As a result of assembly, 121,987,386 total reads were used for sequencing, aligned reads in the reference genome were 2,315,382, assembled reads were 2,300,724, and average organelle coverage was 2173 ⅹ. The complete chloroplast genome size of H. sinosyriacus was 160,892 bp. The chloroplast genome structure is divided into four regions: large single-copy (LSC), two inverted repeats (IRs), and small single-copy (SSC). The LSC was 89,747 bp, IRb and IRa were 25,742 bp each, and the SSC was 19,661 bp. In the total annotation, 131 genes, comprising 86 coding sequences, 37 tRNAs, and 8 rRNAs, were present. The complete chloroplast genome of H. sinosyriacus was registered in GenBank under accession number MZ367751.
The results of the phylogenic tree analysis and comparison of variation differences indicated that H. sinosyriacus showed a clear difference from its close species. In the phylogenetic tree analysis, the complete chloroplast nucleotide sequences of 10 species were compared. A phylogenetic tree of the compared sequences was constructed using the maximum likelihood method and the Jones-Taylor-Thornton matrix-based model (Jones et al. 1992). Bootstrapping was performed with 1000 replicates using MEGA X (Kumar et al., 2018). The outer species, Arabidopsis thaliana, was well differentiated from the early branches, and it was shown that genus Hibiscus last differentiated from the genus Gossypium. H. sinosyriacus and H. syriacus split the latest (Fig. 1). Therefore, we compared the variations between them. Several variations were observed between the two chloroplast sequences. The total number of variants was 453 bp, comprising 274 bp, an insert of 140 bp, and a substitution of 39 bp in various regions (Table 1). In particular, we found 11 single nucleotide polymorphisms and 149 indels in 12 intergenic regions (matK, trnK-UUU, atpF, rpoC1, pscC, trnT-UGU, ndhK, rps18, clpP, ycf2, ndhA, and ycf1). These mutations, including those in the intergenic regions, can be presented as the main basis explaining the differentiation from allied species (Hamilton et al. 2003).
Table 1
Overview of substitutions and insertion/deletions in the total nucleotide sequences of H. sinosyriacus.
Region
|
Number of base substitution events
|
Number of bases
in insertion events
|
Number of bases
in deletion events
|
Number of
total bases
|
Large single copy
|
35
|
121
|
79
|
235
|
Invert repeat
|
1
|
-
|
6
|
7
|
Small single copy
|
1
|
19
|
39
|
59
|
Invert repeat
|
2
|
-
|
150
|
152
|
Total bases
|
39
|
140
|
274
|
453
|