Pedigree and molecular data were used to evaluate genetic diversity in the Polish populations of the Polish primitive horse (also known as Polish Konik) and Hucul breeds over the time period of 30 years (1980–2011). Based on genotypes in 12 microsatellite loci (for 3865 Polish primitive horses and 1627 Huculs), as well as on pedigree data derived from over 7000 individuals (both breeds), several indices describing structure of the analysed populations were estimated. For both analysed breeds, we observed an increasing trend of inbreeding since 1980 which seems to be much more stable (oscillating around 10 % in the Polish primitive horse and 5 % in Hucul) since the beginning of 2000s when they were included in conservation programs in Poland. We observed that generally, indices related to genetic diversity are higher in the Hucul breed. Our study indicated that genetic diversity in the Polish primitive horse and Hucul breeds in Poland is still relatively high and conservation programs should be continued to keep it on the “safe” level in the future.
Preservation of endangered species is one of the most important goals for
the present biological sciences, especially in the context of natural
ecosystems stability. In the case of domestic animals, conservation programs
are usually initiated for breeds which present a unique genetic and
phenotypic value. It is well known that the traditional animal breeding
programs are mainly based on strong selection to improve important
phenotypic traits. Naturally, the side effect of such an approach is
depletion of the gene pool represented by a given species/breed. Thus,
conservation programs are needed to preserve breeds in which a significant
part of given species' genetic diversity is still present
(
In Poland, there are six different horse breeds managed with conservation programs: Polish primitive horse (Polish Konik), Hucul, Silesian, Wielkopolska, Malopolska and Polish Heavy horse (Sztumski and Sokolski types). Among them, only two can be considered as pure breeds (Polish primitive horse and Hucul) because their studbooks have been closed since the 1980s and currently no outside blood is accepted. Thus, in these two breeds monitoring of genetic diversity seems to be particularly important to avoid the potential negative effects of inbreeding.
Both breeds included in this study are primitive type horses which
putatively maintained many features of the wild Tarpan (
The aim of this study was to estimate genetic diversity of the Polish populations of the two endangered horse breeds (Polish primitive horse and Hucul) using pedigree and molecular data.
Pedigree analysis was conducted for all Hucul and Polish primitive horses recorded in the studbooks between 1980 and 2011. Pedigree data, received from the Polish Horse Breeders Association, was screened for errors and inconsistencies. Doubtful records were removed from the analysis. Additionally, genetic diversity assessment in both breeds was performed using 12 microsatellite markers (AHT4, AHT5, ASB2, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG1 and VHL20). All of the genotypes were derived from the Horse Genetic Markers Laboratory's database (Poznan University of Life Sciences, Poland), from the routine parentage control. Microsatellite markers were genotyped without the use of commercial kits, according to the procedure described previously by Iwańczyk et al. (2006). A detailed structure of the animal groups used in the present study is shown in Table 1.
Genealogical analysis was initially performed for the whole population of
the Polish primitive horse and Huculs breed in Poland. Subsequently, in order
to characterise the present genetic diversity of the breeds, a reference
population was created for each breed containing individuals born between
2002 and 2011. Pedigree completeness in the analysed breeds was assessed
with complete generations equivalent. The number of equivalent generations
traced was computed as the sum over all known ancestors of the terms
(1/2)
This leads to the following formula:
Animal groups.
Effective population size (“realized”
Effective number of founders (Lacy, 1989) was calculated as
Additionally, effective number of ancestors (Boichard et al., 1997), which
accounts for possible bottlenecks, was calculated as
The amount of genetic diversity (GD) in the population taking into account
the loss of diversity due to genetic drift and unequal founder contribution
was calculated according to Lacy (1995):
Allele frequencies were estimated by direct allele counting. Allelic
richness per locus and population was calculated according to Petit et al. (1998)
as
Homozygosity by loci was calculated using an R package Rhh (Alho et al., 2010)
according to the method proposed by Aparicio et al. (2006):
Heterozygosity for each microsatellite marker was calculated using a
standard formula and then observed and expected heterozygosyties were
compared with the application of
Monitoring of genetic diversity is important especially for the management of small, endangered livestock breeds (Hasler et al., 2011). Among horse breeds remaining in conservation programs in Poland, two (Polish primitive horse and Hucul) should be monitored particularly carefully because their studbooks are closed and the risk of increased inbreeding is relatively high. To date, there have been a limited number of reports focused on genetic diversity of the Polish population of Polish primitive horses and Huculs, especially taking into account both pedigree and molecular data.
