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Revista MVZ Córdoba

Print version ISSN 0122-0268

Rev.MVZ Cordoba vol.19 no.2 Córdoba May/Aug. 2014

 

ORIGINAL

Genetic characterization of the domestic pig (Sus scrofa domestica) in Cerete-Colombia, using microsatellite markers

 

Caracterización genética del cerdo doméstico (Sus scrofa domestica) en Cereté - Colombia, usando marcadores microsatélites

 

Iván Meléndez G,1* M.Sc, Enrique Pardo P,2 Ph.D, Teodora Cavadia M,2 M.Sc.

1Universidad de Pamplona. Faculty of Basic Sciences. Department of Biology. Laboratory of Genetics. Ciudad Universitaria. Pamplona, Colombia.
2Universidad de Córdoba. Department of Biology. Genetics Section. Carrera 6 No. 76-103. Monteria. Colombia.

*Correspondence: imgelvez@hotmail.com

Received: April 2013; Accepted: December 2013.


ABSTRACT

Objective. The purpose of this study was to characterize a population of domestic pig (Sus scrofa domestica) in Cereté, Córdoba, using 20 microsatellite; calculate heterozygosity per locus and average heterozygosity. Materials and methods. Hair samples were collected from 62 specimens. DNA was extracted by proteinase K digestion and phenol-chloroform purification. Information from 20 microsatellites was selected out of those recommended for swine biodiversity studies. PCR products were separated by a vertical polyacrylamide gel electrophoresis. The bands were visualized by staining with silver nitrate. Results. All microsatellites used were polymorphic. Between 3 (SW1067) and 15 (IFNG) alleles were detected with an average number of 6.7 and a total de 134 alleles. The average expected and observed heterozygosities were 0.5278 and 0.5479, respectively. PIC values ranged between 0.1999 and 0.8300 for loci SW1067 and SW911, respectively. Conclusions. Levels of observed and expected heterozygosity found in the present study indicate that the domestic pig (Sus scrofa domestica) in Córdoba Cereté show high degree of genetic variability.

Key words: Alleles, genetic diversity, heterozygosity (Source: DeCS).


RESUMEN

Objetivo. El objetivo del presente estudio fue caracterizar una población de cerdo doméstico (Sus scrofa domestica) en Cereté, Córdoba utilizando 20 microsatélites; calcular la heterocigosidad por locus y la heterocigosidad media. Materiales y métodos. Se recolectaron muestras de bulbos de pelo de 62 ejemplares. El ADN se extrajo mediante digestión con proteinasa K y una purificación con fenol-cloroformo. Se utilizó la información proporcionada por 20 marcadores microsatélites de los recomendados para estudios de biodiversidad porcina. Los productos de PCR se separaron mediante electroforesis vertical en gel de poliacrilamida. Las bandas se visualizaron por tinción con nitrato de plata Resultados. Todos los microsatélites utilizados fueron polimórficos. Se detectaron, entre 3 (SW1067) y 15 (IFNG) alelos, con un número medio de alelos de 6.7 y un total de 134. Las heterocigosidades media esperadas y observadas fueron 0.5278 y 0.5479 respectivamente. Los valores del PIC oscilaron entre 0.1999 y 0.8300 para los loci SW1067 y SW911 respectivamente. Conclusiones. Los niveles de heterocigosidad observada y esperada encontrados en el presente estudio, indican que el cerdo doméstico (Sus scrofa domestica) en Cereté Córdoba muestran alto grado de variabilidad genética.

Palabras clave: Alelos, diversidad genética, heterocigosidad (Fuente: DeCS).


INTRODUCTION

The domestic pig (Sus scrofa domestica) is a four-legged mammal belonging to the group Suinos of the genus Sus (Family Suidae), which is characterized by having four toes ending in the shape of a hoof; the second and third toes serve as support and the first and fourth are not used for support. The fact that it has four hooves places it in the order Artiodactyla.

Its head is long and thick, in common races it is pyramid-shaped. Its skull is solid; in young animals the nose and frontal regions are straight and eventually become concave. The snout is cylindrical in shape with a bulky edge; hairless and smooth; the nostrils are small and its eyes have round pupils.

