УДК 636.4:636.082.4:575.174.015.3


N.A. Zinovieva1, V.R. Harzinova1, 2, T.I. Logvinova1, 2, E.A. Gladyr’1, 2, E.I. Sizareva3, Yu.I. Chinarov2

The approaches of using of microsatellites for confirmation of breed purity and evaluation of heterogeneity degree of parents’ pairs were developed. 100 per cent of breeding pigs of large white (Yorkshire), landrace and duroc breeds were correctly assigned to own breed based on microsatellites’ profiles analysis. Blood groups application made it possible to differentiate 91.7, 66.7 and 8.3 per cent animals of duroc, large white and landrace breeds, respectively. AFLP markers were not suitable to perform the pig breed assignment. The increasing in levels of productive traits (the per cent of piglets born alive, the number of piglets at 21 day and at weaning, the weight of one piglet at birth, at 21 day and at weaning, and the little weight at 21 day and at weaning) by decreasing of their variability with the increasing of genetic likeness evaluated using microsatellites was shown.

Keywords: genetic markers, microsatellites, pure breeding, pig breeds, heterogeneity of parents’ pairs.


The progress of genomic research provides techniques and algorithms allowing to use the molecular genetic information in animal breeding. One of the topical problems of pig breeding is developing the techniques for control of animals’ breed purity. Modern technologies of industrial pork production are based on obtaining the heterosis effect by crossing specified breeds (1). The best heterosis for reproductive qualities can be formed by crossing pure lines (2). At the same time, it requires new methods for assessing heterogeneity of selected parental pairs to ensure a stable transfer of productive properties to offspring while reducing its diversity.
In this regard, one of promising techniques is using microsatellites – tandemly located short non-coding repetitive DNA sequences, evenly spaced throughout the genome (3). Microsatellites are highly polymorphic structures inherited on Mendelian type, which makes them the perfect DNA markers in mammals (4, 5). The applied importance of microsatellites has been proved by population-genetic studies, pedigree tests, assessing the degree of heterozygosity, as well as using them as markers of the gene pool and its changes over time and generations along with revealing genetic distinctions of various breeds, types and lines (6-8). The study performed on Girolando cows obtained by crosses of dairy Gir (bred by crossing Zebu with Taurine breed) with Holstein Black-and-White cattle has shown a high significant correlation between the ancestors’ contribution and microsatellite profiles (r = 0,84, p <0,0001), which suggests microsatellites for establishing the contributions of source breeds in certain individuals (9).
The purpose of this work was to develop an algorithm for using microsatellites as the evaluation criteria of breed purity and heterogeneity of parent pairs in pig breeding, along with studying the effects of different heterogeneity degrees calculated upon microsatellite profiles on commercially valuable properties of pigs.
Technique. The objects of study were samples of blood (anticoagulant - sodium citrate) and tissue (ear notches, preservative -96% ethanol) of pedigree boars of different breeds from pig breeding enterprises of Russia. Informativity of the microsatellite test-system revealing animals’ breed purity was studied in comparison with blood groups and AFLP-markers (amplified fragment length polymorphism) on pigs the breeds Large White (n = 12), Landrace (n = 12) and Duroc (n = 12). Subsequent experimental testing was carried out on Large White (n = 82), Landrace (n = 59) and Duroc (n = 60) pigs from three independent enterprises. Using microsatellites as the criteria of heterogeneity of parental pairs was assessed on boars and sows the breeds Yorkshire (n = 16) and Duroc (n = 23) (JSC "Trosnyansky bekon," Orel province).
Microsatellite analysis was performed on 12 loci (S0155, S0355, S0386, SW24, SW72, SW951, S0101, SW240, SW857, S0228, SW911, and SW936), using the developed test system for DNA-testing of pigs. Blood groups were analyzed for 20 erythrocyte antigens of 10 systems (A, B, D, E, F, G, H, K, L and M) using conventional techniques. Determination of population-genetic parameters was carried out using the profiles for 16 erythrocyte antigens of 6 closed systems (B, D, E, F, G and L); the degree of heterogeneity of selected parental pairs was evaluated upon the complete analysis of 20 antigens. AFLP-markers were analyzed for three types of polymorphism (E33/T47, E46/T48 and 33/T62) using own developed techniques. The data on each animal’s alleles were summarized in Microsoft Excel table; the resulting matrix of genotypes was used in statistical analysis of results.
Processing of data was performed according to B. Weir (10). Variability at individual, intrabreed and interbreed levels were assessed by AMOVA (analysis of molecular variance) using the software GenAlEx (v. 6.4). Individuals’ breed purity was determined using the method of J.K. Pritchard et al. (11) and the software Structure (v. 2.3.1). The analysis was performed considering the most probable number of populations (k = 3 or k = 2) without introducing the prior data about individuals’ breed. The threshold value of breed identity was established at 75% level of exclusion (Q criterion).
Genetic similarity of selected parental pairs was assessed by Rst parameter calculated using AMOVA function for pairwise comparisons (smaller Rst values indicate greater genetic similarity of parents). Variability of productive traits was evaluated by coefficient of variation (Cv).
Results. Genetic diversity in three breeds of pigs was assessed by fixation index (Rst - for microsatellites, Fst - for blood groups, PhiPT - for AFLP-markers); the obtained results were calculated using AMOVA function (Table 1).

