Genetic structure of Antioquia Holstein from two SNPs and association with dairy traits

Objective. Analyze the structure and genetic differentiation of a population of Antioquia Holstein cows from the polymorphisms A192G of INHA and A-320T of FSHR, and explore the association of the genotypic combinations with milk traits. Materials and methods. 1240 lactations of 356 animals from 9 herds in 6 municipalities of Antioquia were analyzed. Genotyping was performed by PCR-RFLP. Structure and genetic diversity parameters were determined using GenAlex software. The association of genotypes combinations with productive and reproductive traits was explored through a linear mixed model. Results. SNP A192G showed a frequency of 0.534 and 0.466 for A and G alleles respectively and SNP A-320T had a frequency of 0.660 far A allele and 0.339 for T allele, this way the population is in HWE. The FST, FIS and FIT values were 0.059, 0.285 and 0.328 respectively indicating a moderate genetic differentiation between subpopulations. The A-320T SNP showed significant effect on milk yield. Fat and protein percentage, calving interval and services per conception were not affected by these polymorphisms or their interaction. Conclusions. Phenotypic selection made on this population has not been strong enough to generate noticeable changes in allele frequencies of these polymorphisms or deviations from Hardy-Weinberg equilibrium. The interaction of these polymorphisms has no significant effect on the characteristics of zootechnical interest, so its use in programs of molecular marker assisted selection is not recommended.


INTRODUCTION
The genetic variability of a population is influenced by the number of founding individuals, genetic drift, strategy and selection intensity (1), while the population structure can be affected by the interchange of genetic material among populations, the reduction of population size, and the decrease in the number of male reproducers, among other factors (2).
The breeding programs implement in developing countries based on importation of genetic material, but the majority of this comes from populations with low genetic variability and high levels of endogamy due to using the few genetically superior individuals that are used for mating, putting at risk the sustainability of the improvement programs (2).
In the dairy herds in Antioquia, artificial insemination is implemented as the principal tool for genetic improvement.Techniques such as multiple ovulation and embryo transfer and producing embryos in vitro are implemented with lesser proportion in order to obtain individuals with high genetic merit that improve the productive levels of those systems.
Among the existing methodologies to select superior individuals we find marker assisted selection (MAS), which are sites in the genome where differences exist in the nucleotide sequence between individuals of the same species, generating single nucleotides polymorphism (SNP) that can be identified by the PCR-RFLP (3).These polymorphisms can also be used to know the structure and diversity of a population (4).The SNP A192G is found in the exon 1 of the inhibin alpha gene (INHA), and since it is a synonymous mutation it does not change the protein amino acid sequence, but rather generates an additional restriction site for the MspI endonuclease (5,6).The SNPA-320T is found upstream from the follicle stimulating hormone receptor gene (FSHR) and although it is not found in any codifying region, it can modulate gene expression and also change the restriction pattern obtained in the Taq1 enzyme (7,8).

