Comparative analysis of Chloroplast genomes in 48 samples from 5 species and 2 varieties of Agropyron Gaertn....

Agropyron belongs to Triticeae of the Poaceae family; its plants are rich in nutrients and have good palatability, serving as important forage grasses in the temperate zone of the Northern Hemisphere. The accurate identification of Agropyron germplasm resources is essential for breeding. However, the morphological variation of Agropyron is complex, making identification on the basis of traditional morphological characteristics difficult. Molecular markers are effective tools for the rapid and accurate identification of many forage plants. Therefore, in this study, second-generation high-throughput sequencing, chloroplast genome assembly, chloroplast genome comparison, and phylogenetic analysis were conducted on 48 Agropyron samples from 5 species and 2 varieties.

Using next-generation sequencing reads, the chloroplast genome was assembled and annotated. Analyses of repetitive sequences, codon usage bias, and nucleotide diversity revealed high conservation: the genome length ranged from 135,321 to 135,564 bp, the GC content was stable at 38%, and all the genomes encoded 132 consistent genes. Simple repeat sequences (SSRs) were mainly of the A/T type (21-28), and direct repeats were predominant in long repeat sequences (29-31). Each sample harbored 29-34 tandem repeats, with Agropyron mongolicum Keng (0506) showing the maximum (34). The long repeat sequence included 13 palindromic and 29-31 forward repeats in most samples, with no reverse or complementary repeats detected. The codon endings were predominantly A/U (RSCU > 1 codons accounted for 48.4%). Nucleotide diversity analysis revealed 6 hypervariable regions (e.g., psbA and rpl32), but the hypervariable regions did not contain enough informative sites to distinguish the five species of Agropyron. In the phylogenetic tree, all Agropyron samples formed a highly supported monophyletic clade (bootstrap = 100), but the intragenus relationships among the five species and two varieties remained unresolved, most likely due to insufficient phylogenetically informative sites because there were only 178 phylogenetically informative sites in the 135,978 chloroplast effective sites (0.13%). We also did not find species-specific chloroplast genome SNPs or indels. PCA revealed that different species could not be separated on the first two principal components. STRUCTURE analysis based on Angiosperms353 markers showed that K = 2 was the optimal grouping result, but the genetic grouping did not match the taxonomy of Agropyron, which was consistent with the results of analysis based on the chloroplast genome.

Our study comprehensively revealed the chloroplast genome information of Agropyron plants and its consistency across the genus.