目的: 利用传统四大鉴定、DNA条形码分子鉴定以及染色体倍性鉴定不同来源的五叶型半夏种质资源。方法: 利用组织培养对收集的13份资源进行组培扩繁,按照《中华人民共和国药典》 （2020年版,一部）对13份资源的基原、性状、显微特征进行鉴定;以三氯甲烷 ∶ 甲醇=6 ∶ 1为展开剂,10%硫酸乙醇为显色剂,对13份资源进行薄层鉴定;利用PCR扩增不同资源的ITS2核酸序列,用Snapgene和Mega软件进行剪切比对,并构建系统发育树;利用染色体计数法对五叶型半夏的倍性进行鉴定。结果: 五叶型半夏资源均与《中国植物志》中半夏描述相符,初步鉴定为半夏资源;五叶型半夏资源性状、显微特征与《中华人民共和国药典》 （2020年版,一部）半夏描述一致,薄层鉴定结果显示传统半夏与五叶型半夏斑点一致,且可以很好与虎掌区分开来。ITS2的扩增、测序成功率均为100%,基于NCBI数据库内天南星科半夏属资源ITS2序列构建的NJ系统发育树,五叶型半夏与半夏属资源亲缘关系更近,且与传统半夏资源聚为一类。染色体计数结果表明来自云南的3份种质资源SY-3、WY-8和WY-9为六倍体（2n=6x=78）;来自湖北9份种质资源（SY-1、SY-2、WY-1、WY-2、WY-3、WY-4、WY-5、WY-6和WY-7）为八倍体（2n=8x=104）;1份虎掌资源为二倍体（2n=2x=26）。结论: 五叶型半夏为半夏属半夏资源,明确半夏五叶突变体的倍性水平,为其新种质培育提供重要依据。
Objective: To identify different sources of Pinellia ternata (Thunb.) Breit. five-leaf germplasm resources using traditional four major identifications, DNA barcode molecular identification, and ploidy identification. Methods: 13 germplasm resources were collected and propagated in vitro through tissue culture. The origin, traits, and microscopic characteristics of the 13 resources were identified according to the Chinese Pharmacopoeia (2020 Edition, Volume Ⅰ). Thin-layer chromatography (TLC) was conducted using chloroform-methanol (6 ∶ 1) as the developing agent and 10% sulfuric acid ethanol as the color reagent for identification. ITS2 nucleic acid sequences of the resources were amplified by PCR, and sequence alignment and phylogenetic tree construction were performed using Snapgene and Mega software. Chromosome counting was used to identify the ploidy of the Pinellia ternata five-leaf. Results: All five-leaf resources were consistent with the description of Pinellia ternata in the “Flora of China,” and were preliminarily identified as Pinellia ternata. The traits and microscopic characteristics of the five-leaf also aligned the description of Pinellia ternata in the “Chinese Pharmacopoeia” (2020 Edition, Volume Ⅰ). TLC analysis showed that the traditional Pinellia ternata and the five-leaf had the same chromatographic profiles and exhibited clear differentiation from Rhizoma Arisaematis (tiger palm). The amplification and sequencing success rate of ITS2 was 100%. Phylogenetic analysis based on the ITS2 sequences in the NCBI database revealed that the five-leaf was closely related to Pinellia ternata and clustered together with traditional Pinellia ternata resources. Chromosome counting indicated that three germplasm resources from Yunnan (SY-3, WY-8, and WY-9) were hexaploid (2n=6x=78), while nine germplasm resources from Hubei (SY-1, SY-2, WY-1, WY-2, WY-3, WY-4,WY-5, WY-6, and WY-7) were octaploid (2n=8x=104). One tiger palm resource was diploid (2n=2x=26). Conclusion: The five-leaf is identified as a Pinellia ternata resource,and the determination of its ploidy levels provides an important basis for developing new germplasm.
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