## 2 Materials and Methods
### DNA Extraction, Amplification and Sequencing  
Approximately 20 mg of the silicon-dried leaves were rubbed for 2 min at 30 times/s in a Fast Prep bead mill (Retsch MM400, Haan, Germany). The total genomic DNA (gDNA) was isolated from the crushed materials using the Plant Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions and stored at \(-20\)\({}^{\circ}\)C. The ITS2 sequences from each plant were amplified in a reaction containing 2 \(\mu\)L of gDNA (approximately 30 ng), 12.5 \(\mu\)L of 2\(\times\) Taq Mix Master, and 1 \(\mu\)L each of the 2.5 \(\mu\)M upstream (S2F: 5\({}^{\prime}\)-ATGCGATACTTGGTGTAAT-3\({}^{\prime}\)) and downstream (S3R: 5\({}^{\prime}\)-GACGCTTCTCAGACTACAAT-3\({}^{\prime}\)) primers, with ddH\({}_{2}\)O to a volume of 25 \(\mu\)L. The PCR program consisted of an initial denaturation step at 94 \({}^{\circ}\)C for 5 min, followed by  
\begin{table}
\begin{tabular}{c c c c} \hline
**Species Name** & **Amount of Samples** & **Sample No. or GenBank Accession Number** & **Sample Sources** \\ \hline  & 1 & CH-3 & Liandu, Zhejiang \\  & 6 & CH-4, CH-18 *, CH-23-24, CH-26, RC\_CH-4 & Kaihua, Zhejiang \\  & 4 & CH-16, CH-17 *, CH-25, RC\_CH-16 & Songyang, Zhejiang \\ _Climonanthus_ & 1 & CH-22 & Chun’an, Zhejiang \\ _salciflius_ & 2 & CH-5, RC, CH-5 & Wuyuan, Jiangxi \\  & 1 & CH-21 & Dexing, Jiangxi \\  & 2 & CH-6, RC\_CH-6 & Xi’an, Shaanxi \\  & 1 & CH-19 & Xiuming, Anhui \\  & 5 & JQ781681-85 & GenBank \\ \hline  & 2 & CH-7, CH-33 & Liandu, Zhejiang \\  & 1 & CH-27 & Songyang, Zhejiang \\  & 1 & CH-28 & Qingtian, Zhejiang \\  & 1 & CH-29 & Yunne, Zhejiang \\ _C. zhejiangensis_ & 1 & CH-30 & Jingning, Zhejiang \\  & 1 & CH-31 & Qingyuan, Zhejiang \\  & 1 & CH-32 & Longquan, Zhejiang \\  & 1 & CH-34 & Taishun, Zhejiang \\  & 1 & CH-9 & Shouning, Fujian \\  & 5 & JQ781668-72 & GenBank \\ \hline C. _praccox_ & 1 & CH-1 & Liandu, Zhejiang \\  & 9 & JQ781691-95, AY786095-98 & GenBank \\ \hline C. _nitens_ & 1 & CH-11 & Yangshou, Guangxi \\  & 5 & JQ781664-67, AY786094 & GenBank \\ \hline C. _campanulatus_ & 2 & CH-13, RC\_CH-13 & Liandu, Zhejiang \\  & 5 & JQ781686-90 & GenBank \\ \hline _Calycanthus_ & 3 & CH-15, CH-36, RC\_CH-15 & Liandu, Zhejiang \\ _chinensis_ & 2 & KF547941, AY524084 & GenBank \\ \hline _Ca. floridus_ & 1 & CH-14 & Liandu, Zhejiang \\  & 4 & AY524078, AY786084-86 & GenBank \\ \hline \end{tabular}
* Commercial sample.  
\end{table}
Table 1: Sample detail information of Shi-Liang tea and its closely related species.  
40 cycles of 94 \({}^{\circ}\)C for 30 s, 56 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 45 s, with a final extension step at 72 \({}^{\circ}\)C for 10 min [28]. The amplified products were separated on 1.2% agarose gels and sequenced after gel extraction. All 71 ITS2 sequences were aligned with Clustal-W, and genetic distances were computed with MEGA 6.0 [28] according to the Kimura 2-parameter (K2P) model. A neighbor-joining (NJ) tree was built, and the bootstrap test (1000 replications) was applied to determine the reliability of each branch, and _Certophyllum echinatum_ from Calycanthaceae were used as the outgroup. The secondary structures of the ITS2 sequences were predicted according to the ITS2 database by [PERSON] et al. [24, 29, 30]. The two-dimensional DNA barcode images were obtained by encoding the Latin names of the Shi-Liang tea species and closely related species, as well as the ITS2 sequences, using the coding program and the open source PHP QR Code. Mobile terminals (such as Android and iPhone devices) can be used as QR code scanners to read the information. Finally, the information was made available online ([[http://www.tcmbarcode.cn](http://www.tcmbarcode.cn)]([http://www.tcmbarcode.cn](http://www.tcmbarcode.cn))) to identify and analyze ITS2 sequences [27, 31].