Characterization Of Genome Wide SSRs In C. Canephora And Analysis Of Genetic Diversity In Coffee Using SSR And ISSR Markers

Coffee is an important beverage crop in the world and has a significant contribution to many developing economies like Kenya.The overall genetic diversity of Coffea arabica is low due to history of domestication and genetic bottleneck associated with domesticated crops.Genetic analysis provides initial step for breeders to improve or develop new cultivars.To achieve this,we analyzed the genetic diversity and population structure of key commercial varieties in Kenya and China.Our study also surveyed and characterized the distribution and frequency of perfect microsatellites in the 710 Mbp assembled C.canephora genome sequence to unravel its genome architecture.A total of 159,041 SSRs were identified,with an overall density of 308 SSRs per Mb across all the eleven chromosomes and the unanchored scaffold unknown.Tetra-nucleotide repeats are the most abundant,accounting for 32%of the total SSRs.Dinucleotide repeats(DNRs),tri-nucleotide repeats(TNRs),penta-nucleotide repeats(PNRs),and hexa-nucleotide repeats accounted for 29.2,21.4,11.3,and 6.1%of all SSRs,respectively.An overall decrease in the number of SSR with an increase in the number of repeat unit pattern was observed in all the SSRs motifs.Generally,most SSRs(99.3%)were less than 30 bp in length.The predominant DNRs,TNRs,TtNRs,PNRs,and HNRs had 6,4,3,3,and 3 repeat units,respectively.AT-rich motifs are dominant across all SSR repeat units,while GC-rich motifs were generally rare.A set of 100 SSRs primers were selected to screen 96 coffee accessions,including cultivars and 10 wild accessions collected from Mt.Marsabit(Kenya)with an aim to assess the genetic diversity and population genetic structure in Kenya.Of these SSRs,33%generated clear polymorphic bands among all tested accessions,generating a total of 129 alleles with an average of 3.9 alleles per SSR locus and an average polymorphism information content(PIC)of 0.54.The number of effective number of alleles(Ne)ranged from 1.286 to 2.364 while the pairwise population differentiation matrix FST ranged from 0.047 to 0.454.In addition,the observed and expected heterozygosity ranged from 0.170 to 0.480 and from 0.216 to 0.447 respectively with an average Shannon index of 0.577.Wild coffee species from Mt.Marsabit showed a close genetic similarity with cultivated accessions in Kenya,suggesting that the wild species in Mt.Marsabit played an important role in the domestication of cultivated coffee in Kenya.Significantly low pairwise genetic divergence was observed between cultivated and wild accessions in Kenya,suggesting a relatively narrow level of genetic basis among coffee germplasm in Kenya.This was further demonstrated by population structure and principle coordinates(PCoA)analyses,which separated the genotypes into two major groups.Significantly low genetic differentiation coefficients and closer genetic relationships by cluster analysis were observed among all accessions from Kenya suggesting that the genetic basis was narrow.In addition,cultivated and wild coffee accessions in Kenya show a great divergence from those in other countries.In contrast significant divergence between cultivars from Kenya and those from other countries could be exploited for germplasm improvements.Our results not only provide molecular tools for genetic studies in coffee but are also helpful for conservation of genetic resources and for progress in coffee breeding programs in Kenya.Population genetic analysis is crucial for understanding the degree of linkage disequilibrium in a germplasm collection for association mapping and breeding strategies.We analyzed population structure patterns and genetic differentiation in coffee germplasm collections in China using 19 IS SR markers.No evidence of isolation by distance,but a clustering pattern correlating with predefined lineages was observed for all the tested accessions.C.liberica and C.excelsa Chevalier showed a close genetic association,but significantly deviated from C.canephora and C.arabica.Significant differentiation was also detected within C.arabica accessions,separating into three genetic subgroups supported by pairwise differentiation index(Fsr)and Nei's standard genetic distance tests.The FST outlier test for deviation from expected distribution between FST and expected heterozygosity identified five putative loci under selection.