Domestic pigs in the Americas have their origin in the Iberian pigs brought by Columbus on his second voyage (1). Years later by imposition of Carlos V, the expedition of Rodrigo Bastidas who left La Española and founded Santa Marta in 1525, brought 300 pigs (2). It is possible that the pigs brought by Bastidas were the first that came to Colombia. It was initially raised outdoors with very rudimentary nutrition. It was looked after because of its fertility, meat and fat. Therefore, in each trip of the Spaniards to the American continent the number of pigs increased considerably, until occupying much the Colombian geography. Thus, it became an indispensable factor in the family feeding of natives.

The Department of Cordoba is one of the regions in Colombia with the largest populations of domestic swine (Sus scrofa domestica), made up mostly by the mixture of the creole race called zungo with other races. It seems that the first pigs were introduced into the Department of Cordoba around 1500-1550, at the time of the Conquest and they came from the Spanish breeds known as hairless (3).

The PCR (Polymerase Chain Reaction) is the most used technique for the study of polymorphic markers such as microsatellites or SSRs (Simple Sequence Repeats), formed by DNA segments between two and seven pairs of bases repeated in tandem as neutral markers of high informative value due to their abundance throughout eukaryotic genomes. They display codominant Mendelian inheritance, featuring high polymorphism, their amplification through PCR as well as the interpretation of the results is simple (4).

This study is justified by the interest in contributing to a wider genetic knowledge of the domestic pig (Sus scrofa domestica) in Cerete, Colombia to reveal the status of genetic variability, a conclusive element in the determination of breeding strategies and genetic conservation programs (4) and the establishment of a database that allows comparing the same with other existing populations.

The objective of this study was to characterize a population of domestic swine (Sus scrofa domestica) in Cerete, Colombia using 20 microsatellite; calculating their heterozygosity per locus, their average heterozygosity and compare this information with that obtained in other populations with the same genetic markers.

MATERIALS AND METHODS

Study site. The samples were collected in Cerete, Córdoba (08°53’08” N and 75°47’48” W), Colombia.

Sample collection. Hair samples of 62 specimens were collected. The specimens come from family farms, therefore they lack genealogical records.

Experimental procedure. The DNA of each one of the samples was extracted by means of a modification to the protocol described by Sambrook and Russell (5). The DNA was extracted from the hair bulb by digestion of proteins with the enzyme Proteinase K and phenol-chloroform purification.

The 20 microsatellites used in the study belong to the panel of those recommended by the FAO / ISAG (International Society of Animal Genetics) (6) for swine biodiversity studies. The amplification of the same was carried out using a thermal cycler Mycycler Bio-Rad® in a final volume of 25 mL which included 10 µL of 100 µM dNTPs, 2.5 µL of a 10X buffer, 1.0 µL of MgCl2 25 mM, 3.0 µL of specific primers for each locus of 10 pmol, 0.3 µL of the enzyme Taq DNA polymerase at a concentration of 1 U/µL, 4.0 µL of genomic DNA at a concentration of 50 ng/µL and 4.2 µL of sterile bidistilled water.

The PCR reaction consisted of a denaturalization phase at 94°C for 30 seconds, followed by 30 denaturation cycles at 94°C for 30 seconds, hybridization at 56°C for 30 seconds and elongation at 72°C for 30 seconds. PCR products were separated by vertical gel electrophoresis of polyacrylamide in a Mini-Protean II Biorad® chamber. The bands were visualized by staining with silver nitrate (7).

An allelic ladder was used to determine the size of the alleles and the allelic allocation was made by an adjustment to a linear regression curve developed from the migration distances of fragments of a known size.

Computer software and statistical analysis. The allele frequencies, heterozygosities, the value of FIS (8), the existence of the Hardy-Weinberg (HW) equilibrium, the allelic richness and the consanguinity coefficient were evaluated using the software GENEPOP v. 4.0.6 (9). Additionally, the Polymorphic Information Content (PIC) of each microsatellite was calculated using the software CERVUS v. 3.0.3 (10).

RESULTS

All the microsatellites used showed a high degree of polymorphism, evidenced in the average number of alleles per locus, detecting a total of 134 alleles with a range between 3 (SW1067) and 15 (IFNG) per locus (Table 1).

Table 1

The statistics FIS (Table 2) ranged from -0.414 for SW911 to 0.304 for the marker SWR345. Eleven of the 20 markers show a positive sign and 9 have a negative sign. The average FIS found was -0.033 (Table 3).

