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.

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.

Prairie grass (Bromus catharticus Vahl) is an important grass species that could be used in the production systems of certified seed and high-quality forage for grazing ruminants. In the present research, a sequence-related amplified polymorphism (SRAP) marker was employed to detect the genetic variability and structure of 80 prairie grass accessions from all over the world. Altogether, 460 reliable bands were amplified from 47 SRAP primer pairs with 345 (75%) polymorphic bands. The average values of discrimination power (DP) and polymorphic information content (PIC) were 0.753 and 0.317, respectively. Both the UPGMA clustering and PCoA analyses grouped the 80 accessions into five clusters, whereas the STRUCTURE analysis showed that 80 prairie grass accessions possessed three genetic memberships (K = 3). The results of the Mantel test showed that the distance matrix has a moderately positive correlation between the morphological and molecular data sets (r = 0.524). A poor genetic differentiation (Fst = 0.045) was discovered among the six geo-groups of accessions. Besides, the highest intragroup genetic diversity was found in the North America group (He = 0.335). This study provides a genetic structure and diversity case for prairie grass, and supplies new clues for the study and utilization of prairie grass.

Kengyilia is a newly established genus. Most species in this genus survive in hash environment, which might be an indicator of an acquirement of stress resistance genes and the potential for molecular breeding in Triticeae species. Quantitative real-time PCR (qRT-PCR) is a widely used technique with varied sensitivity heavily dependent on the optimal level of the reference genes. K. melanthera is a typical psammophyte species which has high drought resistance. The reference genes of K. melanthera are not yet reported. This study aims to evaluate the expression stability of 14 candidate reference genes (EF1A, GAPDH, ACT1, UBI, TUBB3, TIPRL, CACS, PPP2R1B, TUBA1A, EIF4A1, CYPA3, TCTP, ABCG11L, and FBXO6L) under five treatments (drought, heat, cold, salt, and ABA) and find the most stable and suitable one even upon stressed conditions. The software NormFinder, GeNorm, BestKeeper, and RefFinder were used for data analysis. In general, the genes CACS and PPP2R1B are concluded to have the best overall performance under the various treatments. With the ABA treatment, TCTP and TIPRL show the best stability. CACS and TCTP, as well as TIPRL and CYPA3, were most stable under the treatments of cold and salt, respectively. CACS and FBXO6L were ranked the highest with the heat treatment and drought treatment, respectively. Finally, the Catalase-1 (CAT1) gene was used to verify the reliability of the above reference genes. Accordingly, CAT1’s expression pattern remained unchanged after normalization with stable reference genes. This study provides beneficial information about the stability and reliability of potential reference genes for qRT-PCR in K. melanthera.