Alfalfa is the most widely cultivated forage legume and one of the most economically valuable crops in the world. The large size and complexity of the alfalfa genome has delayed the development of genomic resources for alfalfa research. Second-generation Illumina transcriptome sequencing is an efficient method for generating a global transcriptome sequence dataset for gene discovery and molecular marker development in alfalfa. More than 28 million sequencing reads (5.64 Gb of clean nucleotides) were generated by Illumina paired-end sequencing from 15 different alfalfa tissue samples. In total, 40,433 unigenes with an average length of 803 bp were obtained by de novo assembly. Based on a sequence similarity search of known proteins, a total of 36,684 (90.73%) unigenes were annotated. In addition, 1,649 potential EST-SSRs were identified as potential molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs of PCR primers were randomly selected to validate the assembly quality and develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to amplify sequences in initial screening tests, and 27 primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 10 alfalfa accessions. The present study provided global sequence data for autotetraploid alfalfa and demonstrates the Illumina platform is a fast and effective approach to EST-SSR markers development in alfalfa. The use of these transcriptome datasets will serve as a valuable public information platform to accelerate studies of the alfalfa genome.

Alfalfa is the most extensively cultivated forage legume. Salinity is a major environmental factor that impacts on alfalfa’s productivity. However, little is known about the molecular mechanisms underlying alfalfa responses to salinity, especially the relative contribution of the two important components of osmotic and ionic stress. In this study, we constructed the first full-length transcriptome database for alfalfa root tips under continuous NaCl and mannitol treatments for 1, 3, 6, 12, and 24 h (three biological replicates for each time points, including the control group) via PacBio Iso-Seq. This resulted in the identification of 52,787 full-length transcripts, with an average length of 2551 bp. Global transcriptional changes in the same 33 stressed samples were then analyzed via BGISEQ-500 RNA-Seq. Totals of 8861 NaCl-regulated and 8016 mannitol-regulated differentially expressed genes (DEGs) were identified. Metabolic analyses revealed that these DEGs overlapped or diverged in the cascades of molecular networks involved in signal perception, signal transduction, transcriptional regulation, and antioxidative defense. Notably, several well characterized signalling pathways, such as CDPK, MAPK, CIPK, and PYL-PP2C-SnRK2, were shown to be involved in osmotic stress, while the SOS core pathway was activated by ionic stress. Moreover, the physiological shifts of catalase and peroxidase activity, glutathione and proline content were in accordance with dynamic transcript profiles of the relevant genes, indicating that antioxidative defense system plays critical roles in response to salinity stress. Overall, our study provides evidence that the response to salinity stress in alfalfa includes both osmotic and ionic components. The key osmotic and ionic stress-related genes are candidates for future studies as potential targets to improve resistance to salinity stress via genetic engineering.

Elymus sibiricus as an important forage grass and gene pool for improving cereal crops, that is widely distributed in West and North China. Information on its genetic diversity and relationships is limited but necessary for germplasm collection, conservation and future breeding. Start Codon Targeted (SCoT) markers were used for studying the genetic diversity and relationships among 53 E. sibiricus accessions from its primary distribution area in China. A total of 173 bands were generated from 16 SCoT primers, 159 bands of which were polymorphic with the percentage of polymorphic bands (PPB) of 91.91%. Based upon population structure analysis five groups were formed. The cluster analysis separated the accessions into two major clusters and three sub-clusters, similar to results of principal coordinate analysis (PCoA). The molecular variance analysis (AMOVA) showed that genetic variation was greater within geographical regions (50.99%) than between them (49.01%). Furthermore, the study also suggested that collecting and evaluating E. sibiricus germplasm for major geographic regions and special environments broadens the available genetic base and illustrates the range of variation. The results of the present study showed that SCoT markers were efficient in assessing the genetic diversity among E. sibiricus accessions.