Análisis de estructura y diferenciación poblacional.
El ADN genómico se obtuvo a partir de muestras de sangre con el método de Salting out modificado (10).Para la genotipificación del SNP A192G (GenBank n u m b e r : r s 4 1 2 5 7 1 1 6 ) s e a m p l i f i c ó u n fragmento de 249 pb del exón 1 utilizando los primers F: 5'-GCCCTGTTTCTGGATGCC-3' y R: 5'-ATTCAACCCAACCTGCCTA-3' (5).El volumen final de reacción fueron 30 µl, se empelaron 3.8 µl de buffer de reacción 10X (200 nM (NH 4 ) 2 SO 4, 750 mM Tris-HCl pH 8.8 a 25°C), 2.5 mM de MgCl 2 , 15 pmol de primers, 0.4 mM de dNTP (desoxirribonucleótidos trifosfato), 1.5 undidades de Taq-ADN polimerasa y 100 ng de ADN genómico como muestra.Las condiciones de la PCR fueron 5 min a 95°C, 34 ciclos de 94°C por 45 s, 62°C por 45 s para el alineamiento de los primers, 72°C por 45 s y 72°C por 10 min para la extensión final.La digestión se realizó con 15 µl de producto de PCR, 7 U de enzima MspI y The stimulating follicle hormone (FSH) has an essential role in oogenesis, allowing recruitment and follicular development, and acts exclusively through its receptor (FSHR) which is found on the surface of granulosa cells (7,9).Inhibin regulates FSH levels affecting folliculogenesis (5).These polymorphisms have been associated with the response to superovulation and seminal quality, and it has been found that the INHA G allele exercises a positive effect on both traits, while the FSHR A allele improves seminal traits (5,7,9).However, there are no studies on the effect of the principal parameters evaluated in the specialized dairy systems.The objective of this research was to analyze the structure and genetic diversity of a population of Holstein cattle based on A192G and A-320T polymorphisms of INHA and FSHR genes, and to determine the association of its interaction with the main productive and reproductive parameters estimated in the specialized dairy systems.

MATERIALS AND METHODS
Population study.The phenotypic information and the DNA samples were obtained from 356 Holstein cows (1240 lactation periods) from nine herds in seven municipalities of Antioquia (Table 1).Analysis of structure and population differentiation.The genomic DNA was obtained from blood samples using the modified Salting out method (10).To genotype the SNP A192G (GenBank number: rs41257116), a fragment of 249 bp of exon 1 was amplified using the primer F: 5'-GCCCTGTTTCTGGATGCC-3' and R: 5'-ATTCAACCCAACCTGCCTA-3' (5 Las frecuencias alélicas se determinaron mediante las fórmulas y donde N A y N a son el número de alelos A y a en la población y N es el número de individuos.Las frecuencias genotípicas se determinaron con las fórmulas , y donde N AA, N Aa y N aa son el número de copias de los genotipos AA, Aa y aa (11).
La estructura poblacional se determinó mediante los estadísticos F de Wright; , , donde H o y H e son la heterocigosidad observada y esperada entre subpoblaciones y H T es la heterocigosidad total esperada.Estos se obtuvieron utilizando el software GenAlex mediante el método de Análisis de Varianza Molecular (AMOVA).El flujo genético (triphosphate deoxyribonucleotide), 1.5 units of Taq-DNA polymerase and 100 ng of genomic DNA as a template.The PCR conditions were 5 min at 95°C, 34 cycles of 94°C for 45 s, 62°C for 45s for annealing, 72°C for 45s and 72°C for 10 min for the final extension.Digestion was done with 15 µl of PCR product, 7 U of MspI enzyme and 2.0 µl of buffer in a final volume of 30 µl for 8 hours at 37°C.The amplified fragment contained two common restriction sites and one mutant (GGGAC) for the enzyme in positions 31, 153 and 75, respectively, generating three fragments (123, 95 and 31 bp) for allele A and four fragments (95, 79, 44 and 31 bp) for allele G.The digestion fragments were visualized using electrophoresis in agarose gel at 4%.
To genotype the SNP A-320T (GenBank number: rs43676359) a fragment of 970 bp was amplified with the primers F: 5'-AGTTCGACCGCATCCCTG-3' and R: 5'-AATTCATTTGTGCCAGCATC-3' (7).The final reaction volume was 25 µl, and 3.1 µl was used as buffer reaction 10X, 2.7 nM of MgCl 2 , 5 pmol of primers, 0.24 mM of dNTP, 1.5 units of Taq-DNA polymerase and 100 ng of genomic DNA as a template.The conditions of the PCR were 5 min at 95°C, 35 cycles of 94°C for 30 s, 58°C for 30 s for annealing, 72°C for 30 s and 72°C for 8 min for the final extension.Digestion was done with 12 µl of PCR product, 7 U of TaqI and 1.6 µl of buffer in a final volume 25 µl for 16 hours at 65°C.The amplified fragment contained four restriction sites (T´CG_A) for the enzyme, resulting in five fragments (446, 293, 140, 86 and 5 bp) for A allele.Allele T causes the loss of one of the restriction sites, generating four fragments (586, 293, 86 y 5 bp).The digestion fragments were visualized using electrophoresis in agarose gel at 3%.The allelic frequencies were determined using formulas y where N A and N a are the number of A and a alleles, in the population and N is the number of individuals.The genotype frequencies were determined with formulas , y where N AA, N Aa and N aa are the number of copies of genotypes AA, Aa and aa (11).
Genetic differentiation was determined comparing the observed heterozygosity (Ho) and the expected heterozygosity (He), calculated with GenAlex software (12).The Hardy-Weinberg state of equilibrium (HWE) was determined based on allelic and genotypic frequencies observed and expected using GenAlex software (12).
The population structure was determined using Wright's F statistics: , , where H o and H e are the observed and expected expresado como el número de migrantes (Nm) se determinó con la formula (13).
El desequilibrio de ligamiento se evaluó a partir del coeficiente desequilibrio de ligamiento el cual se calculó con el software GenAlex mediante la fórmula (11) donde f AB es la frecuencia de la combinación de alelos AB y f A y f B es la frecuencia alélica de A y B respectivamente; y mediante el coeficiente de correlación mediante pares de loci, con la fórmula (11).(H*MP) q es el efecto fijo de la interacción entre heterozygosity among subpopulations and H T is the total expected heterozygosity.These were obtained using GenAlex software with the Molecular Variance Analysis (AMOVA) method.
The genetic flow expressed as the number of migrants (Nm) was determined with the formula (13).
With the F ST values paired among subpopulations, the UPGMA tree was built using MEGA 6 software (14).
The linkage disequilibrium was evaluated using a linkage disequilibrium coefficient which was calculated with GenAlex software using the formula (11) where f AB is the frequency of the combination of AB alleles and f A and f B is the allelic frequency of A and B respectively; and using the correlation coefficient through sets of loci with the formula (11).(H*MP) q is the fixed effect of the interaction between herd and birth month; (AP*MP) r is the fixed effect of the interaction between birth month and year; (H*AP*MP) s is the fixed effect of the interaction between herd, birth year and month; A(INHA*FSHR) t is the random effect of the interaction of the nested genotypes with the animal and e ijklmnopqrst is the random error.In addition to these effects, the real production of milk (PREAL) and the length of lactation (DL) were included as covariates in the PP and FT models.For SCS, IEP and SPC, the PAJUS was also included as a covariate, and additionally for SPC the IEP was included, that corresponded to the analyzed lactation as a covariate.

