桔梗特异DNA条形码筛选、种质资源鉴定及遗传多样性分析

Specific DNA barcodes screening,germplasm resource identification,and genetic diversity analysis of Platycodon grandiflorum

中药材桔梗为桔梗科植物桔梗的干燥根,具有多种药理作用,是常用的大宗中药材。本研究利用Illumina HiSeq X Ten平台对6份来自不同产地的桔梗进行叶绿体基因组测序,筛选特异性DNA条形码,基于特异性DNA条形码对不同产区的桔梗样品的种质资源和遗传多样性进行分析。6份桔梗叶绿体基因组全长为172260~172275 bp,均呈现典型的环状四分体结构,编码141个基因。根据比较基因组学分析和扩增效率分析发现trnG-UCC和ndhG_ndhF可作为潜在的桔梗种内种质资源鉴定的特异性DNA条形码。对来自9省15个产地305份桔梗样品的trnG-UCC和ndhG_ndhF进行PCR扩增和序列分析,结果表明trnG-UCC和ndhG_ndhF分别有5和11个变异位点,分别鉴定到5和7个单倍型;两段序列联合分析鉴定13个单倍型(Hap1~Hap13),其中占比最多的是Hap4,其次是Hap1。3个产地拥有的特异单倍型,可作为该产地的DNA分子标签与其他产地的桔梗种质资源进行区分。单倍型多样性、核苷酸多样性和遗传距离分别为0.94、4.79×10^(-3)和0.0000~0.0203,表明桔梗在物种水平上有较高的遗传多样性,种内各单倍型之间亲缘关系比较接近。本研究为桔梗产地鉴定、种质资源保护利用和分子育种等工作奠定基础。

Platycodonis Radix is the dry root of Platycodon grandiflorum of Campanulaceae,which has a variety of pharmacological effects and is a commonly used bulk Chinese medicine.In this study,the chloroplast genome sequences of six P.grandiflorum from different producing areas has been sequenced with Illumina HiSeq X Ten platform.The specific DNA barcodes were screened,and the germplasm resources and genetic diversity were analyzed according to the specific barcodes.The total length of the chloroplast genome of 6 P.grandiflorum samples was 172260−172275 bp,and all chloroplast genomes showed a typical circular tetrad structure and encoded 141 genes.The comparative genomics analysis and results of amplification efficiency demonstrated that trnGUCC and ndhG_ndhF were the potential specific DNA barcodes for identification the germplasm resources of P.grandiflorum.A total of 305 P.grandiflorum samples were collected from 15 production areas in 9 provinces,for which the fragments of trnG-UCC and ndhG_ndhF were amplificated and the sequences were analyzed.The results showed that trnG-UCC and ndhG_ndhF have 5 and 11 mutation sites,respectively,and 5 and 7 haplotypes were identified,respectively.The combined analysis of the two sequences formed 13 haplotypes(named Hap1-Hap13),and Hap4 is the main genotype,followed by Hap1.The unique haplotypes possessed by the three producing areas can be used as DNA molecular tags in this area to distinguish from the germplasm resources of P.grandiflorum from other areas.The haplotype diversity,nucleotide diversity and genetic distance were 0.94,4.79×10^(-3) and 0.0000−0.0203,respectively,suggesting that the genetic diversity was abundant and intraspecific kinship was relatively close.This study laid a foundation for the identification of P.grandiflorum,the protection and utilization of germplasm resources,and molecular breeding.