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.

Low temperature is an important limiting factor in the environment that affects the distribution, growth and development of warm-season grasses. Transcriptome sequencing has been widely used to mine candidate genes under low-temperature stress and other abiotic stresses. However, the molecular mechanism of centipedegrass in response to low-temperature stress was rarely reported. To understand the molecular mechanism of centipedegrass in response to low-temperature stress, we measured physiological indicators and sequenced the transcriptome of centipedegrass under different stress durations. Under cold stress, the SS content and APX activity of centipedegrass increased while the SOD activity decreased; the CAT activity, POD activity and flavonoid content first increased and then decreased; and the GSH-Px activity first decreased and then increased. Using full-length transcriptome and second-generation sequencing, we obtained 38.76 G subreads. These reads were integrated into 177,178 isoforms, and 885 differentially expressed transcripts were obtained. The expression of AUX_IAA and WRKY transcription factors and HSF transcription-influencing factors increased during cold stress. Through KEGG enrichment analysis, we determined that arginine and proline metabolism, plant circadian rhythm, plant hormone signal transduction and the flavonoid biosynthesis pathways played important roles in the cold stress resistance of centipedegrass. In addition, by using weighted gene coexpression network analysis (WGCNA), we determined that the turquoise module was significantly correlated with SS content and APX activity, while the blue module was significantly negatively correlated with POD and CAT activity. This paper is the first to report the response of centipedegrass to cold stress at the transcriptome level. Our results help to clarify the molecular mechanisms underlying the cold tolerance of warm-season grasses.

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.

Phylogenetic origin of the Y haplome present in allopolyploid Triticeae species remains unknown. Here, we report the 10.47 Gb chromosome-scale genome of allohexaploid Elymus nutans (StStYYHH). Phylogenomic analyses reveal that the Y haplome is sister to the clade comprising V and Jv haplomes from Dasypyrum and Thinopyum. In addition, H haplome from the Hordeum-like ancestor, St haplome from the Pseudoroegneria-like ancestor and Y haplome are placed in the successively diverged clades. Resequencing data reveal the allopolyploid origins with St, Y, and H haplome combinations in Elymus. Population genomic analyses indicate that E. nutans has expanded from medium to high/low-altitude regions. Phenotype/environmental association analyses identify MAPKKK18 promoter mutations reducing its expression, aiding UV-B adaptation in high-altitude populations. These findings enhance understanding of allopolyploid evolution and aid in breeding forage and cereal crops through intergeneric hybridization within Triticeae.