Single Nucleotide Polymorphisms and Haplotype Analyses in Tilapia Fish Inferred from mtDNA D-loop and Cyt-b Regions

Abstract

Objective: This research was aimed at analysing single nucleotide polymorphisms and haplotypes on D-loop and Cyt-b regions of the mitochondrial DNA of tilapia fish. Methods: Fifteen and thirteen tilapia fish were obtained from two populations, south-south (Domita farm) and south-west (Odeda farm). DNA extraction from fish tissue was done using Quick-gDNATM mini Prep kit after which PCR amplification was carried out. Sequencing of the two mtDNA regions were done using forward primer 5’- GGATTYTAACCCYTRCCCC- 3’ and reverse 3’-AGTAAGTCAGGACCAAGCC-5’ for D-loop and 5’-GGATTTTAACCCTTACCCC-3’ and 3’-AGTAAAGTCAGGACCAAGCC-5’ for Cyt-b region. Statistical analyses were carried out on the aligned sequenced data using MEGA version 6.06, Dnasp 5.1, Codon code aligner 6.06 as well as NETWORK 4.6.1.1. Results: mtDNA polymorphism was highest in the D-loop of South-South (SS) population with 176 polymorphic sites, while South-West (SW) population had 162 polymorphic sites translating to 176, 162 and 144 SNPs with higher non-synonymous substitutions than synonymous substitutions.  Haplotype diversities (Hd) were 1.00 ± 0.024 and 1.00 ± 0.030 while nucleotide diversities were 0.168 ± 0.086 and 0.161 ± 0.084 for D-loop of SS and SW populations, respectively. For Cyt b region, haplotype and nucleotide diversities were 0.91 ± 0.003 and 0.051 ± 0.016. Positive selection was more on mtDNA D-loop of tilapia sampled from SS than those from the SW as well as Cyt-b region of tilapia fish from SS. 28 haplotypes were identified among the tilapia from SS and SW with no shared haplotypes while 9 haplotypes were identified from the Cyt-b region with haplotypes 4, 5, 6 and 7 shared between species. Median-joining network analysis revealed population-based clustering pattern. The demographic expansion was not significant using Tajima's D and Fu's F statistics.  Conclusion: Higher SNPs were revealed in mtDNA D-loop when compared with mtDNA Cyt-b region of tilapia fish.