Genome-Wide Identification and Expression Analysis of the SWEET Gene Family in Annual Alfalfa (Medicago polymorpha).

SWEET (Sugars will eventually be exported transporter) proteins are a group of sugar transporters that are involved in sugar efflux, phloem loading, reproductive development, plant senescence, and stress responses. In this study, 23 SWEET transporter members were identified in the Medicago polymorpha genome, heterogeneously distributed on seven chromosomes. These MpSWEET genes were divided into four subfamilies, which showed similar gene structure and motif composition within the same subfamily. Seventeen MpSWEET genes encode seven transmembrane helices (TMHs), and all MpSWEET proteins possess conserved membrane domains and putative serine phosphorylation sites. Four and three pairs of MpSWEET genes were predicted to be segmentally and tandemly duplicated, respectively, which may have contributed to their evolution of M. polymorpha. The results of microarray and RNA-Seq data showed that some MpSWEET genes were specifically expressed in disparate developmental stages (including seedling stage, early flowering stage, and late flowering stage) or tissues such as flower and large pod. Based on protein network interaction and expression patterns of MpSWEET genes, six MpSWEET genes were selected for further quantitative real-time PCR validation in different stress treatments. qRT-PCR results showed that MpSWEET05, MpSWEET07, MpSWEET12, MpSWEET15, and MpSWEET21 were significantly upregulated for at least two of the three abiotic stress treatments. These findings provide new insights into the complex transcriptional regulation of MpSWEET genes, which facilitates future research to elucidate the function of MpSWEET genes in M. polymorpha and other legume crops.

Metagenomics analysis reveals the performance of homo- and heterofermentative lactic acid bacteria in alfalfa silage fermentation, bacterial community, and functional profiles.

Alfalfa (Medicago sativa L.) is a kind of roughage frequently utilized as an animal feed but challenging to be ensiled due to its low water-soluble carbohydrate (WSC), high water content, and elevated buffering capacity, thus requiring the application of lactic acid bacteria (LAB) to improve its fermentation. This study employed high-throughput metagenomic sequence technology to reveal the effects of homofermentative LAB, Lactobacillus plantarum (Lp), or Pediococcus pentosaceus (Pp), and heterofermentative LAB, L. buchneri (Lb), or their combinations (LbLp or LbPp) (applied at 1.0 × 109 colony forming units (cfu) per kilogram of alfalfa biomass fresh material) on the fermentation, microbial community, and functional profiles of alfalfa silage after 7, 14, 30, and 60 ensiling days. The results indicated a reduction (P < 0.05) in glucose and pH and higher (P < 0.05) beneficial organic acid contents, xylose, crude protein, ammonia nitrogen, and aerobic stability in Lb-, LbPp-, and LbLp-inoculated alfalfa silages after 30 and 60 d. Also, higher (P < 0.05) WSC contents were recorded in LbLp-inoculated alfalfa silages after 30 d (10.84 g/kg dry matter [DM]) and 60 d (10.92 g/kg DM). Besides, LbLp-inoculated alfalfa silages recorded higher (P < 0.05) LAB count (9.92 log10 cfu/g) after 60 d. Furthermore, a positive correlation was found between the combined LAB inoculants in LbLp-inoculated alfalfa silages and dominant LAB genera, Lactobacillus and Pediococcus, with fermentation properties after 30 and 60 d. In addition, the 16S rRNA gene-predicted functional analyses further showed that the L. buchneri PC-C1 and L. plantarum YC1-1-4B combination improved carbohydrate metabolism and facilitated further degradation of polysaccharides in alfalfa after 60 d of ensiling. These findings reveal the significant performance of L. buchneri and L. plantarum in combination with dominant LAB species in suppressing the growth of Clostridia, molds, and yeasts and improving the fermentation characteristics and functional carbohydrate metabolism of alfalfa after 60 d ensiling, thus suggesting the need for further studies to uncover the diverse performance of the LAB combination and their consortium with other natural and artificial inoculants in various kinds of silages.

Current studies are aimed towards utilizing certain lactic acid bacteria (LAB) strains with high-yielding beneficial organic acid production for enhancing the quality of forages during preservation (otherwise known as ensiling) and to improve animal feed production. This study addresses the challenges of ensiling alfalfa (a kind of roughage frequently utilized as an animal feed in the livestock industries), such as low water-soluble carbohydrate, high water and fiber content, and high buffering capacity, by unraveling the positive interaction mechanisms of mixed LAB inoculants (homo-and heterofermentative LAB strains) and dominant epiphytic LAB in altering the development of pathogenic microorganisms and consequently improving the fermentation characteristics, chemical compositions, bacterial community, and functional profile of alfalfa after 60 d of ensiling.

Genome-Wide Identification and Expression Analysis of the Dof Transcription Factor in Annual Alfalfa Medicago polymorpha.

