in M. falcata and lay a foundation for molecular studies and genetic breeding of legume crops in future research.

Born in the mitochondrion and raised in the nucleus: evolution of a novel tandem repeat family in Medicago polymorpha (Fabaceae).

Plant nuclear genomes harbor sequence elements derived from the organelles (mitochondrion and plastid) through intracellular gene transfer (IGT). Nuclear genomes also show a dramatic range of repeat content, suggesting that any sequence can be readily amplified. These two aspects of plant nuclear genomes are well recognized but have rarely been linked. Through investigation of 31 Medicago taxa we detected exceptionally high post-IGT amplification of mitochondrial (mt) DNA sequences containing rps10 in the nuclear genome of Medicago polymorpha and closely related species. The amplified sequences were characterized as tandem arrays of five distinct repeat motifs (2157, 1064, 987, 971, and 587 bp) that have diverged from the mt genome (mitogenome) in the M. polymorpha nuclear genome. The mt rps10-like arrays were identified in seven loci (six intergenic and one telomeric) of the nuclear chromosome assemblies and were the most abundant tandem repeat family, representing 1.6-3.0% of total genomic DNA, a value approximately three-fold greater than the entire mitogenome in M. polymorpha. Compared to a typical mt gene, the mt rps10-like sequence coverage level was 691.5-7198-fold higher in M. polymorpha and closely related species. In addition to the post-IGT amplification, our analysis identified the canonical telomeric repeat and the species-specific satellite arrays that are likely attributable to an ancestral chromosomal fusion in M. polymorpha. A possible relationship between chromosomal instability and the mt rps10-like tandem repeat family in the M. polymorpha clade is discussed.

Genome-wide analysis of the Glutathione S-Transferase family in wild Medicago ruthenica and drought-tolerant breeding application of MruGSTU39 gene in cultivated alfalfa.

KEY MESSAGE: Transformation of MruGSTU39 in M. ruthenica and alfalfa enhanced growth and survival of transgenic plants by up-regulating GST and glutathione peroxidase activity to detoxify ROS under drought stress. Glutathione S-transferases (GSTs) are ubiquitous supergene family which play crucial roles in detoxification of reactive oxygen species (ROS). Despite studies on GSTs, few studies have focused on them in perennial, wild plant species with high tolerance to environmental stress. Here, we identified 66 MruGST genes from the genome of Medicago ruthenica, a perennial legume species native to temperate grasslands with high tolerance to environmental stress. These genes were divided into eight classes based on their conserved domains, phylogenetic tree and gene structure, with the tau class being the most numerous. Duplication analysis revealed that GST family in M. ruthenica was expanded by segmental and tandem duplication. Several drought-responsive MruGSTs were identified by transcriptomic analyses. Of them, expression of MruGSTU39 was up-regulated much more in a tolerant accession by drought stress. Transformation of MruGSTU39 in M. ruthenica and alfalfa (Medicago sativa) enhanced growth and survival of transgenic seedlings than their wild-type counterparts under drought. We demonstrated that MruGSTU39 can detoxify ROS to reduce its damage to membrane by up-regulating activities of GST and glutathione peroxidase. Our findings provide full-scale knowledge on GST family in the wild legume M. ruthenica with high tolerance to drought, and highlight improvement tolerance of legume forages to drought using genomic information of M. ruthenica.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives.

BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

Transcriptome profiling unveils the mechanism of phenylpropane biosynthesis in rhizome development of Caucasian clover.

Caucasian clover is the only perennial herb of the genus Leguminous clover with underground rhizomes. However, we know very little about its development process and mechanism. Transcriptome studies were conducted on the roots of Caucasian clover without a rhizome (NR) at the young seedling stage and the fully developed rhizome, including the root neck (R1), main root (R2), horizontal root (R3), and rhizome bud (R4), of the tissues in the mature phase. Compared with the rhizome in the mature phase, NR had 893 upregulated differentially expressed genes (DEGs), most of which were enriched in 'phenylpropanoid biosynthesis', 'phenylalanine metabolism', 'DNA replication' and 'biosynthesis of amino acids'. A higher number of transcription factors (AP2/ERF, C2H2 and FAR1) were found in NR. There were highly expressed genes for R4, such as auxin response factor SAUR, galacturonosyltransferase (GAUT), and sucrose synthase (SUS). Phenylpropanoids are very important for the entire process of rhizome development. We drew a cluster heat map of genes related to the phenylpropanoid biosynthesis pathway, in which the largest number of genes belonged to COMT, and most of them were upregulated in R4.

Annual and perennial Medicago show signatures of parallel adaptation to climate and soil in highly conserved genes.

