1. Collection and production of plant specimens and acquisition of DNA material

  Plant specimens were collected in the field with collection number plates bolted on, and the coordinates of the collection site and habitat were recorded in the field. In order to ensure the consistency between the voucher specimens and DNA materials, healthy leaves without obvious diseases or insect pests were collected directly from the voucher specimens in the process of pressing the specimens, and placed in small paper bags with very thin paper, and the numbers on the paper bags were consistent with the specimen collection numbers. After screening and removing decayed specimens, a total of 1,300 complete specimens were obtained, which were pressed dry and then put on the counterpane. the DNA material was permanently preserved in the refrigerator at -80℃.

  2. Image data acquisition of plant specimens

  The specimen images were captured with a high-resolution camera, and the image brightness was adjusted and sharpened to form the image data of the plant specimens.

  3. DNA barcode generation

  Plant DNA is extracted by magnetic bead method or CTAB method, and after extraction, a small amount of sample is taken to detect the quality and integrity of DNA by agarose gel electrophoresis. PCR was used to amplify the 5 DNA barcodes required by the project. The amplified products were handed over to a professional sequencing company for sequencing, and in order to ensure that the correct sequence was obtained, they were sequenced through from both ends respectively. Sequencing was performed using an ABI series automated sequencer (Applied Biosystems, Foster City, California, USA). Sequencing primers used PCR amplification primers.

  After obtaining the peak maps returned by the sequencing company, the DNA sequences were manually aligned using Geneious or Chromas software to remove low-quality base sequences at both ends, and the forward and reverse sequences were spliced and edited to obtain the DNA sequences and saved as FASTA format files. To verify the accuracy of sequencing or whether the material was contaminated, the spliced DNA barcode sequences of each sample were checked through the BLASTn function on the NCBI website. If a sample's barcode sequence (query sequence) matches the highest score value (subject sequence) for the same family, genus or species, it is determined that the sample in the material collection and experimental process did not make mistakes, its barcode sequence can be initially determined as correct.

  4. Generation of DNA barcode information table

  Sampling location, geographic coordinates, elevation and habitat information is compiled, and at the same time, the sample number is connected with the image data of the plant specimen, and the sample collection number is connected with the physical specimen, which ultimately forms the DNA barcode description information table.