Table 2

Table 3

The polymorphic information content (PIC) obtained (Table 2) varied between 0.1999 (SW1067) and 0.8300 (SW911), values that corresponded to markers that showed the lesser and the greater number of alleles. The high degree of polymorphism was also evidenced by the average number of alleles of 6.7 found in the population (Table 3).

Of the microsatellites studied, three were not found to be in the Hardy-Weinberg equilibrium, (SW2083, S0215 and SWR345) which indicates an excess of homozygotes.

DISCUSSION

Previous studies on the genetic diversity of pigs reported greater and lower average values of alleles per locus as follows: between 5 and 13 alleles; (11, 12) and values between 2 and 3 alleles (13).

After applying the statistics FIS to 20 markers, 11 show a positive sign indicating an excess of homozygotes and 9 show a negative sign. The average FIS of -0.033 indicate a low value of exogamy, but with a slight excess of heterozygotes.

According to Liu et al (14), of the 20 markers analyzed, 15 can be considered as very informative (PIC>0.5) at the time of detecting the genetic variability in the population of domestic pigs in Cerete, Colombia, 3 markers are fairly informative (PIC>0.25) and 2 markers are little informative (PIC<0.25). The average PIC value in this study was lower in comparison with the data previously published in a study conducted with pigs from Portuguese and European breeds (15) and similar to those reported by the studies carried out in Mamellado of Uruguay and native pigs from China (11, 16, 17).

Of the total number of microsatellites analyzed, seventeen were found in the Hardy-Weinberg equilibrium, so it may indicate that the population is genetically stable (Table 2). In principle this could show that the mating in the population occurred randomly (in relation to the markers taken into consideration) or that, if there are new animals that have recently joined this population, these come from other populations with the same gene pool of the individuals in the population under analysis (18).

Three loci showed a significant deviation from the H-W equilibrium (SW2083, S0215 and SWR345) revealing an excess of homozygotes. The excess of homozygotes in a population may be the result of inbreeding events within the same (19). However, inbreeding equally affects the entire genome so it would be expected that if this phenomenon were to be the most transcendent, all the markers used should show an excess of homozygotes, which is not the case.

Likewise, the existence of a possible genetic structure by subdivision (Wahlund Effect) may also occur. If so, this would mean that there are marked differences between close populations of domestic swine for the markers (SW2083, S0215 and SWR345), but not for the other markers. If these differences for these markers have not been removed it is because the gene flow among nearby populations is limited (aspect not showed by other markers), or that these markers (SW2083, S0215 and SWR345) are linked to genes undergoing selection natural that act differentially at micro or macro-spatial level. Another possibility is the presence of null alleles in such loci (20), an event that can be discarded in this study as it was not found. A founder effect (few reproducers arrived that then multiplied a lot) could have also occurred.

In this study, the average heterozygosity expected of 0.527 and observed of 0.547 (Table 3) shows a high degree of variability, since it is considered as such when values exceeded 0.5. This value is similar to that reported in previous studies carried out in creole pigs in Uruguay, Cuban creole pigs, the Mexican hairless pigs and Argentinian creole pigs (21-24).

The percentage of heterozygous individuals was over 50%, reaching values of 54.79% for the average heterozygosity observed and 52.78% for the average heterozygosity expected. These values are similar to those reported in Denmark and the Netherlands and are exceeded by those reported in China, Brazil and Thailand (25-28).

The parameters mean number of alleles per locus and allelic richness indicate that this population exhibits certain degree of variability.

Since this is the first study where it is attempted to learn the population genetic diversity of Sus scrofa domestic in Cerete, Colombia and there is no prior information allowing to explore the possibility of loss of genetic diversity over time; the possibility of inbreeding processes for this population of the Colombian Caribbean region cannot be ruled out.

The results of this study allow concluding that the microsatellites used in the population of domestic swine (Sus scrofa domestica) in Cerete, Colombia revealed a high degree of polymorphism. In addition, the high percentage of markers with a high PIC would facilitate the implementation and optimization of this technique for other studies on this race such as genealogical research and allocation of individuals to populations. Similarly, the heterozygosity levels observed and expected found in this study indicate that the domestic pig (Sus scrofa domestica) in Cerete, Colombia show a high degree of genetic variability.

 

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