1. Molecular variance (AMOVA) assessed upon microsatellites, blood groups and AFLP-markers in a group of three pig breeds (Large White, Landrace and Duroc).  


Value of molecular variance


blood groups


Criterion of evaluation




Between populations




Within populations, including:




between individuals




within individuals




Note. AFLP — amplified fragments length polymorphism; empty cells – for the dominant type of markers, i.e. AFLP-markers, the corresponding parameters weren’t calculated

As it shows Table 1, the largest interpopulation differences between the studied groups of pigs was observed for blood groups, which apparently reflects the relationship between productivity parameters of pigs and some alleles for blood groups (12).


Breed identity of pedigree boars assessed using different markers: A – microsatellites (12 polyalle loci), B – blood groups (8 diallel loci), C – AFLP-markers (amplified fragments lengths polymorphism, 465 binary loci); a, b and c – respectively, Large White, Landrace and Duroc breeds of pigs.  
abscissa – Tested boars (n = 12)                   from left to right – Large White, Landrace, Duroc,
 ordinate – Breed identity criterion (Q)              А Б В а б в – A B C a b c

Significantly lower values of inter-population variability (2,2% for microsatellites and 1,1% for AFLP-markers) (Table 1) suggest selective neutrality of these DNA markers. The analysis showed that intrapopulation variability of microsatellites is primarily formed by differences between individuals (86,2% vs. 11,6% variation within individuals), while intrapopulation variability of blood groups is mainly provided by differences within individuals (64,1 % vs. 8,6% variation between individuals).
Suitability of various genetic markers for determining breed identity of pigs was compared. Only microsatellites were found to provide correct identification of individuals’ identity to own breed based upon to pedigree account records at the level of exclusion Q equal to 0,75 (Figure).
The analysis of DNA microsatellite profiles allowed to classify 100% Large White, Landrace and Duroc boars as members of different populations, while the value of Q criterion in certain individual ranged from 0,974 to 0,992 in Large White breed, from 0,956 to 0,991 - in Landrace and from 0,978 to 0,991 - in Duroc; average values of Q criterion in these breeds were, respectively, 0,984 ± 0,002; 0,976 ± 0,004 and 0,985 ± 0,002. In the variant with the markers of blood groups, correct determination of individuals’ breed identity at a given level of exclusion was achieved in 91,7% pigs of Duroc, in 66,7% individuals of the Large White breed, and only in 8,3% cases - in the Landrace breed. The values of Q criterion in these breeds varied, respectively, from 0,740 to 0,976; from 0,040 to 0,957 and from 0,065 to 0,951. The analysis of AFLP-profiles didn’t reveal any patterns of individuals’ breed identity.
The test-system based on microsatellites was experimentally approbated in order to confirm breed identity of pigs.  Breed identity of Large White, Landrace and Duroc individuals was confirmed in 100% cases (Table 2). Q criterion values ranged from 0,871 to 0,996 and averaged 0,981 ± 0,004 for pigs of the Large White breed, 0,981 ± 0,003 - for Landrace and 0,986 ± 0,002 - for Duroc.

2. Breed identity of pedigree pigs from three populations assesed using microsatellites


The value of Q criterion in investigated pig breeds

Lagre White



Population 1 (k = 3)

n = 16

n = 12

n = 30









Population 2 (k = 3)

n = 26

n = 14

n = 30









Population 3 (k = 2)

n = 40

n = 33








n = 82

n = 59

n = 60





Q (M±m)




Note. Q — criterion of individual’s identity to a particular population calculated according to B. Weir (10) for the most probable number of populations (k). Dashes mean that Population 3 was represented by animals of only two breeds.

