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Comparison of Genetic Diversity between the Ex-Situ Conservation Herd and Smallholders of Turkish Grey Cattle.

Byline: Sleyman Kk


Turkish grey cattle (TGC) are facing the danger of extinction. Intensive breeding program has mainly been used for purebred TGC that are under the conservation of Bandirma Livestock Research Institute (BLRE) ex-situ program. This study aims to compare 3 SNP of the genetic features of purebred TGC that are under the conservation of ex-situ (51 cattle) program with the ones raised by the smallholders (79 cattle) in the villages. According to the estimated average heterozygosity values for ex-situ breeding program and smallholders in the villages, the difference between the cattle were found meaningful (P<0.05). The observed average heterozygosity (Ho) value was calculated as 0.40770.1922, while the expected heterozygosity (He) value was found 0.39090.1663.

The research findings show that the difference between the two TGC groups in terms of Calpastatin gene (CAST) loci (P<0.01) and Calpain gene (CAPN1) loci (P<0.05), gene assortment was found to be meaningful and these are incompatible along with Hardy-Weinberg theory. In addition, significance check was done for the expected heterozygosity results for the two TGC groups (BLRE ex-situ and the smallholders in the villages). For the Fis value, the difference was significant for CAPN1 316 loci (P<0.001) and CAST loci (P0.05) which did not display a significant difference. The Fis inbreeding coefficients being negative in the sample populations for CAST loci imply heterogeneity in CAST loci (PG), Polymerase Chain Reaction-Restriksiyon Fragment Length Polymorphism (PCR-RFLP) method was used based on the work of Schenkel et al. (2006). Identification of Calpain gene CAPN1 316 SNP (GenBank Accession No. AF_252504.2:g.5709C>G) with PCR-RFLP method and CAPN1 4751 SNP (GenBank Accession No. AF_248054.2:g.6545C>T) genotype was identified by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) method was used based on the work of Rinco'n and Medrano (2006). The primers (Sentegen Biotech, Ankara / Turkey) used and fragment sizes that were reproduced (Table I) by Bioneer My Genie 96 Thermal Block PCR device (Bioneer Corporation, South Korea).

For identification of CAST SNP and CAPN 316 SNP genotypes were worked with PCR-RFLP method. CAST PCR products were amplified 523 bp fragments. The PCR products of CAST gene were cut with RsaI (3 U) restriction endonuclease enzyme (REE) (New England Biolabs).

Table I.- The PCR method used, primer sequences and amplification products.

Marker###Sequences of the primers (5' - 3')###bp***









CAPN 316 SNP fragment of 446 bp amplified using the PCR protocol was incubated with BtgI (3 U) REE (New England Biolabs) for 4 h at 37 degC. CAPN 316 SNP fragment sizes and genotypes obtained are shown in Figure 1.

All PCR reactions were performed EmeraldAmp GT PCR master mix (Takara, Japan). Amplification of DNA was run in the 3 % horizontal gel electrophoresis by "Thermo Scientific electrophoresis and power supply" and then was used for the genotyping by "DNR BioImaging Systems Minibis Pro. Jerusalem, Israel" and software was used for molecular analysis (Image Aide from Spectronics Corporation). The characteristics of TGC animal samples and genotyping methods have been used by Kk et al. (2017).

Statistical analyses

Two subgroups were formed, one for the cattle raised in villages and the other one for the in ex-situ conservation herds within the total TGC sample. The groups were examined with regard to the population genetics considering the environmental interaction. For each loci, the Observed (Ho) and the Expected (He) Heterozygosity in the total sample population and the two TGC groups (Bandirma ex-situ and the smallholders in the villages) were calculated with Levene (1949) unbiasedly according to Nei (1987) and the Fixation Index (Fis) values were calculated according to Wright (1965) and the effective allele number (ne) values were calculated according to Kimura and Crow (1978) (Tables II, III, VI).


Where, xi is ith allele freqency, m is Number of alleles, He is the expected heterozygosity ratio in a sample population at Hardy Weinberg equilibrium and Ho is number of observed heterozygotes / number of total sample.

ne = 1/Sxi2

Where, ne is Number of effective alleles per loci and xi is the average allele density at the ith loci.


Where, f(Ai) is the ith allele density, n is Number of individuals in the population, Nii and Nij are the number of Aii and Aij genotypes in the population and m is to represents the number of alleles

Where, Hoj is the rate of heterozygosity observed in jth population, Hej is the expected heterozygosity of the population j and s is for population number.

The significance control for the difference between the estimated average heterozygosity indices for the two TGC groups was held with respect to probability a in t test table. kh2 test (Degree of Freedom = Allele Number - 1) was used for the significant control of the inbreeding coefficient (Fis) was estimated in each loci and whether the populations show Hardy-Weinberg (HW) equilibrium was discussed. The evaluations were made using Popgene32 version 1.31 program (Yeh et al., 2000).


