Characterization of the complete chloroplast genome of Hordeum vulgare L. var. trifurcatum with phylogenetic analysis

Abstract In the present study, the complete chloroplast genome of Hordeum vulgare L. var. trifurcatum was sequenced, assembled and compared with closely related species. The chloroplast genome of Hordeum vulgare L. var. trifurcatum was composed of 84 protein-coding genes (PCG), 8 ribosomal RNA (rRNA) genes, and 38 transfer RNA (tRNA) genes. The Hordeum vulgare L. var. trifurcatum chloroplast genome is 136,485 bp in size, with the GC content of 38.32%. Phylogenetic analysis based on the combined chloroplast gene dataset indicated that the Hordeum vulgare L. var. trifurcatum exhibited a close relationship with Hordeum vulgare subsp. spontaneum and Hordeum vulgare subsp. vulgare.

Tibetan hulless barley; Hordeum; chloroplast genome; phylogenetic analysis; molecular marker Hordeum vulgare L. var. trifurcatum, belonging to the Poaceae family (Soreng et al. 2015;Saarela et al. 2018), is one of the staple foods for Tibetans and an important livestock feed in the Tibetan Plateau (Zeng et al. 2015;Huang et al. 2020). The highland barley (Hordeum vulgare L. var. trifurcatum) shows good environmental tolerance, which can be successfully planted in high altitude, low temperature, and high salinity places (Walia et al. 2006;El-Esawi et al. 2018;Elsawy et al. 2018). In addition, the highland barley also showed good antioxidant activity (Asif et al. 2020). Thus, Hordeum vulgare L. var. trifurcatum is a promising nutritious food source and traditional Chinese medicine grows widely in plateau, which concerning by more and more researchers, just like the Fagopyrum tataricum (L.) Gaertn Xiang et al. 2016; and Stellera chamaejasme L. (Ren et al. , 2021a(Ren et al. , 2021b. The genus Hordeum comprises more than 30 species. Some varieties are also found in this genus (Malysheva-Otto et al. 2006;Forsberg et al. 2019;Hagenblad and Morales 2020;Kumar et al. 2020). Organelle genomes have been widely used in study of taxonomy, evolution and genetics Yang et al. 2019;Wang et al. 2020;Li, He, et al. 2020). However, no complete chloroplast genome of Hordeum vulgare L. var. trifurcatum was reported to date, which limits its breeding and application (Su et al. 2020).
The specimen (Hordeum vulgare L. var. trifurcatum) used for chloroplast genome assembly was collected from Qinghai, China (101.97 E; 53.70 N). A specimen was deposited at Collection Center of Chengdu University (Y. Ren, renyuan-hang@cdu.edu.cn) under the voucher number ZQK_R1. The chloroplast genome of Hordeum vulgare L. var. trifurcatum was sequenced and assembled according to methods previously described . First, we extracted the total genomic DNA of Hordeum vulgare L. var. trifurcatum using a Plant DNA Kit (D3485-00, Omega Bio-Tek, Norcross, GA, USA). Then we purified the genomic DNA using a Gel Extraction Kit (Omega Bio-Tek, Norcross, GA, USA). The purified DNA was stored in Chengdu University (No. DNA_ ZQK_R1). Sequencing libraries of Hordeum vulgare L. var. trifurcatum was constructed using a NEBNextV R Ultra TM II DNA Library Prep Kit (NEB, Beijing, China). We conducted the whole genomic sequencing (WGS) of Hordeum vulgare L. var. trifurcatum using the Illumina HiSeq 2500 Platform (Illumina, SanDiego, CA). The chloroplast genome of Hordeum vulgare L. var. trifurcatum was initially assembled using SPAdes v3.11.0 (Bankevich et al. 2012). The chloroplast sequences obtained in the above steps were used as seed sequences to assemble the complete chloroplast genome of Hordeum vulgare L. var. trifurcatum using NOVOPlasty v4.3.1 with a k-mer size of 35 (Dierckxsens et al. 2017). Approximately 1.10 million reads were assembled into a complete circular chloroplast genome. The average chloroplast sequence coverage was 2,168 Â. The complete chloroplast genome of Hordeum vulgare L. var. trifurcatum was annotated by GeSeq (Tillich et al. 2017) using the chloroplast genome of Hordeum vulgare subsp. spontaneum as the reference (Bdolach et al. 2019).
The complete chloroplast genome of Hordeum vulgare L. var. trifurcatum is 136,485 bp in length, which was larger than Hordeum vulgare subsp. vulgare (136,462 bp) (Zeng et al. 2017) and smaller than Hordeum vulgare subsp. spontaneum (136,536 bp) (Bdolach et al. 2019). The GC content of the Hordeum vulgare L. var. trifurcatum chloroplast genome is 38.32%, which is larger than that of Hordeum vulgare subsp. spontaneum (38.30%). The base compositions of the Hordeum vulgare L. var. trifurcatum chloroplast genome were as follows: A (30.93%), T (30.76%), G (19.22%) and C (19.10%). The complete chloroplast genome of Hordeum vulgare L. var. trifurcatum contains 84 protein-coding genes, 8 ribosomal RNA genes, and 38 transfer RNA (tRNA) genes. The number of protein-coding genes in Hordeum vulgare L. var. trifurcatum chloroplast genome was more than that in two subspecies (Hordeum vulgare L. var. trifurcatum and Hordeum vulgare subsp. vulgare), while the number of tRNA was less than that in the two subspecies. To investigate the phylogenetic status of Hordeum vulgare L. var. trifurcatum, we constructed a phylogenetic tree for 15 species. The protein-coding region of 13 genes conserved in the 15 species was used to construct combined a chloroplast gene set according to previous methods (Li, Xiang, et al. 2019;Wu et al. 2021). Bayesian (BI) analysis method ) was used to construct the phylogenetic tree based on combined protein-coding genes of chloroplast genome as described by previous methods (Li, Yang, et al. 2020;Cheng et al. 2021;). MrBayes v3.2.6 (Ronquist et al. 2012) was used to construct the phylogenetic tree using Bayesian inference (BI) method. Two independent runs with four chains (three heated and one cold) each were conducted simultaneously for 2 Â 10 6 generations. Each run was sampled every 100 generations. We assumed that stationarity had been reached when estimated sample size (ESS) was greater than 100, and the potential scale reduction factor (PSRF) approached 1.0. The first 25% samples were discarded as burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities (BPP) in a 50% majority-rule consensus tree (Li, Ren, et al. 2019). According to the phylogenetic tree (Figure 1), the Hordeum vulgare L. var. trifurcatum exhibited a close relationship with Hordeum vulgare subsp. spontaneum (Bdolach et al. 2019) and Hordeum vulgare subsp. vulgare (Zeng et al. 2017).

Disclosure statement
The authors have declared that no competing interests exist.

Funding
This work was funded by Sichuan Science and Technology Program [2021YJ0477].

Data availability statement
The genome sequence data that support the findings of this study are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under the accession no. MW017635. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA717119, SRR14082750, and SAMN18478699, respectively.