RESULTS
Allelic frequencies and genotypes.The PCR product corresponding to a fragment of the exon 1 of INHA gene presented two common restriction sites and a mutating one for the MspI enzyme generating three or four fragments for the A and G allele, respectively (Figure 1).
The PCR product corresponded to a fragment of the 5'UTR region of the FSHR gene that contained four restriction sites for the TaqI enzyme when the A allele was present, and three restriction sites when the T allele was present, generating five or four fragments, respectively (Figure 2).
For INHA the allelic frequencies were 0.534 and 0.466 for A and G respectively.The genotype frequencies were 0.269, 0.529 and 0.201 for AA, AG and GG respectively.For FSHR, the frequency was 0.660 and 0.339 for the T and A allele respectively.The genotype frequencies found for AA, AT and TT were 0.096, 0.485 and 0.417 respectively (Table 2).
Hardy-Weinberg equilibrium.The total population did not present significant differences among observed (Ho=0.504±0.028)and expected (He=0.466±0.013)heterozygosity for both genes (Table 3).The majority of the subpopulations were found in HWE for both genes, except the subpopulations in Entrerrios and La Unión.The subpopulation in Entrerrios showed a significant reduction in the number of heterozygotes expected for the INHA polymorphism, while the subpopulation of La Unión had an excess number of expected heterozygotes for FSHR polymorphism.
Genetic structure.The F ST statistic for the total population was 0.059 (p=0.001)indicating a low differentiation between the subpopulations.The 0.285 (p=0.010)indicando un tendencia baja a la endogamia dentro de las subpoblaciones, en la población total también se observa una leve tendencia a la endogamia, representado F IS value obtained for the total population was 0.285 (p=0.010),indicating a slight tendency towards endogamy within the subpopulations, in the total population also seen a slight tendency

