Abstract
BackgroundSpecies-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.ResultsA 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.ConclusionThe 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.
Highlights
Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement
The designed primer pairs were standardized for PCR amplification, and validated for utility as genetic markers using panels of elite coffee genotypes, a mapping population for linkage studies, and related taxa of coffee for cross-species transferability
The analysis revealed coffee genome to be enriched in AT type DNRs (AT-DNR), which were estimated to be many fold more than any other Short Sequence Repeats (SSR) motifs
Summary
Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Efforts undertaken globally to improve coffee, though successful, have proven to be too slow and severely constrained owing to various factors The latter includes: genetic and physiological makeup (low genetic diversity and ploidy barrier in arabicas, and self incompatibility/ easy cross-species fertilization in robustas), long generation cycle, requirement of huge land resources, and the dearth of accessible and assayable genetic tools/techniques for screening/selection. The situation warrants recourse to newer, easy, practical technologies that can provide acceleration, reliability and directionality to the breeding efforts, and allow characterization of cultivated/secondary genepool for proper utilization of the available germplasm in genetic improvement programs In this context, development of DNA marker tools and availability of markers-based molecular linkage maps becomes imperative for MASbased accelerated breeding of improved coffee genotypes
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