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Microsatellite-based Sibship Reconstruction and Estimation of Genetic Relatedness in the Endangered Labeo Calbasu (hamilton 1822) (cyprinidae: Cypriniformes)

M. Nahiduzzaman
World Fish Center, Dhaka, Bangladesh

M. Samsul Alam
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202

M. A. R. Hossain
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202

M. RobiulHasan
Department of Fisheries and Marine Science, Noakhali Science and Technology University, Noakhali 3814.

S. Akter
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202

Abstract/ Executive Summary:

The microsatellite genotyping technique was used to reconstruct sibship and estimate pairwise relatedness among individuals of Labeo calbasu collected from four natural and one hatchery stocks. The mean values for the number of alleles, observed and expected heterozygosity and polymorphic information content of the five microsatellite markers used for genotyping 165 individuals were 6.00, 0.622, 0.743 and 0.699 respectively. Deviation from Hardy-Weinberg expectation was detected at four of the five loci. The number of reconstructed half-sib family was lowest in the Hatchery sample. The number of full-sib family was lowest in the Jamuna river sample and highest in the Haor sample. The numbers of reconstructed families were fewer in the real sample compared to the simulated sample of the same number of unrelated individuals. The mean relatedness coefficients r_xyW was found to be highest in the Jamuna river sample and lowest in the Hatchery sample, though no difference was observed in mean r_xyLR values of the five samples. The misclassification rates estimated based on the relatedness coefficients were found to be quite high, ranging from 11.17 (Unrelated-Fullsib) to 32.02% (Halfsib-Fullsib) and more loci need to be analyzed for accurate separation of related individuals from unrelated ones.

Keywords: Polymorphism, Microsatellite, Relatedness, Family Reconstruction, Labeo calbasu

Location: Faculty of Fisheries, BAU

Start Date: 00-00-2013

End Date: 00-00-2013

Program Area: Variety and Species

Commodity: Carp fish

Objectives:

1. To reconstruct sibship and estimate pairwise relatedness among individuals of Labeo calbasu collected from four natural and one hatchery stocks.

Methodology:

Collection of samples and isolation of genomic DNA: Fin clips were collected from a total of 165 mature L. calbasu samples, 33 each from five sources: the Jamuna river, the Padma river, the Halda river, Tola Haor and a hatchery, and preserved in 95% ethanol in separate microfuge tubes and stored at -20ºC. The sampling locations are shown in a map of Bangladesh. Genomic DNA was isolated from each sample following standard protocol of proteinase-K digestion, phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. ?Microsatellite genotyping: We used five heterologous microsatellite markers: Lr10, Lr21, Lr24, Lr26 developed from Labeo rohita and CcatG1 developed from Catla catla. The methods of microsatellite genotyping have been detailed. Statistical analysis of microsatellite data: For quantifying genetic variability we calculated the number of alleles, observed heterozygosity (Ho), expected heterozygosity (He) and the polymorphic information content (PIC) for each locus in the complete set of samples using CERVUS version 3.0.3. The same software was used for exact test for deviation from Hardy-Weinberg expectation at the five loci based on genotype frequencies of 165 individuals. For estimating the degree of genetic relationship among all pairs of individuals we calculated three relatedness coefficients: r_xyW, r_xyLR and r_xyQG using the software COANCESTRY version 1.0.1.2 (Wang, 2011). For evaluating the bias of r_xyW, r_xyLR and r_xyQG values for unrelated, half-sibs and full-sibs categories, the observed allele frequency estimated from the genotype data of all the 165 individuals were used to generate the relatedness coefficient of 5000 pairs of individual under each of the three relationship categories: Full-sibs (FS), Half-sibs (HS) and unrelated (UR). The mean values for the relatedness coefficients obtained from the simulated genotype data and those expected under three different relationship categories- 0.5 (Full-sib), 0.25 (Half-sib) and 0.0 (unrelated) were subjected to two-tailed t-tests to examine if there was any significant difference. The rates of misclassification for assigning individuals to different relationship categories were estimated based on a cut-off point set at the average of the simulated r_xyW, r_xyLR and r_xyQG values for the two levels of relationship categories. As, the relatedness estimates of two samples showing different levels of heterozygosity cannot be directly compared, we made comparisons between the distribution of observed pairwise relatedness (r_xy) values among pairs of individuals within each sample with that expected in a sample of simulated unrelated individuals from a population with the same allele frequencies. For that purpose, we generated the genotypes of 250 unrelated individuals for each population by using the genotype data of the corresponding sample both for population 1 and population 2 in the HYBRIDLAB v1.0 program (Nielsen et al., 2006). We then calculated the relatedness among all the pairs of individuals totaling 31125 [n(n-1)/2] pairs per population. Comparisons between the real and simulated pairwise relatedness coefficients were undertaken using a Mann-Whitney U-test. We reconstructed half- and full-sib families without information on parental genotypes as per maximum likelihood method implemented in the software COLONY 2. We assumed a tentative 2.5% error rate for all loci, both for allelic dropouts and erroneous sizing of alleles. We compared the family configuration of each sample with that of a sample of the same number (n=33) of unrelated individuals simulated from the empirical allele frequency data by using the HYBRIDLAB v 1.0 program. The sibship reconstruction of a sample was considered „significant? if the number of half- and full-sib families was lower in the real samples compared to the samples of simulated unrelated individuals and if there were more individuals in one or more of the full-sib families generated from real individuals than were observed in any of the full-sib „families? generated from the simulated unrelated individuals.

Source of Information: The Journal of Animal & Plant Sciences, 25(3): 2015, Page: 825-835

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The number of reconstructed half-sib family was lowest in the Hatchery sample. The number of full-sib family was lowest in the Jamuna river sample and highest in the Haor sample. The numbers of reconstructed families were fewer in the real sample compared to the simulated sample of the same number of unrelated individuals. The mean relatedness coefficients r_xyW was found to be highest in the Jamuna river sample and lowest in the Hatchery sample, though no difference was observed in mean r_xyLR values of the five samples. The misclassification rates estimated based on the relatedness coefficients were found to be quite high, ranging from 11.17 (Unrelated-Fullsib) to 32.02% (Halfsib-Fullsib) and more loci need to be analyzed for accurate separation of related individuals from unrelated ones.

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