基于叶绿体基因组SNP的天台鹅耳枥谱系结构与分化分析

Genealogical structure and differentiation analysis of Carpinus tientaiensis based on single nucleotide polymorphism of chloroplast genome

关键词:  天台鹅耳枥, 叶绿体基因组, 单核苷酸多态性, 谱系结构, 系统发育

Key words:  Carpinus tientaiensis, chloroplast genome(cpDNA), single nucleotide polymorphism(SNP), genealogical structure, phylogeny

摘要:

天台鹅耳枥为中国特有的濒危植物,仅间断分布于浙江省境内,种群数量稀少,已处于极危状态。该文通过对6个自然居群(包含所有居群的母株)叶绿体基因组(cpDNA)单核苷酸多态性(SNP)研究,探讨天台鹅耳枥谱系结构与系统分化,以评估濒危状况,并提出相应的保护策略。使用TIANGEN试剂盒法提取基因组DNA,用Illumina NovaSeq 6000进行高通量测序,对获得叶绿体全基因组序列,使用在线程序OGDRAW制作cpDNA图谱,用DnaSP分析核苷酸多样性,用PopART软件进行单倍型网络构建,使用RAxML软件构建极大似然树(ML tree),用MrBayes构建 Bayes tree。结果表明:(1)通过天台鹅耳枥叶绿体全基因组序列分析,发现大多数蛋白质编码基因和氨基酸序列显示出明显的密码子偏好,检测到cpLTR正向重复32个、回文重复25个、反转重复22个; SSR重复序列不同类型87个,其中大多数富含A/T,单核苷酸的数量最多。(2)在cpDNA中鉴定了314条SNPs,单核苷酸取代显示天台鹅耳枥群体属单系,分为天台县居群(THS)和景宁县居群(JST),居群单倍型之间演化关系呈现星状中心辐射。(3)所有居群核苷酸多样性的变异均较低(P_i<0.005),JST居群和THS居群单倍型多样性较低(H_d为0.5～0.6),显示出天台鹅耳枥在历史上遇到瓶颈后曾发生局部扩张,居群间呈现较大的遗传分化,居群内具有较低的遗传变异与居群间较高的分化水平。通过对cpDNA SNP的研究,揭示天台鹅耳枥的遗传多样性和谱系分化,为濒危植物天台鹅耳枥种质资源保护和遗传拯救提供理论依据。

Abstract:

Carpinus tientaiensis is an endangered plant species with sparse population densities and is endemic to China. The distribution of this species is restricted to the territory of Zhejiang Province and its survival rate is very low. Based on data of chloroplast genome(cpDNA)single nucleotide polymorphism(SNP)in six natural populations(including the mother plants of all populations), we investigated genealogical structure and systematic development, assessed how endangered C. tientaiensis is and put forward relevant conservation strategies. The genomic DNA of C. tientaiensis was extracted using TIANGEN kit method and a library was constructed for high-throughput sequencing with Illumina NovaSeq 6000. To obtain the sequence of chloroplast complete genome, cpDNA maps were constructed using the online program OGDRAW. Nucleotide diversity was analyzed by DnaSP software while PopART was used for haploid network construction and RAxML was adopted for maximum likelihood(ML)tree construction. Finally, MrBayes software was used for construction of Bayes tree. The results were as follows:(1)According to chloroplast complete genome sequence analysis of C. tientaiensis, most of the protein-coding genes and amino acid sequences showed obvious codon preference. Moreover, chloroplast long-term relationship(cpLTR)revealed 32 forward repeats, 25 palindromic repeats and 22 reverse repeats. We detected 87 simple sequence repeats(SSR)in different types, most of which were A/T rich and the number of single nucleotides was the largest.(2)A total of 314 SNPs was identified in the cpDNA of C. tientaiensis. Single-nucleotide substitution indicated that C. tientaiensis was monophyletic and could be categorized into Tiantai County(THS)and Jingning County(JST)populations. The evolution of the haplotypes of the population manifested stellar radiation.(3)The variation of nucleotide diversity was low in all populations(P_i<0.005)and the haplotype diversity of THS and JST populations was also low(H_d 0.5-0.6). This indicated that C. tientaiensis had undergone historically local expansions after experiencing bottlenecks across its evolution history, with lower genetic variation within populations and higher level of diversity among populations. The results of SNP of the cpDNA reveal the genetic diversity and lineage differentiation of C. tientaiensis, and provide theoretical basis for conservation of the genetic resources as well as rescue of this endangered plant species.