Genetic differentiation.
The paired F ST were between 0.006 and 0.174 (Table 4), presenting significant differences indicating degrees of differentiation between the subpopulations from low to medium.The studied subpopulations can be grouped in two branches; the subpopulations of Bello and Belmira are those that are farthest from the genetic tree, while the subpopulations of Entrerrios, La Unión, Medellín and San Pedro de los Milagros show less genetic differentiation (Figure 3).Linkage disequilibrium.For the analyzed Holstein population, the most frequent combination of genotypes was the double heterozygote AGAT with a frequency of 24.45%, while the less frequent combination of genotypes was the double homozygote AAAG with a frequency of 1.31% (Table 5).
The total population showed D=0.016 and r 2 =0.066 but these values were not significant (p>0.05),indicating that the presence of alleles in one locus is independent of the presence of alleles in another locus.
A pesar que en la población total se observa un mayor número de individuos heterocigotos que el esperado, esta diferencia no es significativa por lo cual la población total se encuentra en HWE.Solamente dos poblaciones, La Unión y Entrerrios, no se encontraron en HWE, la primera por un aumento significativo de los animales heterocigotos indicando una tendencia a la exogamia, y la segunda por una reducción significativa de los mismos mostrando una tendencia a la endogamia.Dentro de las poblaciones existen varias fuerzas que pueden generar desviaciones del HWE, como lo son el apareamiento selectivo, la endogamia, la estructura poblacional y la selección, pero son la endogamia y la estructura poblacional las principales razones que pueden generar Protein percentage.The PREAL had a significant effect on this trait (p<0.05), the other variables did not have a significant effect (p>0.05).

Fat percentage. None of the variables included
in the model presented a significant effect on the trait (p>0.05).

Association of INHA-FSHR genotypes with health traits. Somatic cells scores.
None of the variables included in the model presented a significant effect on this trait (p>0.05).

Association of INHA-FSHR genotypes with reproductive traits.
Calving interval.The AP*MP interaction had a significant effect on the trait (p<0.05).The other variables did not have a significant effect (p>0.05).

Services per conception.
None of the variables included in the model presented a significant effect on this trait (p>0.05).
The mean of each trait for each combination of genotypes is shown in table 6.

