Dna plantzol reagent

According to the Flora of China (http://www.efloras.org/index.aspx), C. yanhusuo has two synonyms: C. turtschaninovii Besser f. yanhusuo Y. H. Chou & C. C. Hsu and C. tertici (Nakai) Nakai f. yanhusuo (Y. H. Chou & C. C. Hsu) Y. C. Zhu. In this study, we collected leaves of approximately 15 to 20 individuals of wild C. yanhusuo from the 11 known natural populations (Figure 1; Table 1). The distances between neighboring plants exceeded 15 m. In addition, we obtained leaves and rhizomes from 14 to 20 individuals of C. yanhusuo from 6 cultivated populations representing the major C. yanhusuo-producing regions in China (CG, GZ, HQ, HZ, JS, and QX; Figure 1, Table 1). Leaf tissues were ground in liquid nitrogen, and genomic DNA was extracted using DNA Plantzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The rhizomes from cultivated populations were dried at 60° C to a constant weight and prepared for alkaloid analysis.

We collected a total of 160 H. chrysotricha individuals from eight islands and eight adjacent mainland populations in the TIL region, and 110 individuals from seven islands and four adjacent mainland populations in the ZA region (Table 1 and Table 2; Figure 5). In each population, almost equal numbers of individuals with long-styled (L-morph) or short-styled (S-morph) flowers were collected separately. We extracted the total genomic DNA from the dry leaf material of each sample using DNA Plantzol Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. We then checked the quality of the DNA using 1% agarose gel electrophoresis and measured the DNA concentration using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Finally, 256 of the 270 DNA samples met the minimum quality requirements and were retained for subsequent sequencing and genotyping.

Fresh leaves from three Menispermum species and Sinomenium acutum were collected and then dried in silica-gel. In total twelve samples of M. canadense, four of M. dauricum, and one Sinomenium acutum were included. Voucher specimens were mostly deposited at the Herbarium of Zhejiang University (HZU), with the two M. mexicanum specimens (one of which is an isotype specimen) were sampled from the Gray Herbarium (GH) and Arnold Arboretum Herbarium (A) of Harvard University (Table 1). Total genomic DNA was extracted using DNA Plantzol Reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. Agarose gel electrophoresis and an ultraviolet spectrophotometer (K5800, KAIAO, Beijing, China) were used to check the quality and quantity of genomic DNA, respectively.