The Dof transcription factor is a plant-specific transcription gene family that plays various biological functions in plant development and stress response. However, no relevant research has been conducted on Medicago polymorpha. Here, 36 MpDof genes were identified in the M. polymorpha genome and further divided into 10 groups based on the comparative phylogenetic analysis. The essential information of MpDof genes, such as chromosomal localization, gene structure, conserved motifs, and selective pressures were systematically analyzed. All 36 MpDof genes were predicted to contain more cis-acting elements related to hormone response. MpDof24 and MpDof25 were predicted to interact with MpDof11 and MpDof26 to involve in the photoperiod blooms process. The MpDof genes showed a diverse expression pattern in different tissues. Notably, MpDof29 and MpDof31 were specifically expressed in the large pod and root, respectively, suggesting their crucial role in the pod and root development. qRT-PCR analysis indicated that the expression levels of MpDof10, MpDof25, MpDof26, and MpDof29 were obviously up-regulated under drought, salt, and cold stress. Collectively, genome-wide identification, evolutionary, and expression analysis of the Dof transcription gene family in M. polymorpha will provide new information to further understand and utilize the function of these Dof genes in Medicago plants.

Screening and identification of multiple abiotic stress responsive candidate genes based on hybrid-sequencing in Vicia sativa.

Common vetch is an important leguminous forage for both livestock fodder and green manure and has a tremendous latent capacity in a sustainable agroecosystem. In the present study, a comprehensive transcriptome analysis of the aboveground leaves and underground roots of common vetch under multiple abiotic stress treatments, including NaCl, drought, cold, and cold drought, was performed using hybrid-sequencing technology, i. e. single-molecule real-time sequencing technology (SMRT) and supplemented by next-generation sequencing (NGS) technology. A total of 485,038 reads of insert (ROIs) with a mean length of 2606 bp and 228,261 full-length nonchimeric (FLNC) reads were generated. After deduplication, 39,709 transcripts were generated. Of these transcripts, we identified 1059 alternative splicing (AS) events, 17,227 simple sequence repeats (SSRs), and 1647 putative transcription factors (TFs). Furthermore, 640 candidates long noncoding RNAs (lncRNAs) and 28,256 complete coding sequences (CDSs) were identified. In gene annotation analyses, a total of 38,826 transcripts (97.78%) were annotated in eight public databases. Finally, seven multiple abiotic stress-responsive candidate genes were obtained through gene expression, annotation information, and protein-protein interaction (PPI) networks. Our research not only enriched the structural information of FL transcripts in common vetch, but also provided useful information for exploring the molecular mechanism of multiple abiotic stress tolerance between aboveground and underground tissues in common vetch and related legumes.

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.

Full-length transcriptional analysis reveals the complex relationship of leaves and roots in responses to cold-drought combined stress in common vetch.

Plant responses to single or combined abiotic stresses between aboveground and underground parts are complex and require crosstalk signaling pathways. In this study, we explored the transcriptome data of common vetch (Vicia sativa L.) subjected to cold and drought stress between leaves and roots via meta-analysis to identify the hub abiotic stress-responsive genes. A total of 4,836 and 3,103 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Transcriptome analysis results showed that the set of stress-responsive DEGs to concurrent stress is distinct from single stress, indicating a specialized and unique response to combined stresses in common vetch. Gene Ontology (GO) enrichment analyses identified that "Photosystem II," "Defence response," and "Sucrose synthase/metabolic activity" were the most significantly enriched categories in leaves, roots, and both tissues, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis results indicated that "ABC transporters" are the most enriched pathway and that all of the genes were upregulated in roots. Furthermore, 29 co-induced DEGs were identified as hub genes based on the consensus expression profile module of single and co-occurrence stress analysis. In transgenic yeast, the overexpression of three cross-stress tolerance candidate genes increased yeast tolerance to cold-drought combined stress. The elucidation of the combined stress-responsive network in common vetch to better parse the complex regulation of abiotic responses in plants facilitates more adequate legume forage breeding for combined stress tolerance.

Molecular Characterization of the miR156/MsSPL Model in Regulating the Compound Leaf Development and Abiotic Stress Response in Alfalfa.