DISCUSSION
The A192G and A-320T polymorphisms have been studied in other populations, such as the Holstein China population (5,7,9).For cows of the Holstein China population, have reported that the A and G alleles of SNP A192G have a frequency of 0.55 y 0.45 respectively, similar to the frequencies found for the Antioquia Holstein population.In the Holstein cows of both populations, the most frequent genotype was AG, however for the population of Holstein China bulls the most common genotype was the AA (5,7).For the Holstein China bulls a frequency of 0.691 was reported for the A allele and 0.309 for the T allele (5,7).
Respecting the A-320T polymorphism, for the Holstein China population a frequency was found for the A and T alleles of 0.301 and 0.699 in females, while in males it was 0.321 and 0.679, respectively.For this population, the most common genotype is TT, in males and females, while in Antioquia is more common the heterozygous (7,9).
La estructura y diferenciación de la población Holstein del departamento de Antioquia también ha sido evaluada a partir de polimorfismos de otros genes como lactoferrina (LTF) y la hormona de crecimiento bovina (bGH).Con base en el gen LTF se encontró que la población de vacas Holstein se encontraba en HWE, y a nivel de subpoblación solo el municipio de San Pedro de los Milagros presentó desviaciones del HWE posiblemente debido a una intensa selección, ya que este es uno de los municipios In spite of the fact that in the total population a greater number of heterozygote individuals was found than was expected, this difference is not significant, since the total population is found in HWE.Only two populations, La Unión and Entrerrios, were not in HWE, the first due to a significant increase of heterozygote animals, indicating a tendency to exogamy, and the second due to a significant reduction of the same, showing a tendency towards endogamy.Within the populations there are several factors that can generate HWE deviations, such as selective pairing, endogamy, population structure and selection, but endogamy and population structure are the main factors that generate HWE deviations due to small differences in the allelic frequencies between subpopulations (11).However, for populations in La Unión and Entrerrios, it is expected that HWE deviations are due to selection processes on traits that can be associated with these polymorphisms, causing an indirect selection and therefore affecting the allelic and genotypic frequencies.
The endogamy coefficient of a subdivided population (F IT ) can be divided using Wright's F statistics between the component due to non-random pairing within subpopulations (F IS ) and the subdivision between populations (FST) (13).For the analyzed population, the F IT value obtained (0.328) reflects a low tendency to endogamy; in the same way, the F IS value obtained (0.285) indicates a slight tendency to homozygosity due to non-random pairing between individuals in each subpopulation.The F ST value for the total population (0.059) indicates that a moderate differentiation between the populations exists, which represents a reduction in the heterozygosity due to population subdivision.
The Wright F ST statistic measures the subdivision of the population and determines if differences exist in the allelic frequencies among the subpopulations.The populations that showed a greater degree of differentiation were Medellín and Belmira, while the populations in La Unión and San Pedro had less differentiation.This can be explained by the technical level implemented in each of these municipalities, with higher levels in the municipalities of Medellín, San Pedro and La Unión in comparison to Belmira, and therefore among the populations with a higher technical level; in terms of genetic improvement strategies that are implemented, mainly artificial insemination, the F ST values are less than 0.05, indicating little or no differentiation.Due to this differentiation, the subpopulations in Antioquia do not act like one population.más importantes para el sector lechero en el departamento (17).Esta investigación reporta para la población un F IS =-0.0717 indicando una tendencia a la exogamia.Al evaluar esta misma población con base en el gen bGH también se encontró en HWE.Aunque para la población total se reportó un F ST =0.0068, se encontró cierto grado de diferenciación entre las subpoblaciones, siendo el municipio de Marinilla el que presentaba la mayor diferenciación genética (4).
La media para producción de leche fue mayor que el valor reportado en otra investigación (4.482 L/ The structure and differentiation of the Holstein population in the department of Antioquia has also been evaluated using polymorphisms of different genes such as lactoferrine (LTF) and the bovine growth hormone (bGH).Based on the LTF gene, the Holstein cow population was found to have HWE, and at the subpopulation level only the municipality of San Pedro de los Milagros presented HWE deviations, possibly due to an intense selection, since this is one of the most important municipalities in the dairy sector in the department (17).This investigation reports for the population a F IS =-0.0717, indicating a tendency to exogamy.Upon evaluating this population based on the bGh gene, it was also found to be in HWE.Although the total population reported a F ST =0.0068, a certain degree of differentiation was found among the subpopulations, the municipality of Marinilla being the one that presented the greatest genetic differentiation (4).
