Cloning species-specific DNA fragment and SCAR marker development

The DNA fragment (515 bp) specific to T. caries produced by the ISSR 827 primer was isolated from the gel and purified with a TIANgel Purification Kit (TianGen Biochemical Technology Co., Ltd., China). Then, a pCloone007 Versatile Simple Vector Kit (Tsingke Biotechnology Co., Ltd., China) was used to ligate the specific DNA fragment with the pUC19 vector and transform it into chemically competent Escherichia coli DH5α cells (Tsingke Biotechnology Co., Ltd.). The cloned fragment was sequenced with primers M13F and M13R by Tsingke Biotechnology Co., Ltd. The E. coli plasmid was then extracted by a TIANprep Mini Plasmid Kit (TianGen Biochemical Technology Co., Ltd., China), and the concentration calculated by the NanoDrop 3300 fluorospectrometer (Biotech, USA) was 240 ng/μL. Based on the sequencing results, SCAR marker primers Erc19F and Erc19R were designed by Primer Premier 6 and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China).