Plant leaf patterns and shapes are spectacularly diverse. Changing the complexity of leaflet numbers is a valuable approach to increase its nutrition and photosynthesis. Alfalfa (Medicago sativa) is the most important forage legume species and has diversified compound leaf patterns, which makes it a model species for studying compound leaf development. However, transcriptomic information from alfalfa remains limited. In this study, RNA-Seq technology was used to identify 3746 differentially expressed genes (DEGs) between multifoliate and trifoliate alfalfa. Through an analysis of annotation information and expression data, SPL, one of the key regulators in modifiable plant development and abiotic stress response, was further analyzed. Here, thirty MsSPL genes were obtained from the alfalfa genome, of which 16 had the putative miR156 binding site. A tissue expression pattern analysis showed that the miR156-targeted MsSPLs were divided into two classes, namely, either tissue-specific or widely expressed in all tissues. All miR156-targeted SPLs strongly showed diversification and positive roles under drought and salt conditions. Importantly, miR156/MsSPL08 was significantly suppressed in multifoliate alfalfa. Furthermore, in the paralogous mutant of MsSPL08 isolated from Medicago truncatula, the phenotypes of mutant plants reveal that miR156/MsSPL08 is involved not only involved the branches but also especially regulates the number of leaflets. The legume is a typical compound leaf plant; the ratio of the leaflet often affects the quality of the forage. This study sheds light on new functions of SPL genes that regulate leaflet number development.

The genome of Medicago polymorpha provides insights into its edibility and nutritional value as a vegetable and forage legume.

Medicago polymorpha is a nutritious and palatable forage and vegetable plant that also fixes nitrogen. Here, we reveal the chromosome-scale genome sequence of M. polymorpha using an integrated approach including Illumina, PacBio and Hi-C technologies. We combined PacBio full-length RNA-seq, metabolomic analysis, structural anatomy analysis and related physiological indexes to elucidate the important agronomic traits of M. polymorpha for forage and vegetable usage. The assembled M. polymorpha genome consisted of 457.53 Mb with a long scaffold N50 of 57.72 Mb, and 92.92% (441.83 Mb) of the assembly was assigned to seven pseudochromosomes. Comparative genomic analysis revealed that expansion and contraction of the photosynthesis and lignin biosynthetic gene families, respectively, led to enhancement of nutritious compounds and reduced lignin biosynthesis in M. polymorpha. In addition, we found that several positively selected nitrogen metabolism-related genes were responsible for crude protein biosynthesis. Notably, the metabolomic results revealed that a large number of flavonoids, vitamins, alkaloids, and terpenoids were enriched in M. polymorpha. These results imply that the decreased lignin content but relatively high nutrient content of M. polymorpha enhance its edibility and nutritional value as a forage and vegetable. Our genomic data provide a genetic basis that will accelerate functional genomic and breeding research on M. polymorpha as well as other Medicago and legume plants.

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass.

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species.

Proteomics analysis of alfalfa response to heat stress.

The proteome responses to heat stress have not been well understood. In this study, alfalfa (Medicago sativa L. cv. Huaiyin) seedlings were exposed to 25 °C (control) and 40 °C (heat stress) in growth chambers, and leaves were collected at 24, 48 and 72 h after treatment, respectively. The morphological, physiological and proteomic processes were negatively affected under heat stress. Proteins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and differentially expressed protein spots were identified by mass spectrometry (MS). Totally, 81 differentially expressed proteins were identified successfully by MALDI-TOF/TOF. These proteins were categorized into nine classes: including metabolism, energy, protein synthesis, protein destination/storage, transporters, intracellular traffic, cell structure, signal transduction and disease/defence. Five proteins were further analyzed for mRNA levels. The results of the proteomics analyses provide a better understanding of the molecular basis of heat-stress responses in alfalfa.

Mutation of cellulose synthase gene improves the nutritive value of rice straw.

Rice straw is an important roughage resource for ruminants in many rice-producing countries. In this study, a rice brittle mutant (BM, mutation in OsCesA4, encoding cellulose synthase) and its wild type (WT) were employed to investigate the effects of a cellulose synthase gene mutation on rice straw morphological fractions, chemical composition, stem histological structure and in situ digestibility. The morphological fractions investigation showed that BM had a higher leaf sheath proportion (43.70% vs 38.21%, p<0.01) and a lower leaf blade proportion (25.21% vs 32.14%, p<0.01) than WT. Chemical composition analysis showed that BM rice straw was significantly (p<0.01) higher in CP (crude protein), hemicellulose and acid insoluble ash (AIA) contents, but lower in dry matter (DM), acid detergent fiber (ADFom) and cellulose contents when compared to WT. No significant difference (p>0.05) was detected in neutral detergent fiber (NDFom) and ADL contents for both strains. Histological structure observation indicated that BM stems had fewer sclerenchyma cells and a thinner sclerenchyma cell wall than WT. The results of in situ digestion showed that BM had higher DM, NDFom, cellulose and hemicellulose disappearance at 24 or 48 h of incubation (p<0.05). The effective digestibility of BM rice straw DM and NDFom was greater than that of WT (31.4% vs 26.7% for DM, 29.1% vs 24.3% for NDFom, p<0.05), but the rate of digestion of the slowly digested fraction of BM rice straw DM and NDF was decreased. These results indicated that the mutation in the cellulose synthase gene could improve the nutritive value of rice straw for ruminants.