The complete chloroplast genome sequence of Salix kochiana Trautv. and its phylogenetic analysis

Abstract Salix kochiana Trautvetter 1837 is one of the highest value shrubs present in northern China with important economic and ecological benefits. This study revealed the structural characteristics and phylogenetic relationships of chloroplast genes in S. kochiana Trautv. The results showed that the length of the complete chloroplast genome was 155,657 bp, which was a typical circular double-stranded structure, including an 84,458 bp large single-copy region (LSC), a 16,221 bp small single-copy region (SSC) and a 27,489 bp pair of inverted repeat regions (IRA and IRB). The chloroplast genome contains 48,757 A bases, 28,017 G bases, 49,843 T bases, and 29,040 C bases, with a GC content of 36.66%. Through bioinformatics annotation, a total of 126 genes were found in the chloroplast genome, including 81 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis showed that S. kochiana Trautv. was closely related to S. triandroides.

The Salix genus is composed of 350-520 species that are distributed worldwide (Zhou et al. 2021). In China, the Salix genus contains 257 species, including 122 varieties and 33 variants, and they are distributed across provinces (Wang et al. 2012). Salix kochiana Trautv., an important species of the Salix genus, is mainly distributed in the Heilongjiang and Jilin provinces of China and is also found in Mongolia and Russia. S. kochiana Trautv. can grow in sand dunes and river wetlands, so it can serve as an important tree species for windbreaks, sand fixation, and soil conservation in northern China. With its fast growth, luxuriant foliage, and slender and soft branches, S. kochiana Trautv. can be made into a variety of woven goods. Its twigs and leaves can also be used as animal feed. Therefore, S. kochiana Trautv. has high economic value and provides high ecological benefits. However, the available genetic and genomic resources for S. kochiana Trautv. are very limited. In addition, due to the highly efficient crossing rate among Salix species, the classification of the Salix genus is still unclear (Chen et al. 2010). Here, we revealed the structural characteristics and phylogenetic relationships of chloroplast genes in S. kochiana Trautv. These results are of great significance for the taxonomic study of Salix and even Salicaceae and can also be used to evaluate the breeding availability of Salix at the genomic level (Chen and Liu 2008;Ren and Li 2021).
The test materials were collected from Mao'er Mountain of Heilongjiang Province, China (44 29 0 29 00 N, 127 17 0 41 00 E). The specimens were stored in Room 60708 Biotechnology Building (University sample room), Nanjing Forestry University, China (Xiaoping Li, xpli@njfu.edu.cn, voucher number: MRSSQL2017_1451). The modified CTAB method (cetyl trimethylammonium bromide) (Doyle and Doyle 1987) was used to extract whole-genome DNA from the leaves of S. kochiana Trautv., and DNA concentration and purity were detected by 1% agarose gel electrophoresis and a NanoDrop spectrophotometer (Niu et al. 2020). Qualified DNA fragment libraries were constructed with an average insert size of 300 bp using the Illumina Nextera kit and sequenced by the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA). A total of 3.5 G raw data were obtained and filtered to eliminate low-quality reads by Fastp (Chen et al. 2018). Finally, approximately 3.2 G of high-quality clean reads were harvested and used to assemble the chloroplast genome with NOVOPlasty (version 4.1) (https://github.com/ndierckx/ NOVOPlasty/) (Dierckxsens et al. 2017). The chloroplast genes of S. kochiana Trautv. were aligned and annotated with the chloroplast genome of the reference species S. triandroides (MW929215.1) by PGA (Plastid Genome Annotator) (Qu et al. 2019). After manual correction by Geneious v11.0.3 (Kearse et al. 2012), the resulting annotation of the complete chloroplast DNA was obtained and submitted to NCBI online by BankIt (GenBank accession number: OL339478.1).
The annotated results showed that the complete chloroplast genome length was 155,657 bp, which was a typical circular double-stranded structure, including an 84,458 bp large single-copy region (LSC), a 16,221 bp small single-copy region (SSC), and a 27,489 bp pair of inverted repeat regions (IRA and IRB). The chloroplast genome contains 48,757 A bases, 28,017 G bases, 49,843 T bases, and 29,040 C bases, with a GC content of 36.66%. A total of 126 genes were identified, including 81 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Among them, 7 protein-coding genes, 7 tRNA genes and 4 rRNA genes were repeated in the inverted repeat region.
To reveal the relationship between S. kochiana Trautv. and other species in the Salix genus, we downloaded the chloroplast genome sequences of 19 published Salix species from the NCBI database and performed phylogenetic analysis. Comparison of the S. kochiana Trautv. plastome to previously published data shows a high level of gene synteny with some publicly available Salix sequences (Zhou et al. 2021). Arabidopsis thaliana (NC_000932.1) was used as an outgroup. MAFFT v7 software (https://mafft.cbrc.jp/alignment/software/) (Katoh and Standley 2013) was used to perform multiple weight ratio pairs for the common sequences of chloroplast genomes of these 21 species. A phylogenetic tree was constructed based on the maximum likelihood (ML) method with the general time reversible model and 1000 bootstrap replicates using MEGA11 (Koichiro et al. 2021). The results showed that S. kochiana Trautv. was most closely related to S. triandroides (Figure 1). This finding was similar to those of a previous Salix whole-plastome phylogenetic study (Wang and Li 2021;Chen et al. 2019). These results explain the systematic origin and molecular phylogenetic location of S. kochiana Trautv., which can be used for molecular identification and resource exploitation. Overall, this study lays a foundation for Salix phylogenetics in the future.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Author contributions
Xiaoping Li contributed to the conception of the study; Jing Wang, Zicheng Yu, Xu Yao, Jie Wan and Zhengxuan Wang performed the experiment; Jing Wang contributed significantly to analysis and manuscript preparation; Jing Wang performed the data analyses and wrote the manuscript; Xiaoping Li helped perform the analysis with constructive discussions and revised it critically for intellectual content. All authors approve the version to be published and agree to be accountable for all aspects of the work.