Paternity in Brazilian goats through the use of DNA microsatellites

A total of 292 animals from three breeds (Alpine and Saanen dairy breeds, and the Brazilian naturalized breed Moxotó) were genotyped, comprising 276 paternity cases. Statistical analyses were carried out by using TFPGA and CERVUS programs. Heterozygosis ranged from 0.542 (ILSTS005) to 0.825 (INRA006), with an average of 0.717 for all loci. Polymorphic information content (PIC) was 0.676 and 0.542, and combined exclusion probabilities (EP) were 0.999591 and 0.988375 for known and unknown maternal genotypes, respectively. The microsatellite system reveals 10% of paternity misidentification in evaluated registers.


Introduction
Goats are the most widely spread domestic species in the world and play an important economical role in developing countries.Brazil has the tenth largest world goat herd with about ten million animals (IBGE, 2006).In Brazil, goat breeding ranges from an intensive system in the dairy basins in the southern and southeastern regions to an extensive raising system at the Northeast region, where a large number of herds is found, contributing to sustainable small scale agriculture.Goat genetic resources are very diverse, with foreign European breeds like Alpine and Saanen being the most important resources for dairy production.On the other hand, at the Northeast region, a large number of herds has no defined breed, or is composed by naturalized groups, such as Moxotó, Canindé, Marota, and Repartida (Machado, 2000).
There isn't an official National Genetic Goat Improvement Program in Brazil.Genealogical data are registered at the Associação Brasileira de Criadores de Caprinos (ABCC) and its branches.The few initiatives to create a goat database are isolated efforts of breeders and research institutions without substantial results.All breeding programs are based on non official information and considering the losses on selection accuracy and on genetic gain generated by genealogical misidentification the need for a correct paternal identification is a major concern for the evaluation of genetic parameters and the success of breeding programs.
Statistical methods for pedigree checking in natural populations or those on which data are taken on a large scale may differ in focus from commercial laboratories, whose goal is to confirm genetic compatibility of the alleged father, mother and progeny trio using parentage exclusion.The goal of the present study is to use eleven microsatellites loci to solve genealogy and to verify the accuracy of the registered pedigrees in Brazilian goat herds.

Material and Methods
Pedigree register for 121 Alpine and 94 Saanen goats from Universidade Federal de Viçosa, 36 Alpine and 19 Saanen goats from a private herd, and 22 Moxotó goats from preservation herd of Embrapa, were analyzed.Samples were distributed in existing sire families of each herd to obtain a progeny group for each male breeding stock.Known mothers were genotyped within the herd.
For Alpine and Saanen breeds, blood was collected from the jugular vein, using a vacuum system, and stored at 4°C for 6 h.DNA was extracted by following the phenol: chloroform protocol (Sambrook et al., 1989).DNA samples from the Moxotó herd were obtained from hair follicles, following the cetiltrimetilammonium bromide protocol, as described by Ferreira & Grataplagia (1998).
Reverse primers were fluorescently labeled with Tet, 6-Fam, and Hex dyes, so amplified fragments did not overlap, and multiplexed electrophoresis could be used.Amplified fragments were submitted to capillary electrophoresis at 9 V, 60ºC for 22 minutes and analyzed with ABI 310 Genetic Analyzer from Applied Biosystem TM , utilizing Genescan ® software (Table 1).
Gene frequencies were obtained for each allele by direct count.The polymorphic information content was used to quantify polymorphism when one progenitor genotype was know (PIC 1 ) (Bolstein et al., 1980) and when both progenitors genotypes were unknown (PIC 2 ) (Da & Lewin, 1995).
Exclusion probability was calculated with TFPGA software (Miller, 1997), firstly for cases in which one progenitor was confirmed, and genotypes were known for the alleged father, mother and progeny (EP 1 ), and secondly when just the father and progeny genotypes were known (EP 2 ) (Jamieson & Taylor, 1997).
The maximum likelihood method by the CERVUS program (Marshall et al., 1998) was applied to check pedigree mismatching.CERVUS program is used to calculate allelic frequencies in a simulation approach and to determine the confidence in parentage assignments by calculating critical values of the log likelihood statistics Δ (Delta).
The exact test of Hardy-Weinberg proportion was performed by TFPGA software (Miller, 1997), using the method developed by Guo and Thompson (1992) of Markov Chain (10 batches, 5.000 permutations, and 1.000 dememorization steps).

Table 1 -
Locus, chromosome localization, annealing temperature, MgCl 2 concentration, primer concentration, dye, number of alleles observed, and allele size n locus no genetic or RH mapped.