Development of new genomic microsatellite markers from robusta coffee (Coffea canephora Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies

Abstract

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.

Figure 1

Bar-graph showing comparative distribution of: (A) number of alleles (NA) amplified, and (B) PIC values of the new SSR markers in the tested sets of genotypes of arabica and robusta coffee. Note: in case of PIC the plotted values represent normalized proportions of only the total polymorphic markers (which were 41 for robustas, 36 for arabicas, and only 23 in case of Arabica after removing the possible duplicate loci).

Figure 2

Relative position of the nine new SSR markers (20% of the total tested) mapped on a robusta coffee map [12]. The reference map was generated using pseudo-testcross mapping population derived from a cross of 'CxR' (a commercial robusta hybrid) and Kagganahalla (a local selection from India). Note that the new mapped markers are distributed randomly across different linkage groups. The value at the base of each LG refers to its relative length in centiMorgans (cM).

Figure 3

NJ tree showing relationship within and between arabica and robusta germplasm based on the allelic diversity generated using the new SSR markers.

Figure 4

NJ tree showing relationship between 14 Coffea and two Psilanthus taxa based on the allelic diversity generated using the new SSR markers.