Nm represents the absolute number of migrant organisms that enter each subpopulation in each generation.This flux of individuals causes a rapid decrease in the fixation index.Thus, migration exercises an important force against genetic divergence between subpopulations (18).For the population analyzed, a total of four migrants per generation was found.Although it is affirmed that if Nm>1 the subpopulations evolve as just one population, it is more correct to affirm that there is no population subdivision if Nm>10 (11).This could explain the genetic differentiation found among subpopulations in the department of Antioquia; although there is genetic flux between them, it has not been sufficient to homogenize the allelic frequencies, although it could be thought that the populations are in a process of homogenization.In other studies (4,19), the number of migrants that has been reported is much greater than what was found in this investigation.Based on a SNP, the growth hormone gene (bGH) was reported for the Holstein population in Antioquia at Nm=36 (4), while based on the SNP of the lactoferrine gene (LTF), Nm=25 has been reported for the same population (19).This high value could be due to implementing artificial insemination that eliminates geographic barriers and increases the genetic flux between populations (4,19).
The polymorphisms analyzed are found in linkage equilibrium, that is, the observed frequency to combine genotypes does not significantly differ from the expected frequency.In this way, it can be asserted that the presence of each allele for each gene analyzed is independent of the presence of any other.Since in bovines the INHA and FSHR genes are found in different lac ) para la población Holstein de Antioquia (20).Sin embargo en otros estudios han encontraron un valor mayor (7.155 L/lac) de producción de leche para esta población (21).El PP encontrado en esta investigación fue menor que el encontrado por otros investigadores (3.16%), por otro lado, la media encontrada para PG fue mayor que el valor reportado en la literatura (3.37%) (20).En cuanto a los parámetros reproductivos, los promedios de SPC e IEP fueron muy similares al promedio reportado en una investigación previa, donde se encontró una media de 1.67 y 453 días, respectivamente (22).
El menor IEP lo tuvieron los individuos AAAT mientras que el mayor fue para los GGAA, esta diferencia puede deberse a los alelos de INHA ya que el alelo A se asocia con un menor IEP mientras que los individuos GG presentan mayores valores para esta característica.El menor número de SPC fue para los individuos GGTT y el mayor para los AGAT, pero no se puede afirmar que alguno de los alelos analizados presente una tendencia a aumentar o disminuir el valor de esta característica.chromosomes, 2 and 11 respectively, these have a maximum recombinant rate, that is, 0.5, confirming their independent segregation according to the second law of Mendel (19).
The mean milk yield was greater than values reported in other investigations (4.482 L/lac ) for the Holstein Antioquia population (20).However, in other studies a greater value (7.155 L/lac) for milk production has been found for this population (21).The PP found in this investigation was less than that found by other investigators (3.16%), and on the other hand, the mean found for PG was greater than the value reported in literature (3.37%) (20).Regarding reproductive parameters, the SPC and IEP averages were very similar to the average reported in a previous investigation, where a mean of 1.67 and 453 days, respectively, was found (22).
The interaction of the studied polymorphisms did not present a significant effect on the majority of the traits that are of Zootechnical interest, however this interaction makes the A-320T polymorphism have a significant effect on the PAJUS, which had not been discovered when analyzing it independently (data not shown).For A-320T polymorphism, the AA individuals evidenced a greater productive level (5.902 L/ lac), with 193 and 381 L/lac more than the AT and TT individuals respectively.The AA individuals also showed a lesser IEP value in comparison with the other genotypes (403 days) (data not shown).Since AA individuals present greater reproductive efficiency, reflected in a lower IEP, it would be expected that these individuals present shorter lactation periods, which when they adjusting to 305 days show improvement and a greater production level is obtained.When analyzing the combination of alleles for these genes, the GGAA and AAAA individuals are those that present the greatest productive levels, confirming that the A allele for FSHR is associated with greater production.Regarding the milk solid content, the AGTT individuals present the greatest percentages of fat and protein.In parallel studies that were done, it was found that the G allele in INHA is associated with greater PG, while the FSHR T allele is related to greater PG and PP, since the FSHR T allele is also related to a greater volume of milk, causing a minor dilution effect on milk solids.
A lower IEP value was found in AAAT individuals, while a greater value was found in GGAA, a difference that could be due to INHA alleles, since the A allele is associated with a lesser IEP while GG individuals have greater values for this trait.The lesser SPC number was for GGTT individuals and the greater one for AGAT, but it cannot be Con base en esta información se puede concluir que la selección basada en el fenotipo que se realiza en la población de vacas Holstein de Antioquia no ha sido lo suficientemente fuerte para generar desviaciones del equilibrio de Hardy-Weinberg ni ha logrado disminuir de manera notable la frecuencia de ninguno de los alelos de los polimorfismos estudiados, por lo cual existe gran diversidad entre las poblaciones.Esta diversidad genética es de gran importancia ya que permite a los individuos responder de forma más adecuada a diversos retos ambientales, y además es el punto de partida para realizar los programas de mejoramiento genético que buscan aumentar la eficiencia productiva y reproductiva en los sistemas de producción animal.Son necesarios más estudios para determinar si estos polimorfismos pueden ser útiles en los programas de selección asistida por marcadores moleculares y para estos se recomienda realizar un mejor control de los factores ambientales con el fin de evidenciar más fácilmente el efecto que estos polimorfismos puedan tener sobre las características zootécnicas evaluadas.

