DNA barcoding analysis and phylogenetic relationships of Indian wild coffee species

: Wild coffee species are the reservoirs of genetic diversity and could play a critical role in the genetic improvement of coffee. However, the conservation and genetic assessment of wild coffee species has been largely neglected. In the present study, DNA barcoding approaches were employed to assess the phylogenetic relationships between five indigenous wild coffee species from India and compared with two cultivated, and four wild coffee species of African origin. The efficacy of three barcoding loci namely matK , rbcL , and trnL-trnF was investigated using PCR amplification and sequence characterization. The intergenic spacer trnL-trnF is the highly polymorphic loci followed by matK and rbcL chloroplast gene. Among the three barcoding loci, the matK locus has the maximum number of parsimony informative sites, whereas the trnL-trnF locus contains maximum singleton variable sites. Although all the three loci contain a few unique fixed nucleotides (UFNs), no individual barcode locus has the critical nucleotide sequence tags for all the five Indian wild coffee species that help in species discrimination. However, the multilocus combinations are efficient in discriminating the species due to the presence of SNPs and specific sequence tags. The phylogenetic tree constructed using the maximum likelihood analysis of the combined barcoding loci separated all the Indian wild coffee species from African wild coffee species compared to phylogeny inferred using individual barcoding loci. Our study supports the utility of DNA barcoding as a useful tool for coffee species identification, which can be used for conservation purposes.