基于叶绿体基因组解析贝母类药材基原物种鉴定研究

Fritillariae cirrhosae bulbus (Chuan Bei Mu) is a commonly used multi-origin precious medicine in China, there are 5 types of Fritillaria bulbs recorded in the Pharmacopoeia of the People’s Republic of China (2015 edition) with 11 Fritillaria species, while effectiveness and safety for clinical use Fritillaria bulbs are threatened because it is difficult to identify original species of Fritillaria bulbs which are similar in morphology and various in type. However, significant limitations are presented in original species identification of Fritillaria bulbs by the conventional barcode sequences. In this proposal, we intends to obtain chloroplast genome of 11 original species of Fritillaria bulbs using Illumina high-throughput sequencing technology, analyze mutation rate of genes and intergenic regions by comparative genomics, and reveal the nucleotide variation between the chloroplast genome of original species of Fritillaria bulbs. Then we will evaluate success rate of PCR amplification using more than 100 samples of original species of Fritillaria bulbs and closely related species, examine the identification ability, and compare identification success rate, obtain screened hypervariable molecular markers with comprehensive analysis. Last we will establish a rapid identification detection system for authentication original species of Fritillaria bulbs, to solve the difficult problem of identification in original species of Fritillaria bulbs. Meanwhile, this study will provide a new effective method for species identification in multi-origin medicine which is difficult to identify using common barcodes.

川贝母为常用多基原名贵中药材，仅2015年版《中国药典》收载的贝母药材就有五类，基原多达11种。贝母属药用植物种类繁多，且形态相似，基原物种鉴定困难，导致混用乱用，从而严重影响其临床用药的有效性和安全性。然而，常规通用DNA条形码序列在解决贝母属药用植物鉴定时仍存在很大的局限性。本研究拟收集常用的11种贝母类药材基原物种，采用Illumina高通量测序技术获得其叶绿体全基因组，通过比较基因组学分析各基因及间隔区的变异率，揭示贝母类药材基原物种间叶绿体基因组高变异区域；以贝母类药材基原物种及其近缘物种的100余份样品来评估候选高变区序列扩增成功率，并考查鉴定能力和计算鉴定成功率，综合考虑以上评价指标获得特异性高变条形码；建立快速鉴定贝母类药材基原物种的检测体系，解决贝母类药材基原物种鉴定困难问题。同时，本研究还将为传统DNA条形码技术鉴定困难的多基原药材基原鉴定提供有效的研究方法。

川贝母是一种常用名贵中药，属于典型的多基原药材，贝母类药材入药和流通较为混乱，不同基原物种之间相互混用，以次充好，严重影响贝母类药材的用药有效性和安全性，因此亟需开发准确有效的贝母类药材基原物种鉴定方法。本研究采用叶绿体基因组作为超级条形码对贝母类药材基原物种进行了分子鉴定研究。累计收集了2015年和2020年版《中国药典》收载的五大类贝母药材（川贝母、浙贝母、平贝母、伊贝母、湖北贝母）的10种基原物种。对10个贝母属物种的26个叶绿体基因组进行了比较分析，贝母属物种叶绿体基因组在基因组结构、基因类型和基因含量上较为保守。经过贝母属各物种叶绿体基因序列之间的多重比对发现，贝母属物种叶绿体基因组相似度较高。LSC区的变异程度要明显高于IR区和SSC区。本研究筛选出10个基因间区的种间变异程度较大，对这10个基因间区以及相关文献报道的其它47个片段进行鉴定效率测试，构建系统发育树，发现这57个片段均不能有效鉴别贝母属10个物种。将10个贝母属物种的26个叶绿体基因组序列作为超级条形码构建最大似然法和最大简约法进化树，MP树和ML树的拓扑结构完全一致。树图中可以明显看出，叶绿体全基因组可以成功区分这10个贝母属物种，且各物种的不同样本均可单独聚集成一支。对于这五类贝母类药材，川贝母与浙贝母和湖北贝母的亲缘关系更近。《中国药典》中同一贝母类药材下的不同基原物种所有样本分别聚集成簇并与其它贝母类中药材分开，且各分支的支持率均较高。结果表明，基于叶绿体基因组序列的超级条形码可以将贝母属10种药用植物完全区分开。叶绿体基因组可以帮助鉴别贝母属近缘物种，并有作为近缘物种鉴定的通用超级条形码的潜力。