The seed dispersion caused by pod shattering is a form of propagation used by many wild species. Loss of seeds from pod shattering is frequent in the common vetch (Vicia sativa L.), an important self-pollinating annual forage legume. However, pod shattering is one of the most important defects that limits the reproduction of the vetch in the field and the usage as a leguminous forage crop. To better understand the vetch pod shattering mechanism, we used high-throughput RNA sequencing to assess the global changes in the transcriptomes of the pod ventral sutures of shattering-susceptible and shattering-resistant vetch accessions screened from 541 vetch germplasms. A total of 1,285 significantly differentially expressed unigenes (DEGs) were detected, including 575 up-regulated unigenes and 710 down-regulated unigenes. Analyses of Gene Ontology and KEGG metabolic enrichment pathways of 1,285 DEGs indicated that 22 DEGs encoding cell wall modifications and hydrolases associated with pod shattering were highly expressed in shattering-susceptible accessions. These genes were mainly enriched in "hydrolase activity", "cytoplasm" and "carbohydrate metabolic process" systems. These cell wall modifications and hydrolases genes included β-glucosidase and endo-polygalacturonase, which work together to break down the glycosidic bonds of pectin and cellulose, and to promote the dissolution and disappearance of the cell wall in the ventral suture of the pod and make the pod more susceptible to shattering. We demonstrated the differences in gene transcription levels between the shattering-susceptible and shattering-resistant vetch accessions for the first time and our results provided valuable information for the identifying and characterizing of pod shattering regulation networks in vetch. This information may facilitate the future identification of pod shattering-related genes and their underlying molecular mechanisms in the common vetch.

Water stress seriously constrains plant growth and yield. Long non-coding RNAs (lncRNAs) serve as versatile regulators in various biological regulatory processes. To date, the systematic screening and potential functions of lncRNA have not yet been characterized in Cleistogenes songorica, especially under water stress conditions. In this study, we obtained the root and shoot transcriptomes of young C. songorica plants subjected to different degrees of water stress and recovery treatments by Illumina-based RNA-seq. A total of 3397 lncRNAs were identified through bioinformatics analysis. LncRNA differential expression analysis indicated that the higher response of roots compared to shoots during water stress and recovery. We further identified the 1644 transcription factors, 189 of which were corresponded to 163 lncRNAs in C. songorica. Though comparative analyses with major Poaceae species based on blast, 81 water stress-related orthologues regulated to lncRNAs were identified as a core of evolutionary conserved genes important to regulate water stress responses in the family. Among these target genes, two genes were found to be involved in the abscisic acid (ABA) signalling pathway, and four genes were enriched for starch and sucrose metabolism. Additionally, the 52 lncRNAs were predicted as target mimics for microRNAs (miRNAs) in C. songorica. RT-qPCR results suggested that MSTRG.43964.1 and MSTRG.4400.2 may regulate the expression of miRNA397 and miRNA166, respectively, as target mimics under water stress and during recovery. Finally, a co-expression network was constructed based on the lncRNAs, miRNAs, protein-coding genes (PCgenes) and transcription factors under water stress and during recovery in C. songorica. In C. songorica, lncRNAs, miRNAs, PCgenes and transcription factors constitute a complex transcriptional regulatory network which lncRNAs can regulate PCgenes and miRNAs under water stress and recovery. This study provides fundamental resources to deepen our knowledge on lncRNAs during ubiquitous water stress.

Cultivated alfalfa (Medicago sativa L.) is one of the most important perennial legume forages in the world, and it has considerable potential as a valuable forage crop for livestock. However, the molecular mechanisms underlying alfalfa responses to cold stress are largely unknown. In this study, the transcriptome changes in alfalfa under cold stress at 4 °C for 2, 6, 24, and 48 h (three replicates for each time point) were analyzed using the high-throughput sequencing platform, BGISEQ-500, resulting in the identification of 50,809 annotated unigenes and 5283 differentially expressed genes (DEGs). Metabolic pathway enrichment analysis demonstrated that the DEGs were involved in carbohydrate metabolism, photosynthesis, plant hormone signal transduction, and the biosynthesis of amino acids. Moreover, the physiological changes of glutathione and proline content, catalase, and peroxidase activity were in accordance with dynamic transcript profiles of the relevant genes. Additionally, some transcription factors might play important roles in the alfalfa response to cold stress, as determined by the expression pattern of the related genes during 48 h of cold stress treatment. These findings provide valuable information for identifying and characterizing important components in the cold signaling network in alfalfa and enhancing the understanding of the molecular mechanisms underlying alfalfa responses to cold stress.