极小种群植物川柿叶绿体基因组特征与系统发育分析（附录）

川柿(Diospyros sutchuensis)为极小种群和国家重点保护野生植物,分布范围狭窄,种群数量极少。目前,川柿基因组信息缺乏,在柿属(Diospyros)中的系统亲缘关系不明确。该研究通过Illumina平台对川柿叶绿体基因组进行测序,应用Getorganellev1.7.3.4和PGA软件对基因组进行组装和注释,使用DnaSP6.12.03软件进行多序列对比分析,并使用REPuter、Tandem Reapeats Finder和MISA软件进行重复序列分析,使用CodonW1.4和EasyCodemL软件分别进行密码子偏好性和选择压力分析。同时,基于4个不同的叶绿体基因组序列数据集,使用IQtree软件分析川柿与11个柿属物种的系统发育关系。结果表明:(1)川柿叶绿体基因组全长157 917 bp,包含1对26 111 bp的反向重复区、大单拷贝区(87 303 bp)和小单拷贝区(18 392 bp),GC碱基含量为37.4%。(2)川柿叶绿体基因组共注释到113个基因,包括79个蛋白编码基因、30个tRNA基因和4个rRNA基因; 共检测到49个长重复序列、27个串联重复序列和34个简单重复序列; 蛋白编码基因中高频密码子31个,多数密码子末位碱基为A或U,编码亮氨酸的密码子使用最多; 基因组编码区比非编码区更为保守,10个高变热点区域可作为潜在的分子标记; 蛋白编码基因中有8个基因(ndhB、ndhG、ndhI、rbcL、rpoB、petB、petD、rps12)受到正选择压力。(3)系统发育分析显示,川柿与老鸦柿(D. rhombifolia)和乌柿(D. cathayensis)亲缘关系最为密切,它们与海南柿(D. hainanensis)共同形成一个单系分支。该研究结果既为川柿及柿属种质资源鉴定、遗传多样性保护以及种群恢复等提供了叶绿体基因组资源,也为阐明川柿的系统进化提供了重要的分子信息。

Characteristics of chloroplast genome and phylogenetic analysis of Diospyros sutchuensis with extremely small populations

Diospyros sutchuensis is a national protected wild plant species with narrow distribution and extremely small population in southwestern China. At present, the genomic information of this species is lacking and its phylogenetic relationships among Diospyros remain unclear. In this study, the chloroplast genome of D. sutchuensis was sequenced by Illumina platform, assembled and annotated by Getorganelle v1.7.3.4 and PGA, and analyzed by DnaSP 6.12.03 for sequence comparison, REPuter, Tandem Reapeats Finder, MISA for repetitive sequences, CodonW1.4 for codon usage bias, and EasyCodemL for selection pressure. Meanwhile, based on four different chloroplast genome sequence datasets, the phylogenetic relationships between D. sutchuensis and 11 Diospyros species were analyzed using IQtree. The results were as follows:(1)The chloroplast genome of D. sutchuensis was 157 917 bp in length, including two inverted repeats(IRs)of 26 111 bp, which was separated by large single copy(LSC)and short single copy(SSC)of 87 303 bp and 18 392 bp, respectively. The GC content was 37.4%.(2)The genome contained 113 genes, including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. A total of 49 long repeats, 27 tandem repeats, and 34 simple sequence repeats were identified. There were 31 high-frequency codons in protein-coding genes, most of them ended in A or U, and the most used codons were the ones encoding leucine. The coding regions were more conserved than the non-coding ones, and 10 regions were identified as most divergent hotspots for potential molecular markers. There had been positive selection on ndhB, ndhG, ndhI, rbcL, rpoB, petB, petD and rps12 among protein-coding genes.(3)Phylogenetic analyses showed that D. sutchuensis was closely related to D. rhombifolia and D. cathayensis. Together with D. hainanensis, these four species formed a monophyletic group. The phylogenetic tree constructed from the chloroplast genomes had the highest support values, indicating that the chloroplast genomes with most variable and informative sites are more suitable for phylogenetic studies of Diospyros. The results provide useful chloroplast genomic resources for germplasm identification, genetic diversity conservation, repopulation, and phylogenetic analysis of D. sutchuensis as well as Diospyros.