A simple and versatile authenticity assay of coffee products by single-nucleotide polymorphism genotyping

Interspecific single-nucleotide polymorphisms (SNPs) in the rbcL DNA barcode have been strictly validated and adopted as a designed SNP genotyping maker to discriminate between two major coffee species, Coffea arabica and C. canephora, and to estimate the mixing ratio of DNA from C. arabica/C. canephora in this study. The SNP genotyping is applicable to not only green (unroasted) coffee beans, but also processed coffee products (roasted coffee beans and instant coffee powder), in which genomic DNA is degraded, because the genotyping developed in this study requires only 10 copies of 63-bp-long DNA fragments of rbcL gene. The authenticity assay established in this study has several advantages: a high versatility to DNA sample conditions; simple and rapid procedures (only two steps; DNA extraction and SNP genotyping); the feasibility in coffee business for practical use to prevent false advertising and provide quality control. Abbreviations: SNP: single-nucleotide polymorphism; SBS: single base substitution; ISR: intergenic spacer region; INDEL: insertion-deletion. SNP Genotyping: Required only 10 copies of 63-bp-long DNA fragment of rbcL gene.

本研究针对rbcL DNA条形码中的种间单核苷酸多态性（single-nucleotide polymorphism, SNP）进行了严格验证，并将其作为设计的SNP基因分型标记，用于区分阿拉比卡咖啡（Coffea arabica）与刚果咖啡（C. canephora）这两种主要咖啡树种，同时可估算二者的DNA混合比例。本研究开发的SNP基因分型技术不仅适用于生（未烘焙）咖啡豆，同样适用于基因组DNA已发生降解的加工咖啡制品（烘焙咖啡豆与速溶咖啡粉），因为该方法仅需rbcL基因63bp长度DNA片段的10个拷贝即可完成检测。本研究建立的真伪鉴定方法具备多项优势：对DNA样品状态具有高度通用性；操作流程简便快捷（仅需DNA提取与SNP基因分型两个步骤）；可在咖啡产业中实际应用，用于防范虚假宣传并实现质量管控。缩写说明：SNP：单核苷酸多态性（single-nucleotide polymorphism）；SBS：单碱基替换（single base substitution）；ISR：基因间隔区（intergenic spacer region）；INDEL：插入缺失（insertion-deletion）。SNP基因分型：仅需rbcL基因63bp长度DNA片段的10个拷贝即可完成。