Trifolium L., which belongs to the IR lacking clade (IRLC), is one of the largest genera in the Leguminosae and contains several economically important fodder species. Here, we present whole chloroplast (cp) genome sequencing and annotation of two important annual grasses, Trifolium alexandrinum (Egyptian clover) and T. resupinatum (Persian clover). Abundant single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels) were discovered between those two species. Global alignment of T. alexandrinum and T. resupinatum to a further thirteen Trifolium species revealed a large amount of rearrangement and repetitive events in these fifteen species. As hypothetical cp open reading frame (ORF) and RNA polymerase subunits, ycf1 and rpoC2 in the cp genomes both contain vast repetitive sequences and observed high Pi values (0.7008, 0.3982) between T. alexandrinum and T. resupinatum. Thus they could be considered as the candidate genes for phylogenetic analysis of Trifolium species. In addition, the divergence time of those IR lacking Trifolium species ranged from 84.8505 Mya to 4.7720 Mya. This study will provide insight into the evolution of Trifolium species.

Drought is one of the most important factors affecting plant growth and production due to ongoing global climate change. Elymus sibiricus has been widely applied for ecological restoration and reseeding of degraded grassland in the Qinghai–Tibetan Plateau (QTP) because of its strong adaptability to barren, salted, and drought soils. To explore the mechanism of drought resistance in E. sibiricus, drought-tolerant and drought-sensitive genotypes of E. sibiricus were used in metabolomic studies under simulated long-term and short-term drought stress. A total of 1091 metabolites were detected, among which, 27 DMs were considered to be the key metabolites for drought resistance of E. sibiricus in weighted gene co-expression network analysis (WGCNA). Ten metabolites, including 3-amino-2-methylpropanoic acid, coniferin, R-aminobutyrate, and so on, and 12 metabolites, including L-Proline, L-histidine, N-acetylglycine, and so on, showed differential accumulation patterns under short-term and long-term drought stress, respectively, and thus, could be used as biomarkers for drought-tolerant and drought-sensitive E. sibiricus. In addition, different metabolic accumulation patterns and different drought response mechanisms were also found in drought-tolerant and drought-sensitive genotypes of E. sibiricus. Finally, we constructed metabolic pathways and metabolic patterns for the two genotypes. This metabolomic study on the drought stress response of E. sibiricus can provide resources and a reference for the breeding of new drought-tolerant cultivars of E. sibiricus.

The primary approach for variety distinction in Italian ryegrass is currently the DUS (distinctness, uniformity and stability) test based on phenotypic traits. Considering the diverse genetic background within the population and the complexity of the environment, however, it is challenging to accurately distinguish varieties based on DUS criteria alone. In this study, we proposed the application of high-throughput RAD-seq to distinguish 11 Italian ryegrass varieties with three bulks of 50 individuals per variety. Our findings revealed significant differences among the 11 tested varieties. The PCA, DAPC and STRUCTURE analysis indicated a heterogeneous genetic background for all of them, and the AMOVA analysis also showed large genetic variance among these varieties (Φ_ST = 0.373), which were clearly distinguished based on phylogenetic analysis. Further nucleotide diversity (Pi) analysis showed that the variety ‘Changjiang No.2’ had the best intra-variety consistency among 11 tested varieties. Our findings suggest that the RAD-seq could be an effectively alternative method for the variety distinction of Italian ryegrass, as well as a potential tool for open-pollinated varieties (OPVs) of other allogamous species.

Seed aging has an important effect on the germplasm preservation and industrialized production of Siberian wildrye (Elymus sibiricus) in the Qinghai-Tibet Plateau. However, so far its underlying molecular mechanisms still largely remain unknown. To shed light on this topic, one-year stored seeds of E. sibiricus were exposed to artificial aging treatments (AAT), followed by seed vigor characteristics and physiological status monitoring. Then global proteomics analysis was undertaken by the tandem mass tags (TMT) technique, and the proteins were quantified with liquid chromatography-tandem mass spectrometry on three aging time points (0 h, 36 h and 72 h). Finally, we verified the expression of related proteins by parallel reaction monitoring (PRM). Our results demonstrated that the seed vigor decreased remarkably in response to artificial aging, but the relative ion-leakage and malondialdehyde content, superoxide anion and hydrogen peroxide showed the opposite situation. Proteomic results showed that a total of 4169 proteins were identified and quantified. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that a series of key pathways including carbohydrate metabolism, lipid metabolism, and antioxidant activity were severely damaged by aging treatments. Numerous key proteins such as glyceraldehyde triphosphate glyceraldehyde dehydrogenase, succinate dehydrogenase, lipoxygenase, peroxidase, glutathione-s-transferase and late embryogenesis abundant proteins were significantly down-regulated. However, the up-regulation of the heat shock protein family has made a positive contribution to oxidative stress resistance in seeds. This study provides a useful catalog of the E. sibiricus proteomes with insights into the future genetic improvement of seed storability.

Elymus breviaristatus is a grass species only distributed in the southeast of Qinghai-Tibetan Plateau (QTP), which has suffered from serious habitat fragmentation. Therefore, understanding patterns of genetic diversity within and among natural E. breviaristatus populations could provide insight for future conservation strategies. In this study, sequence-related amplified polymorphism markers were employed to investigate the genetic diversity and hierarchical structure of seven E. breviaristatus populations from QTP, China. Multiple measures of genetic diversity indicated that there is low to moderate genetic variation within E. breviaristatus populations, consistent with its presumed mating system. In spite of its rarity, E. breviaristatus presented high genetic diversity that was equivalent to or even higher than that of widespread species. Bayesian clustering approaches, along with clustering analysis and principal coordinate analysis partitioned the studied populations of E. breviaristatus into five genetic clusters. Differentiation coefficients (F_st, G_ST, etc.) and AMOVA analysis revealed considerable genetic divergence among different populations. BARRIER analyses indicated that there were two potential barriers to gene flow among the E. breviaristatus populations. Despite these patterns of differentiation, genetic distances between populations were independent of geographic distances (r = 0.2197, p = 0.2534), indicating little isolation by distance. Moreover, despite detecting a common outlier by two methods, bioclimatic factors (altitude, annual mean temperature, and annual mean precipitation) were not related to diversity parameters, indicating little evidence for isolation caused by the environment. These patterns of diversity within and between populations are used to propose a conservation strategy for E. breviaristatus.