Human induced environmental change may require rapid adaptation of plant populations and crops, but the genomic basis of environmental adaptation remain poorly understood. We analysed polymorphic loci from the perennial crop Medicago sativa (alfalfa or lucerne) and the annual legume model species M. truncatula to search for a common set of candidate genes that might contribute to adaptation to abiotic stress in both annual and perennial Medicago species. We identified a set of candidate genes of adaptation associated with environmental gradients along the distribution of the two Medicago species. Candidate genes for each species were detected in homologous genomic linkage blocks using genome-environment (GEA) and genome-phenotype association analyses. Hundreds of GEA candidate genes were species-specific, of these, 13.4% (M. sativa) and 24% (M. truncatula) were also significantly associated with phenotypic traits. A set of 168 GEA candidates were shared by both species, which was 25.4% more than expected by chance. When combined, they explained a high proportion of variance for certain phenotypic traits associated with adaptation. Genes with highly conserved functions dominated among the shared candidates and were enriched in gene ontology terms that have shown to play a central role in drought avoidance and tolerance mechanisms by means of cellular shape modifications and other functions associated with cell homeostasis. Our results point to the existence of a molecular basis of adaptation to abiotic stress in Medicago determined by highly conserved genes and gene functions. We discuss these results in light of the recently proposed omnigenic model of complex traits.

Genome-wide identification, phylogenetic, and expression analysis under abiotic stress conditions of LIM gene family in Medicago sativa L.

The LIM (Lin-11, Isl-1 and Mec-3 domains) family is a key transcription factor widely distributed in animals and plants. The LIM proteins in plants are involved in the regulation of a variety of biological processes, including cytoskeletal organization, the development of secondary cell walls, and cell differentiation. It has been identified and analyzed in many species. However, the systematic identification and analysis of the LIM genes family have not yet been reported in alfalfa (Medicago sativa L.). Based on the genome-wide data of alfalfa, a total of 21 LIM genes were identified and named MsLIM01-MsLIM21. Comprehensive analysis of the chromosome location, physicochemical properties of the protein, evolutionary relationship, conserved motifs, and responses to abiotic stresses of the LIM gene family in alfalfa using bioinformatics methods. The results showed that these MsLIM genes were distributed unequally on 21 of the 32 chromosomes in alfalfa. Gene duplication analysis showed that segmental duplications were the major contributors to the expansion of the alfalfa LIM family. Based on phylogenetic analyses, the LIM gene family of alfalfa can be divided into four subfamilies: αLIM subfamily, ßLIM subfamily, γLIM subfamily, and δLIM subfamily, and approximately all the LIM genes within the same subfamily shared similar gene structure. The 21 MsLIM genes of alfalfa contain 10 Motifs, of which Motif1 and Motif3 are the conserved motifs shared by these genes. Furthermore, the analysis of cis-regulatory elements indicated that regulatory elements related to transcription, cell cycle, development, hormone, and stress response are abundant in the promoter sequence of MsLIM genes. Real-time quantitative PCR demonstrated that MsLIM gene expression is induced by low temperature and salt. The present study serves as a basic foundation for future functional studies on the alfalfa LIM family.

The genome of a wild Medicago species provides insights into the tolerant mechanisms of legume forage to environmental stress.

BACKGROUND: Medicago ruthenica, a wild and perennial legume forage widely distributed in semi-arid grasslands, is distinguished by its outstanding tolerance to environmental stress. It is a close relative of commonly cultivated forage of alfalfa (Medicago sativa). The high tolerance of M. ruthenica to environmental stress makes this species a valuable genetic resource for understanding and improving traits associated with tolerance to harsh environments. RESULTS: We sequenced and assembled genome of M. ruthenica using an integrated approach, including PacBio, Illumina, 10×Genomics, and Hi-C. The assembled genome was 904.13 Mb with scaffold N50 of 99.39 Mb, and 50,162 protein-coding genes were annotated. Comparative genomics and transcriptomic analyses were used to elucidate mechanisms underlying its tolerance to environmental stress. The expanded FHY3/FAR1 family was identified to be involved in tolerance of M. ruthenica to drought stress. Many genes involved in tolerance to abiotic stress were retained in M. ruthenica compared to other cultivated Medicago species. Hundreds of candidate genes associated with drought tolerance were identified by analyzing variations in single nucleotide polymorphism using accessions of M. ruthenica with varying tolerance to drought. Transcriptomic data demonstrated the involvements of genes related to transcriptional regulation, stress response, and metabolic regulation in tolerance of M. ruthenica. CONCLUSIONS: We present a high-quality genome assembly and identification of drought-related genes in the wild species of M. ruthenica, providing a valuable resource for genomic studies on perennial legume forages.

Historic exposure to herbivores, not constitutive traits, explains plant tolerance to herbivory in the case of two Medicago species (Fabaceae).

Mechanisms that allow plants to survive and reproduce after herbivory are considered to play a key role in plant evolution. In this study, we evaluated how tolerance varies in species with different historic exposure to herbivores considering ontogeny. We exposed the range-restricted species Medicago citrina and its closely related and widespread species M. arborea to one and two herbivory simulations (80 % aerial biomass loss). Physiological and growth parameters related to tolerance capacity were assessed to evaluate constitutive values (without herbivory) and induced tolerance after damage. Constitutive traits were not always related to greater tolerance, and each species compensated for herbivory through different traits. Herbivory damage only led to mortality in M. citrina; adults exhibited root biomass loss and increased oxidative stress after damage, but also compensated aerial biomass. Despite seedlings showed a lower death percentage than adults after herbivory in M. citrina, they showed less capacity to recover control values than adults. Moderate tolerance to M. arborea herbivory and low tolerance to M. citrina is found. Thus, although the constitutive characteristics are maintained in the lineage, the tolerance of plants decreases in M. citrina. That represents how plants respond to the lack of pressure from herbivores in their habitat.