WITHDRAWN: The complete chloroplast genome sequence of Hibiscus sinosyriacus

Introduction

Hibiscus sinosyriacus Bailey (1922) is a deciduous broad-leaved shrub belonging to the Malvaceae family that is distributed in the temperate and subtropical southern regions of China (Beers and Howie 1992). H. sinosyriacus is valuable as an ornamental tree, along with its allied species, H. syriacus. H. sinosyriacus overlaps with H. syriacus and H. paramutabilis, and the morphological characteristics of its flowers and leaves are intermediate between those of the two species (Yu and Yeam 1972; Kim and Lee 1991). In particular, H. sinosyriacus is morphologically similar to H. syriacus, but with broader leaves and longer epicalyx lobes (van Laere et al. 2007). In this study, the chloroplast genome of H. sinosyriacus was assembled to elucidate the evolutionary and genetic differences between H. sinosyriacus and its allied species.

Materials And Methods

Fresh leaves of H. sinosyriacus ‘Melmauve’ were obtained from the Hibiscus Clonal Archive of the National Institute of Forest Science (37.15 N 126.57 E), Suwon, Korea. Total DNA was extracted using the GeneAll Exgene Genomic DNA Purification Kit (GeneAll Biotechnology, Korea). NGS library construction was carried out by Macrogen Inc., using the TruSeq Nano DNA Kit. Genome sequencing was performed using a NovaSeq 6000 system (Macrogen Inc., Seoul, Korea). The chloroplast genome sequence was assembled using the NOVOPlasity4.1 program, an organelle assembler based on the chloroplast genome of H. syriacus (NC026909) with k-mers of 27, 29, 31, 33, and 35 (Kwon et al. 2016; Dierckxsens et al. 2017). Gene annotation was performed using GeSeq (Katoh and Standley, 2013). Error correction was conducted manually using Sanger sequencing by designing primers around the nucleotides where the error occurred. Alignment analysis of the complete chloroplast genomes of H. sinosyriacus and 9 other species was performed using Clustal omega 1.2.4 (Sievers et al. 2011). The results were used for phylogenetic analysis using Mega X (Kumar et al. 2018). For this, 10 chloroplast complete genomes from five genera of the Malvaceae family were used as an outgroup, including Hibiscus, Gossypium, Theobroma, Talipariti, and Abelmoshus, as well as Arabidopsis thaliana.

Results And Discussion

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

Declarations

Data availability

The genome sequence was deposited in GenBank with the accession number MZ367751. The associated BioProject, BioSample, and SRA numbers are PRJNA789673, SAMN24146414, and SRR17253293, respectively.

Funding

This work was supported by the National Institute of Forest Science (grant number FG0403-2018-01-2021).

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Soon-Ho Kwon, You Lim Jang, and Hae-Yun Kwon. The first draft of the manuscript was written by Soon-Ho Kwon, and all authors commented on the following versions of the manuscript. All authors have read and approved the final manuscript.

Ethical approval

This study was conducted in accordance with the research ethics regulations of the National Institute of Forest Science. The Research Planning and Coordination Division confirmed that no ethical approval is required.

Acknowledgments

This work was supported by the National Institute of Forest Science.

References