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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