Breeding by dividing populations into local groups helps remove the probability of aleatory copulations and reproduction within a herd. For that reason, homozygosity is more likely to be present in the next generation. BLRE ex-situ conservation herd is a closed herd made up of purebred TGC collected from different villages of the various cities in Marmara Region. This herd is expected to be homozygous. Therefore, this herd was regarded as a subgroup and compared to the Grey cattles in the smallholders in the villages.

In order to determine the effect of environmental interaction on BLRE ex-situ conservation herd and the samples taken from smallholders, genotypic results of the two sample groups were statistically evaluated. In both of the sample the herds of TGC under ex-situ conservation and the smallholders in the villages, in order to eliminate the errors in He value, which is the statistical measurement of genetic variation in all three loci, according to Nei (1987) was estimated to be unbiased.

The Fis value varies between 0 and 1 and if it is negative it shows excess for heterozygosity. If it is close to zero, it implies the presence of Hardy-Weinberg equilibrium and if it is positive, it means excessive homozygosity. Homozygosity index (fixation index) is also defined as the positive deviation of the expected heterozygosity in Hardy-Weinberg rates in a population where inbreeding is applied. Negative Fis value is also defined as the heterogeneity rate which is occurring to the result of remote inbreeding in a certain loci (Yeh et al., 2000). Fis value gives us the percentage of which the heterozygous individuals fall below or above the normal amount in a population. The Fis inbreeding coefficient results according to chi-square (kh2) test values presented in Tables II, III and IV are discussed below.

With respect to the inbreeding coefficients computed in the three polymorphic loci in Grey cattle, the differences in CAST (P0.05) and balanced, the difference for CAPN1 316 loci in BLRE ex-situ population was found significant (P<0.05) and non-balanced. The inbreeding coefficients calculated for other populations were not statistically meaningful.

According to Savasi and Atasoy (2016) the Fis value, which was also the indicator of the mean heterozygosity excess determined for TGC, was determined as 5.7% (-0.057) and the population was not-balanced. According to zsensoy et al. (2010) and Altinalan (2005) for TGC the Fis value 0.05524 and 0.11930, respectively, these values are insignificant and the populations are balanced.

Table II.- Results for the effective allele number (ne), the observed (Ho) and the expected (He) heterozygosity indices and the fixation index (Fis) tested in CAST and CAPN1 loci in 79 TGC samples in the smallholders in the villages.

The loci###ne###1Ho###1He###He2###3Fis

CAST###1.9920 0.6329 0.5012 0.4980 -0.2709***

CAPN1 4751###1.8906 0.4557 0.4741 0.4711###0.0327ns

CAPN1 316###1.3311 0.2405 0.2503 0.2488###0.0332ns

Average###1.7379 0.4430 0.4085 0.4059

St. Deviation###0.3559 0.1965 0.1377 0.1368

According to HW equilibrium, while there was upward trend of heterozygous cattle (hyper-heterozygosity ) at 27.09 % in CAST loci (P 0.05) in TGC in the smallholders (Table II).

Table III.- Results for the effective allele number (ne), the observed (Ho) and the expected (He) heterozygosity indices and the fixation index (Fis) tested in CAST and CAPN1 loci in 51 TGC samples in BLRE ex-situ conservation herd.

The loci###ne###1Ho###He1###2He###3Fis

CAST###1.6862 0.4510 0.4110 0.4070 - 0.1082**

CAPN1 4751###1.9969 0.4902 0.5042 0.4992###0.0181ns

CAPN1 316###1.1245 0.1176 0.1118 0.1107 - 0.0625*

Average###1.6026 0.3529 0.3423 0.3390

St. Deviation###0.4422 0.2047 0.2050 0.2030

In the samples TGC in BLRE ex-situ group, according to HW equilibrium, while there were hyper-heterozygosity at 10.82 % and 6.25 % in CAST and CAPN1 316 loci, respectively, there was hypo-heterozygosity at 1.81 % CAPN1 4751. The expected deviations in the HW equilibrium in CAST loci (P<0.01) and CAPN1 316 loci (P<0.05) in BLRE ex-situ conservation herd was found significant (Table III).

In the total TGC sample, the percentage of hyper-heterozygosity was 17.37 % in terms of CAST loci and this difference was significant (P0.05). Therefore, the expected homozygosity in the total TGC sample population for CAST, CAPN1 4751 and CAPN1 316 loci are 51.97 %, 50.67 % and 80.10 %, respectively. The most homogenous genes in the cattle were found in CAPN1 316 loci (Table IV).

Without the effect of sampling error, the average He values in order to estimate the genetic variation in the populations were calculated as 0.40590.1368 for the population that is made up of the cattle of the smallholders, 0.33900.2030 for BLRE ex-situ population and 0.38940.1657 for the total TGC sample population (Tables II, III, IV ).