REFERENCES
stated that one of the analyzed alleles presents a tendency to increase or decrease the value of this trait.
Based on this information, we can conclude that selection based on the phenotype in the population of Holstein Antioquia cows has not been sufficiently strong to generate Hardy-Weinberg equilibrium deviations or diminished in any notable way the frequency of any alleles for the studied polymorphisms, and therefore a great diversity exists among the populations.This genetic diversity greatly important, since individuals can respond adequately to diverse environmental challenges, and additionally it is the starting point to begin genetic improvement programs that seek to increase productive and reproductive efficiency in animal production systems.More studies are needed to determine if these polymorphisms can be useful in molecular marker selection programs and it is recommended that better control over environmental factors be exercised in order to make it easier to detect the effect these polymorphisms could have on the evaluated zootechnical traits.

Figure 1 .
Figure 1.Amplified fragment (249 bp) of the inhibin alpha gene (lines 3 to 5) and the restriction pattern after digesting the MspI enzyme for each genotype (AA lines 7 and 8, GG lines 9 and 10, AG lines 11 and 12); lines 1 and 6 correspond to the molecular weight marker and line 2 corresponds to the negative control (PCR without DNA template).

Figure 2 .
Figure 2. Amplified fragment (970 bp) of follicle stimulating hormone receptor gene (lines 2 to 4) and restriction pattern after digestion of TaqI for each genotype (TT lines 6 and 7, AT line 8 and AA line 9); line 1 corresponds to the molecular weight marker and line 5 to the negative control (PCR without DNA sample).

Figure 3 .
Figure 3. Dendrogram based on the F ST paired for six subpopulations of Holstein cows in the department of Antioquia.

Table 1 .
Distribution of the animals studied by municipality and herd (N), and number of lactations analyzed by herd (N Lac).

Table 2 .
Allelic and genotypic frequencies for inhibin alpha and follicle stimulating hormone receptor genes in a Holstein population belonging to diverse municipalities in the department of Antioquia.Belmira son las más alejadas en el árbol genético, mientras que entre las subpoblaciones de Entrerrios, La Unión, Medellín y San Pedro de los Milagros se presenta menor diferenciación genética (Figura 3).

Table 3 .
Observed heterozygosity (Ho), expected heterozygosity (He) and P value of the Chisquared test for some Holstein populations in the department of Antioquia.

Table 4 .
Statistic for F ST paired among Holstein populations in the department of Antioquia.

Table 5 .
Tukey measurements for the production of milk adjusted (PAJUS), protein percentage (PP), fat percentage (PG), somatic cells score (SCS), calving interval (IEP) and services per conception (SPC) according to the combination of genotypes of inhibin alpha and follicle stimulating hormone receptor for Antioquia Holstein cows.