We have analyzed 7137 samples from 125 different caste, tribal and religious groups of India and 99 samples from three populations of Nepal for the length variation in the COII/tRNALys region of mtDNA. Samples showing length variation were subjected to detailed phylogenetic analysis based on HVS-I and informative coding region sequence variation. The overall frequencies of the 9-bp deletion and insertion variants in South Asia were 1.8% and 0.5%, respectively. We have also defined a novel deep-rooting haplogroup M43 and identified the rare haplogroup H14 in Indian populations carrying the 9bp-deletion by complete mtDNA sequencing. Moreover, we redefined haplogroup M6 and dissected it into two well-defined subclades. The presence of haplogroups F1 and B5a in Uttar Pradesh suggests minor maternal contribution from Southeast Asia to Northern India. The occurrence of haplogroup F1 in the Nepalese sample implies that Nepal might have served as a bridge for the flow of eastern lineages to India. The presence of R6 in the Nepalese, on the other hand, suggests that the gene flow between India and Nepal has been reciprocal.

Background

Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa.

Results

A small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10-6 and 10-12 for arabicas and robustas respectively) and unbiased corrected estimates (10-20 and 10-43 for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee.

Conclusion

The conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.

We have analyzed 7,137 samples from 125 different caste, tribal and religious groups of India and 99 samples from three populations of Nepal for the length variation in the COII/tRNALys region of mtDNA. Samples showing length variation were subjected to detailed phylogenetic analysis based on HVS-I and informative coding region sequence variation. The overall frequencies of the 9-bp deletion and insertion variants in South Asia were 1.9 and 0.6%, respectively. We have also defined a novel deep-rooting haplogroup M43 and identified the rare haplogroup H14 in Indian populations carrying the 9-bp deletion by complete mtDNA sequencing. Moreover, we redefined haplogroup M6 and dissected it into two well-defined subclades. The presence of haplogroups F1 and B5a in Uttar Pradesh suggests minor maternal contribution from Southeast Asia to Northern India. The occurrence of haplogroup F1 in the Nepalese sample implies that Nepal might have served as a bridge for the flow of eastern lineages to India. The presence of R6 in the Nepalese, on the other hand, suggests that the gene flow between India and Nepal has been reciprocal.