Complete chloroplast genome and phylogenetic analysis of a wild grass, Hordeum roshevitzii Bowden.

The complete circular chloroplast (cp) genome of wild Hordeum roshevitzii Bowden was sequenced and reported in this study. The complete chloroplast genome of wild H. roshevitzii was 12,753 bp in length, including a pair of inverted repeat regions (IRA/IRB) of 21,587 bp separated by one small single-copy (SSC) region of 12,753 bp and one large single-copy (LSC) region of 81,138 bp. A total of 133 genes, including 85 protein-coding genes (PCGs), 40 transfer RNA genes, and 8 ribosomal RNA genes were predicted from the cp genomes. The overall GC content of the cp genome was 38.25% and the corresponding values of the IR, SSC, and LSC were 43.89%, 32.19%, and 36.21%. The phylogenetic analysis of H. roshevitzii determined that H. roshevitzii was clustered closely with Hordeum bogdanii.

Wild Hordeum roshevitzii Bowden is an ancient and mysterious plant of the genus Hordeum in family Gramineae, with the common name tall wheatgrass. H. roshevitzii originated with a history of 20,000 years in the Nile River Basin. It is a perennial plant, stems clump erect and smooth, generally 30–70 cm high, with three or four nodes. The base of leaf sheath is longer than the upper part, the length of leaf blade is 3–14 cm, often flat. The spikes are green or purplish in color, generally 4–7 cm in length, and the palea is equal to the lemma. Wild H. roshevitzii is mainly distributed in Inner Mongolia, northern Shanxi, Gansu, Qinghai, and Xinjiang in China. It is usual found in grassland with sandy soil along rivers. It is an important forage grass and also a valuable gene pool for cereal crops (Wang et al. 1991). However, H. roshevitzii is particularly rare and precious because it is extremely difficult cultivate. H. roshevitzii has attracted widespread attention from medical experts and is even seen as a way to prevent cancer due to this grain’s purple chaff, which contains natural anthocyanin glycosides and used as antioxidants. Knowledge of genome constitution of H. roshevitzii was extremely valuable for forage breeding and pharmaceutical development.

Chloroplast genome as independent units of heredity, directly reflects the long-term accumulation of genetic variation of this plant in the process of evolution, and traces the origin of species and migration (Su et al. 2020). Chloroplast genome is widely used in plant phylogeny because chloroplast genome reflects relative interspecific genetic relationship and genetic diversity of plant.

However, there is no research on genome of the wild H. roshevitzii, so obtaining the complete genome of wild H. roshevitzii cp would lay the foundation for the genetic research of this mysterious plant. In our study, we reported and annotated the complete cp genome sequence of wild H. roshevitzii, and submitted to the GenBank with the accession number MW266081.

Fresh sample was obtained from an alpine grazing meadow in Sunan Yugur Autonomous County (Gansu Province, N37°54′24″, E101°48′30″). The plant sample has been deposited in the College of Pastoral Agricultural Science and Technology, Lanzhou University, Lanzhou, China. Genomic DNA extraction and sequencing library construction were conducted by Benagen Technology Services Limited (Wuhan, China), and a 350 bp library of DNA samples was constructed by whole-genome shotgun method, the whole H. roshevitzii chloroplast genome was sequenced with 150 bp double-terminal reads on Illumina NovaSeq (University of Edinburgh 2017) sequencer. A total of 35,359,246 raw reads were obtained, they were filtered and trimmed using the SOAPnuke version 2.1.0 (Chen et al. 2018) to get the clean reads. Then 34,879,360 clean reads were spliced by SPAdes version 3.13.1 (Nicolas et al. 2017), the splicing results and reference genome were compared using the BLAST version 2.2.30+ (Kent 2002) tool with the cp genome of H. bogdanii (GenBank Accession No. NC_043839.1) as a reference. The cp genome sequence of wild H. roshevitzii was annotated by software GeSeq (Michael et al. 2017) with manual correction.

The complete size of wild H. roshevitzii cp genome was 137,065 bp, including a large single-copy region (LSC) with a length of 81,138 bp, and a small single-copy region (SSC) with a length of 12,753 bp, and a pair of inverted repeat regions (IRA/IRB), both of which were 21,587 bp. A total of 133 functional genes were contained in wild H. roshevitzii cp genome, including 85 protein-coding genes (PCGs) (79PCG species), 40 transfer RNA genes (32 tRNA species), and 8 ribosomal RNA genes (4 rRNA species). Among them, trnK-UUU, rps16, atpF, trnG-UCC, trnV-UAC, trnL-UAA rpl2, ndhA, ndhB, trnI-GAU, and trnA-UGC have a single intron, however, two introns are contained in ycf3 and rps12. Most genes appeared in a single copy, whereas nineteen genes occurred as two copies, including seven PCG genes (rps19, rpl2, rpl23, rps12, rps7, ndhB, and rps15), eight tRNA genes (trnH-GUG, trnI-CAU, trnI-GAU, trnL-CAA, trnV-GAC, trnA-UGC, trnR-ACG, and trnN-GUU), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23). The content of CG in whole chloroplast genome was 38.25%, including 36.21% in LSC region, 32.19% in SSC region and 43.89% in IR region.

Phylogenetic analysis was determined with H. roshevitzii cp PCGs and the cp PCGs of 24 species of Gramineae plants based on the maximum likelihood method, PCGs of 25 Gramineae plants were screened out from the complete cp genome, the sequences of PCGs were firstly aligned using MAFFT program (Katoh et al. 2002), and a maximum likelihood tree (Figure 1) was constructed by program RAxML version 8.2.10 (Stamatakis 2014).

Figure 1.

Maximum likelihood tree based on the chloroplast PCGs sequences of H. roshevitzii and other taxa within the Gramineae. The numbers shown next to the nodes are bootstrap support values.

The phylogenetic results showed that 25 species were clustered into two branches, and wild H. roshevitzii was closest to Hordeum bogdanii with strong bootstrap values (Figure 1). Complete cp genome of wild H. roshevitzii provides a theoretical basis for further elucidating the phylogenetic relationships of genus Hordeum.