Genetic distance of haplotypes in joint analysis.

ObjectiveThis study aimed to identify specific DNA barcodes based on the chloroplast genome of Actinidia eriantha Benth. and to utilize these barcodes for the identification of germplasm resources from different geographic origins.MethodsThe chloroplast genome of A. eriantha samples were sequenced using the Illumina NovaSeq PE150 platform. Specific highly variable regions were identified through mVISTA alignment and nucleotide diversity analysis. Haplotypes of samples from various regions were further analyzed based on the selected DNA barcode candidate fragments.ResultsThe complete chloroplast genomes of three A. eriantha from different locations were 156,955–157,100 bp in length and exhibited a typical quadripartite circular structure, with 88 genes annotated in each genome. Comparative analyses with mVISTA and nucleotide diversity indices identified matK, trnK(UUU), ycf1, and the atpH_atpI intergenic spacer as candidate regions for specific DNA barcodes. Among these, trnK(UUU), ycf1, and atpH_atpI were selected for further analysis based on PCR amplification efficiency. Sequencing of these three regions across 223 samples from 21 locations in six provinces revealed 7, 10, and 39 polymorphic sites, respectively, which defined 6, 4, and 6 haplotypes. A combined analysis of the three loci identified 56 polymorphic sites and 12 distinct haplotypes (Hap1-Hap12), with pairwise genetic distances ranging from 0 to 1.96%. Six haplotypes were found to be unique to specific geographic regions, suggesting their potential as molecular markers for tracing the geographic origin of A. eriantha.ConclusionThe chloroplast gene regions trnK(UUU), ycf1, and atpH_atpI, identified through comparative chloroplast genomics, serve as effective DNA barcodes for the intraspecific identification of A. eriantha germplasm. These markers provide a molecular basis for future efforts in geographic origin tracing, germplasm conservation, and breeding of this species.**研究目的** 本研究旨在基于毛花猕猴桃（*Actinidia eriantha* Benth.）的叶绿体基因组（chloroplast genome）筛选特异性DNA条形码（DNA barcode），并利用该类条形码对不同地理起源的毛花猕猴桃种质资源进行鉴定。 **研究方法** 采用Illumina NovaSeq PE150测序平台对毛花猕猴桃样本的叶绿体基因组进行测序；通过mVISTA比对和核苷酸多样性（nucleotide diversity）分析筛选特异性高变区域；基于筛选得到的DNA条形码候选片段，对不同区域样本的单倍型（haplotype）开展进一步分析。 **研究结果** 来自不同产地的3份毛花猕猴桃样本的完整叶绿体基因组长度为156955~157100 bp，均呈现典型的四分体环状结构，每个基因组注释得到88个基因。通过mVISTA比对与核苷酸多样性指数分析，筛选出matK、trnK(UUU)、ycf1以及atpH-atpI基因间隔区作为特异性DNA条形码候选区域。其中，基于PCR扩增效率，最终选取trnK(UUU)、ycf1和atpH-atpI进行后续分析。对来自6个省份21个采样点的223份样本的上述3个区域进行测序，结果显示：trnK(UUU)、ycf1和atpH-atpI分别存在7、10和39个多态性位点，各自定义出6、4和6种单倍型；对3个基因座开展联合分析后，共得到56个多态性位点与12种独特单倍型（Hap1~Hap12），两两遗传距离范围为0~1.96%。其中6种单倍型为特定地理区域所独有，表明其可作为追踪毛花猕猴桃地理起源的分子标记。 **研究结论** 本研究通过比较叶绿体基因组学筛选得到的trnK(UUU)、ycf1和atpH-atpI叶绿体基因区域，可作为毛花猕猴桃种质资源种内鉴定的有效DNA条形码。这些分子标记为后续开展该物种的地理起源溯源、种质资源保护及育种工作提供了分子学基础。