红花变豆菜叶绿体基因组组装与序列特征分析研究

Assembly and sequence analysis of chloroplast genome of Sanicula rubriflora

目的以红花变豆菜Sanicula rubriflora新鲜叶片为试验材料,通过高通量测序手段,对其叶绿体基因组进行组装和序列特征分析,为进一步开展变豆菜属植物的进化和遗传发育提供指导方法。方法实验流程按照Illumina公司提供的标准操作流程执行,包括样品质量检测、文库构建、文库质量检测和文库测序等流程,使用已报道的变豆菜Sanicula chinensis的叶绿体基因组作为参考序列,得到红花变豆菜完整的叶绿体基因组序列并对其进行组装、特征序列分析和进化分析。结果红花变豆菜叶绿体基因组具有典型的环状四分体结构,长度155700 bp,包括1个大的单拷贝区(large single copy,LSC,85979 bp),1个小的单拷贝区(small single copy,SSC,17053 bp),1对相反的序列(inverted repeats sequence,IR,26333 bp);共注释到130个基因,其中蛋白编码的基因86个,tRNA基因36个,rRNA基因8个,AT含量占叶绿体基因组的61.83%;共检测到1个正向长重复序列(forward repeat sequence),3个回文长重复序列(palindromic repeat sequence);此外,还检测到了168个简单重复序列(simple sequence repeats,SSR)位点;与蛋白编码基因对应的密码子偏好使用A/T碱基,编码异亮氨酸的密码子(I)使用次数最高,编码半胱氨酸的密码子(C)使用次数最低;最后与7种已报道的伞形科植物叶绿体基因组和2个外类群物种通过最大似然法(maximum likelihood,ML)构建系统发育树,发现红花变豆菜与变豆菜和直刺变豆菜亲缘关系最为密切。结论红花变豆菜的叶绿体基因组数据为伞形科植物研究提供了更为详细完善的资料,为该物种叶绿体基因工程和系统进化分析提供参考依据。

Objective The chloroplast genome was assembled and the sequence characteristics analyzed with fresh leaves of Sanicula rubriflora as the experimental materials through high-throughput sequencing,so as to provide guidance for the further development of the evolution and genetic development of S.rubriflora.Methods The experimental procedures were carried out according to Illumina’s standard operating procedures,including the sample quality test,the construction of library,the quality test of the sample library and the sequencing of the library,the reported chloroplast genome of Sanicula chinensis was used as a reference sequence,and the complete chloroplast genome of S.rubriflora was obtained and assembled,and the assembly characteristic sequence analysis and evolutionary analysis were performed.Results The chloroplast genome of S.rubriflora had a typical circular quadripartite structure with a size of 155700 bp in length.In which a large single-copy(LSC)of 85979 bp and a pair of inverted repeats(IRs)of 26333 bp were disconnected by a small single-copy(SSC)of 17053bp.A total of 130 genes were annotated,including 86 protein-coding genes(PCGs),36 transfer RNA genes(tRNAs)and eight ribosomal RNA genes(rRNAs).AT content accounted for 61.83%of the chloroplast genome.One forward repeat sequence and three palindromic repeat sequences were detected.In addition,168 simple sequence repeats(SSR)sites were detected.The codon corresponding to the protein coding gene preferred to use A/T base.The codon(I)encoding isoleucine had the highest frequency of use,while the codon(C)encoding cysteine had the lowest frequency.A phylogenetic tree with maximum likelihood(ML)was constructed based on the chloroplast genomes of seven reported Apiaceae species and two other species.It was found that S.rubriflora were most closely related with S.chinensis and S.orthacantha.Conclusion The chloroplast genome of S.rubriflora provides more detailed and complete data for the study of Apiaceae plants,and provides a reference basis for chloroplast genetic engineering and systematic evolution analysis of this species.