Microsatellite Analysis Revealed Potential DNA Markers for Gestation Length and Sub-Population Diversity in Kari Sheep

Abstract

Kari sheep inhabiting the Chitral district of Pakistan show variation in gestation length. In this study, we have analyzed the genetic differences between the three subtypes of Kari sheep (based on variation in gestation length) using microsatellite markers. Kari sheep samples were collected from their breeding tract and were characterized for gestation length and genetic diversity using microsatellite markers. A total of 78 Kari ewes were grouped into three categories based on gestation length (GL), i.e., Kari-S (with a shorter GL), Kari-M (with a medium GL), and Kari-L (with a longer GL). DNA from these samples was used to amplify 31 ovine-specific microsatellite loci through PCR. Of the total 78 Kari specimens, 24 were grouped in Kari-S (GL = 100.7 ± 1.8), 26 were from the Kari-M subtype (GL = 123.1 ± 1.0), and 28 were Kari-L (GL = 143.8 ± 1.5). Microsatellite analysis revealed an association of genotypes at two marker sites (MAF214 and ILSTS5) with variation in GL. A total of 158 alleles were detected across the 22 polymorphic loci with an average of 7.18 alleles per locus. Unique alleles were found in all three subtypes. The highest number of unique alleles was observed in Kari-L (15), followed by Kari-S (10) and Kari-M (8). The results indicated that Kari-S is a genetically distinct subtype (with higher genetic differentiation and distance) from Kari-M and Kari-L. The genetic uniqueness of Kari-S is important for further exploration of the genetic basis for shorter gestation length, and exploitation of their unique values.

Keywords: Kari sheep; bottleneck analysis; genetic diversity; gestation length; microsatellite association.

Figure 1

A representative image of polyacrylamide gel electrophoresis showing microsatellite banding pattern in eight individuals of Kari sheep. The sizes of the PCR product bands in samples (1–8) were calculated from the 50 bp ladder (L). Arrow indicates the primer dimmers. Bracket indicates the bands that came in the size range of the given microsatellite marker. Different size bands within the range were considered as different alleles for the marker. The non-specific bands above the expected region of the gel were not included in the analysis.

Figure 2

Association analysis between microsatellite loci and gestation length in Kari sheep. (A) Principal component plot showing clustering of closely associated parameters. (B) Mean gestation length among different genotypes of highly correlated loci. Labels on the bars (letters a–d) show a comparison among genotypes (p < 0.05).

Figure 3

Ewens-Watterson test of neutrality of markers in the three Kari subtypes. The lines represent the upper and lower 95% confidence intervals. The dots show the loci. Arrows showing the markers (labeled) falling outside of the confidence intervals.

Figure 4

Dendrogram based on Nei’s unbiased genetic distance using the neighbor-joining method.

Figure 5

Summary plot showing the membership probability of individuals in the predefined subtype at different K values. Each bar represents an individual. Note that at K = 3 a cluster made by Kari-M showed a proportion of Kari-S in the ancestry; while an ancestral admixture of Kari-L was observed in the group made by Kari-S.

Figure 6

Allele frequency distribution graph of the three Kari subtypes. A mode shift can be seen in Kari-S subtype, while Kari-M and Kari-L subtypes followed a normal L-shape graph.