木芙蓉三个品种及近缘种的叶绿体基因组比较分析

Comparative analysis of the chloroplast genomes of three cultivars of Hibiscus mutabilis and its related species

关键词:  木芙蓉, 台湾芙蓉, 叶绿体基因组, 分子标记, 系统发育树

Key words:  Hibiscus mutabilis, Hibiscus taiwanensis, chloroplast genome(cpDNA), molecular marker, phylogenetic tree

摘要:

木芙蓉(Hibiscus mutabilis)栽培历史悠久,是原产中国的古老园林树种和药用植物。为了探讨木芙蓉品种及近缘种的进化特征,厘清木芙蓉品种间及其与近缘种间的亲缘关系,以及探究木芙蓉叶绿体基因组(chloroplast DNA, cpDNA)的遗传方式,该文选择了一个杂交组合中的3个木芙蓉栽培品种(‘单瓣白'‘金秋颂'‘牡丹粉'),用高通量测序平台Illumina NovaSeq对其cpDNA进行首次测序。经组装注释后得到3条完整的cpDNA序列,结合该团队已经完成的近缘种台湾芙蓉(H. taiwanensis)和来自基因库的木槿、朱槿的cpDNA,对木槿属4种及木芙蓉种下的3个品种进行了cpDNA组成和结构特征的比较分析,并完成了其系统发育树重建。结果表明:(1)‘单瓣白'‘金秋颂'‘牡丹粉'的cpDNA序列长度分别为160 880、160 879、160 920 bp,基因数目均为130个,其中蛋白编码基因85个、核糖体RNA 8个和转运RNA 37个。(2)比较分析结果显示,木芙蓉的种下3个品种及其近缘种台湾芙蓉在cpDNA上高度保守,反向重复区(IR)均为26 300 bp; 木槿和朱槿在IR区发生了收缩,分别为25 745、25 598 bp。(3)系统发育分析结果显示,种下3个品种先聚成一个单系支,再与台湾芙蓉聚成一个高支持率分支,表明木芙蓉和台湾芙蓉的亲缘关系最近; 相较于木槿和朱槿,木芙蓉、台湾芙蓉2种与海滨木槿、黄槿、大麻槿在亲缘关系上更近。(4)木芙蓉3个品种之间能通过cpDNA序列区分开,在大/小单拷贝区(LSC/SSC)长度上,‘单瓣白'‘金秋颂'‘牡丹粉'分别为89 355 bp/18 925 bp、89 353 bp/18 926 bp、89 400 bp/18 920 bp,并且从重复序列和核苷酸多样性分析中开发出了候选分子标记和DNA条形码,可以作为品种鉴定的分子条码。(5)木芙蓉品种‘单瓣白'与‘金秋颂'cpDNA差异最小,亲缘关系最近,根据两者母本与子代的关系,证明了木芙蓉cpDNA的母系遗传特征。该研究结果有助于更好地了解3个木芙蓉品种及台湾芙蓉cpDNA的进化特征和物种间的系统发育关系,为木芙蓉品种的准确鉴定和优良品种选育提供了cpDNA方面的基础资料。

Abstract:

Hibiscus mutabilis is native to China with a long cultivation history, and is an ancient garden tree species and medicinal plant. In this study, we selected three cultivars of H. mutabilis in a hybrid combination(H. mutabilis cv. Danbanbai, H. mutabilis cv. Jinqiusong, H. mutabilis cv. Mudanfen)to investigate evolutionary characteristics between the cultivars of H. mutabilis and its related species, and clarify the phylogenetic relationship between the cultivars of H. mutabilis and its related species, as well as explore the genetic model of chloroplast genome(cpDNA)of H. mutaibilis at the same time. We first sequenced the three cultivars of H. mutaibilis using Illumina NovaSeq. After assembly and annotation, three complete chloroplast genome sequences were obtained. The cpDNAs of the related species H. taiwanensis from our group, and H. syricus and H. rosa-sinensis from the gene bank. Then we carried out comparative analysis on composition and structure of cpDNAs of four species of Hibiscus and three cultivars of H. mutabilis, and completed its phylogenetic tree reconstruction. The results were as follows:(1)Total sizes of cpDNAs of H. mutabilis cv. Danbanbai, H. mutabilis cv. Jinqiusong, H. mutabilis cv. Mudanfen were 160 880, 160 879, 160 920 bp, respectively, and the total gene number was 130, including 85 protein-coding genes, eight ribosomal RNAs, and 37 transfer RNAs.(2)The comparative analyses showed that the cpDNAs of three cultivars of H. mutabilis and the related species H. taiwanensis were highly conserved, and the inverted repeat regions(IR)were all 26 300 bp; H. rosa-sinensis and H. syriacus shrank to at 25 745 and 25 598 bp, respectively.(3)The phylogenetic analysis revealed that the three cultivars were planted into a monophyletic branch, and then together with H. taiwanensis into a high support branch, indicating that H. mutabilis and H. taiwanensis had the closest relationship; Compared with H. syriacus and H. rosa-sinensis, H. mutabilis and H. taiwanensis were more closely related to H. hamabo, H. tiliaceum and H. canabinus.(4)Three cultivars of H. mutabilis could be distinguished by cpDNA sequence, the length of LSC/SSC of H. mutabilis cv. Danbanbai, H. mutabilis cv. Jinqiusong, H. mutabilis cv. Mudanfen were 89 355 bp/18 925 bp, 89 353 bp/18 926 bp, 89 400 bp/18 920 bp, respectively. And candidate molecular markers and DNA barcodes had been developed from repeat sequence and nucleotide diversity analyses, which could be used as a tool for cultivars identification.(5)The cpDNAs of H. mutabilis cv. Danbanbai and H. mutabilis cv. Jinqiusong showed a minimum difference and had the closest phylogenetic relationship. According to the relationship between their female and offspring, the maternal genetic characteristics of the cpDNAs of Hibiscus were proved. This study will help us to understand the evolutionary characteristics and phylogenetic relationship of cpDNAs of three cultivars of H. mutabilis and H. taiwanensis, and provide basic data on cpDNA for accurate identification of the cultivars of H. mutabilis and breeding of excellent cultivars.