The relationship between reproductive traits of pigs and the degree of parents’ genetic similarity calculated upon microsatellites (using index of fixation Rst, AMOVA, at pairwise comparison) was analyzed (Table 3). The increase of this parameter (reduce in Rst) was reliably correlated with increase in proportion of live piglets at birth, number of piglets on the 21st day and at weaning, conservation of piglets on the 21st day and at weaning, the weight of one piglet at birth, on the 21st day and at weaning, milk production of sows and with weight of a litter at weaning. The degree of parents’ genetic similarity was negatively correlated with number of piglets at birth thereby reflecting a suppression of multiple fetation at higher genetic similarity of parents. Apparently, embryos formed by heterogeneous gametes show higher viability and survival rate than ones resulting homogeneous crosses (individual heterosis).

3. Reliable correlations between the degree of genetic similarity of parents assessed by microsatellites and productivity parameters in Yorkshire and Duroc pigs


Correlation coefficient r



Multiple fetation 



Number of piglets born alive



Proportion of piglets born alive



Weight of a litter at birth



Weight of one piglet at birth



Number of piglets on the 21st day



Conservation by the 21st day



Milk production



Weight of one piglet on the 21st day



Number of  piglets at weaning



Conservation at weaning



Weight of a litter at weaning



Weight of one piglet at weaning



Note. Empty cells – data not shown as not reliable.
*, ** and *** respectively p < 0,005; p < 0,002 and p < 0,001.

Increased genetic similarity of parents significantly reduced variability of productivity parameters (Table 4), which fact was manifested in pigs regardless of breed.

4. Reliable correlations between the degree of parents’ genetic similarity assessed by microsatellites and diversity of productivity parameters in Yorkshire and Duroc pigs


Correlation coefficient r



Multiple fetation



Number of piglets live at birth



Weight of a litter at birth



Number of piglets on the 21st day



Milk production



Number of  piglets at weaning



Weight of a litter at weaning



Note. Empty cells – data not shown as not reliable.
* and ** respectively, p < 0,005 and p < 0,001.

Results of this research confirm suitability of the own developed test system based on microsatellites for determining breed identity of pigs. Analysis of microsatellite profiles allowed correct differentiation of main breeds of pigs used in the hybridization (Large White or Yorkshire, Landrace and Duroc), while markers of blood groups provided correct differentiation of these breeds in, respectively, 66,7; 8,3 and 91,7% of cases. AFLP markers were found to be unsuitable for this purpose. Apparently, microsatellites are best suitable for determining breed identity owing to their higher level of polymorphism than in markers of blood group (total number of alleles per locus  - 4,64 ± 0,24 vs. 1,83 ± 0,12, including effective alleles per locus - 3,11 ± 0,18 vs. 1,39 ± 0,07) along with selection neutrality of most microsatellites (excluding those linked to the loci of economically useful traits) (13). Some alleles for blood groups are linked with productivity traits, which results in selection in favor of certain alleles and possibly leads to lower differentiation of pig breeds for blood groups (14, 15). This assumption was supported by the authors’ findings: the inter-population diversity for blood groups was larger than that for microsatellites (27,3 versus 2,2% of total diversity), along with the higher similarity of genetic profiles of the abovementioned breeds for blood groups than for microsatellite (6).
Modern livestock breeding pays more attention to selection factors based on heredity laws; creation of systems for line-group selection is of the particular importance (16). This study allows assessing heterogeneity of group selection of pigs using the optimal criteria based on microsatellite genotypes of animals. At the purebred breeding of two studied pig breeds (Yorkshire and Duroc) the increase in genetic similarity of parents assessed by Rst (AMOVA) was found to result in increased productivity parameters (proportion of live piglets at birth, number of piglets at weaning, conservation on the 21st day and at weaning, the weight of one pig at birth, on the 21st day and at weaning, milk production of sows, weight of a litter at weaning) while reducing their diversity. The obtained data are consistent with laws of classical genetics and livestock breeding postulating that homogeneous selection is aimed at fixing the parents’ productivity traits in offspring (16), so it can be concluded that microsatellite profiles can be used at purebred breeding pigs as a proper criteria for assessing the degree of genetic heterogeneity of parental pairs.
At the same time, the abovementioned facts should be carefully applied to other breeds, types, or even flocks. Although these findings are scientifically sound and experimentally proven, they require confirmation in larger studies on other breeds and populations.
Thus, microsatellite profiles can be suggested as a relevant criterion for assessing breed identity of swine and serve as a matrix for determining the optimal degree of heterogeneity of parent pairs providing increased level of manifestation of economically valuable traits while reducing their diversity.


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1All-Russia Research and Development Institute for Livestock Husbandry, RAAS,
Moscow province, Podolsk region, Dubrovitsy 142132, Russia,
e-mail: n_zinovieva@mail.ru;
2“Biostrim“ Ltd., Moscow province, Podolsk region, Dubrovitsy 142132, Russia;
3“ Selection-Hybridization Center Znamensky“ Ltd., Orel province, Orel 302030, Russia

Received  July 18, 2011