Some researchers like zbeyaz et al. (1999), Altinalan (2005), zkan (2005), Kurar et al. (2011) and Savasi and Atasoy (2016) reported the Ho average value in TGC they studied as 0.4110.140, 0.433, 0.6823, 0.686 and 0.2788, respectively. Sharma et al. (2009) remarked that it varied between 0.700.10 and 0.650.14 in the 4 different Hind cattle race they examined and that the inadequacy of heterozygous cattle in the populations (Fis) was between 17.7 % and 28.8 %.

Table IV.- Results for the effective allele number (ne), the observed (Ho) and the expected (He) heterozygosity indices and the fixation index (Fis) tested in CAST and CAPN1 loci in the total TGC sample (n = 130).

The loci###ne###1Ho###1He###2He###3Fis

CAST###1.9173 0.5615 0.4803 0.4784 - 0.1737***

CAPN1 4751###1.9664 0.4692 0.4933 0.4914###0.0452ns

CAPN1 316###1.2472 0.1923 0.1990 0.1982###0.0297ns

Average###1.7103 0.4077 0.3909 0.3894

When earlier the Turkish researchers tested the genetic differentiation in TGC population are compared to with the Ho average values (0.40770.1922) specified in my study, the values obtained in this study seems to be higher than the ones obtained by Savasi and Atasoy (2016), similar to the ones in zbeyaz et al. (1999) study while they seem to be lower than the ones obtained in some other research. The cattle samples studied by Altinalan (2005), zkan (2005), Kurar et al. (2011), zsensoy and Kurar (2014) were also more heterogeneity.

The effective allele number and allele density are other criteria to show the spatial heterogeneity in a population. The average effective allele number per loci for TGC samples from the smallholders in the villages, BLRE TGC ex-situ conservation herd and the total population sample were calculated as 1.73790.3559, 1.60260.4422 and 1.71030.4018, respectively. CAPN1 316 loci (1.1245) in BLRE TGC ex-situ conservation herd seems to have the lowest effective allele number while CAST loci (1.992) in TGC in the villages was found to have the highest effective allele number. This shows that there is a linear relationship between Ho and ne. According to the results, it can be said that the herd under BLRE ex-situ conservation program is made up of more homogeneity cattle in terms of CAPN1 316 loci while TGC in the smallholders are more heterogeneity regarding CAST loci.

The significance control for the difference between the estimated average heterozygosity indices for the two subgroups (BLRE ex-situ and the smallholders) was held in t test table with respect to the probability [alpha]. While the expected heterozygosity difference between the two TGC subgroups for CAPN1 316 loci (P<0.001) and CAST loci (P 0.05).


Tables II, III and IV shows a linear relationship between the He values and ne. As is known, as ne in polymorphic loci draws closer to two, it is more likely to see heterogeneity in those loci. The more polymorphic the loci in a species or race are, the more the heterozygosity value will increase. In the total sample, it was found that the Ho (0.5615) and the He (0.4933) values were highest in CAST loci likewise ne value (1.9664) was highest in CAPN1 4751 loci (Table IV). When we look at the average ne, Ho and He values (Tables II and III) in subgroups, it was found that the sample TGC subgroups in the smallholders had the highest ne average (1.73790.3559), the highest He (0.40850.1377) and Ho (0.44300.1965) values. The difference between the average Ho value and the average He value in the total sample and BLRE ex-situ conservation herd being small implies the risk of increasing consanguinity and the beginning of a decrease in the genetic variation (Tables II and IV).

The low He average heterozygosity values of BLRE TGC ex-situ conservation herd can be attributed to the fact that are more closely related to the breeding system. Also the ne supports the increase in the homozygosity. Since the size of the active cattle population was small for breeding and the uncontrolled or faulty breeding programs could cause decrease in genetic variation in BLRE TGC ex-situ conservation herd. The excess in heterozygosity in CAST gene loci (27.09%) in the village group proves that the TGC in the villages are without any selection.

It can be said that there is a risk of decrease in the genetic variation in ex-situ conservation herd which is the result of the increasing consanguinity scaling up the homozygosis. Diminishing productivity, ability to transform feed into meat, and resistance to environmental conditions and illness, and increasing number of deformations are some of the most important negative effects of decreasing genetic variation as a result of increasing homozygosis (Karahan et al., 2007). Therefore, it is important to understand the genetic structures of animal populations to be able to protect the genotypic structure of the races that face the danger of becoming extinct.

According to the data obtained in this study, it can be suggested that cattle breeding herd from different herd be integrated into the herd that are under the ex-situ conservation program and the number of the cattle in the herd be increased. In this case, in this case it is thought that the sustainability of genetic variation will can provide.


This investigation has been supported by the project TUBAP-2013-109 accepted by Commission of Scientific Research Projects of Trakya University in Turkey.

Statement of conflict of interest

Authors have declared no conflict of interest.


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Author:Kk, Sleyman
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Aug 31, 2017
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