Development and Characterisation of Microsatellite Markers for Helmeted Guinea Fowl (NUmida Meleagris) in Ghana

Abstract

The indigenous guinea fowl plays a vital role in the agricultural industry as both its meat and eggs are healthy, tasty and serve as an important protein source for consumers. However, genetic progress on this bird has been partially hindered by the absence of polymorphic markers, especially microsatellites. Therefore, this study developed for the first time original microsatellite markers for this economically important species. The 454 sequencing technique (next-generation sequencing), which is known to eliminate the time consuming cloning step in the traditional microsatellite marker development method, was used in this study. A genomic library was constructed from DNA extracted from the blood of a female guinea fowl, using the next-generation sequencer. A total of 105,015 reads with an average read length of 393 bp containing 1,234 possible microsatellite sites were obtained. One hundred and fifty four primers were designed from the flanking regions of the microsatellites and tested at 55 °C and 60 °C in a polymerase chain reaction using DNA from four unrelated guinea fowls. One hundred and twenty two of these primers showed clear amplification patterns. Polymorphism of 38 of the optimized markers was tested with DNA samples from 32 unrelated individuals and 31 of them were polymorphic. For the 31 polymorphic loci, the observed number of alleles ranged from 2 to 9 (mean 3.39) with allele sizes ranging from 94 bp to 286 bp, while the effective number of alleles ranged from 1.03 to 4.97 (mean 2.04). The observed (HO) and expected heterozygosities (He) ranged from 0.033 to 1.000 (mean 0.396) and 0.033 to 0.799 (mean 0.419), respectively. Nine loci significantly deviated from Hardy-Weinberg Equilibrium (p < 0.05) after Bonferroni correction. The mean fixation index (F) for all 31 loci was 0.052 (-1 to 0.71) while the average probability of identity (PI) was 0.43. Shannon’s Index ranged from 0.085 to 1.821 (mean 0.750). The polymorphism information content (PIC) of the 31 markers averaged 0.3689 (0.0329 to 0.7735) with 29% of them being highly informative (PIC > 0.50), 35.5% being reasonably informative (0.50 > PIC > 0.25), and 35.5% being slightly informative (PIC < 0.25). The results of this study would serve as baseline information for genetic diversity studies, genetic linkage mapping, quantitative trait loci analysis as well as inform breeding strategies for the improvement and conservation in both domestic and wild populations of the species.