The number of horses registered each year increased from 1980 to 2006, when it reached 240 and 173 for Hucul and Polish primitive horse, respectively (Fig. 1). The observed decline in the trend after 2006 is related to the fact that registration of horses for breeding usually takes place between 3–4 years after birth and thus, not all animals born between 2008–2011 were recorded in studbooks at the time when the current study was performed. Increased interest in the Hucul and Polish primitive horse breeding has been observed especially, since the beginning of 2000 when both breeds were included in conservation programs in Poland. The total number of sires used for breeding was very similar in both breeds with 465 and 466 sires in the Hucul and Polish primitive horses, respectively. However between 1980 and 1999, many fewer sires were used for breeding in the Hucul breed (Fig. 2). In more recent years, there were more sires of the Hucul breed than the Polish primitive horse (2000–2009).
Number of Polish primitive horse and Hucul individuals registered between 1980 and 2011 in Poland.
Number of sires used for breeding in the population of Polish primitive horse and Hucul breeds between 1980 and 2011 in Poland.
Pedigree completeness of Polish primitive horse and Hucul individuals registered between 1980 and 2011 in Poland.
Average coefficient of inbreeding calculated for Polish primitive horse and Hucul individuals registered between 1980 and 2011 in Poland.
Genetic diversity loss for Polish primitive horse and Hucul individuals registered between 1980 and 2011 in Poland.
Pedigree completeness in the Polish primitive horses was between 4.6 and 7.6
complete generations and was higher in comparison with the Hucul horses
where it ranged from 3.8 to 7.0 in the analysed period of time (Fig. 3).
It is worth noting that the Hucul breed had some declines of pedigree
completeness between the years 1980 and 1995, which putatively correlates
with the extensive import of foreign individuals in the several years of the
investigated time period. Polish primitive horse also had the highest mean
inbreeding in the analysed time period. It increased from 4.8 % in 1980
to 8.6 % in 2011. A similar but lower trend was also observed for the
Hucul horses, where inbreeding increased from 1.0 % in 1980 to 8.0 %
in 2011 (Fig. 4). Significant regression coefficients (
Effective population size for Polish primitive horse and Hucul individuals registered between 1980 and 2011 in Poland.
The amount of genetic diversity loss in the two breeds since 1980 is
shown in Fig. 5. Overall, the amount of genetic diversity lost (1-GD) was
higher in the Polish primitive horse, where it ranged between 17 and 23 %,
whereas in the Hucul horses it ranged from 11 to 21 %. In the Huculs, the
majority of the genetic diversity loss was due to random genetic drift (GD*
Polish primitive horse had a stable trend for founder genome equivalent
which was around 5. In the case of the Hucul breed, much larger changes of the
coefficient were observed over time. Between 1980 and 1998 it varied between
6 and 10, stabilized afterwards at 7, and declined to 5 genome equivalents
in 2011. Polish primitive horse also had a more stable trend for the
effective population size, which varied between 23 and 37 across the
analysed period of time. It is worth noting that during the analysed period
of time this parameter did not exceed 50, which is the level recommended by
FAO (1998) as a minimum for populations managed under conservation programs.
On the other hand, Hucul horses exhibited a large decline of
Analyses based on microsatellite markers also point towards a higher level of genetic diversity in the Huculs in comparison with the Polish primitive horse. However the differences between the two breeds are smaller than those observed based on their pedigrees. Molecular mean kinship was 0.28 and 0.32 for the Hucul and Polish primitive horse, respectively. In both breeds, trends for homozygosity by loci (HL) fluctuated strongly in the initial period (1980–1995) ranging between 0.05 and 0.37. In some of the analysed years (i.e. 1980, 1988, and 1989 in the Hucul breed), it was impossible to calculate homozygosity because no animals had been genotyped (Fig. 7). In years 1996–2011 HL increased and the trend was more stable with values between 0.22 and 0.36. No major differences between the breeds were detected. Homozygosity by loci (HL), which is considered as a measure of inbreeding based on marker data, did not confirm the difference in levels of inbreeding (based on available pedigrees) between the two breeds as observed in Fig. 4. Therefore, it can be hypothesized that the observed higher inbreeding level of the Polish primitive horse can be partially attributed to higher pedigree completeness of the breed. Heterozygosity calculated separately for 12 microsatellite markers varied between 0.18 (HTG6 in Polish primitive horse) and 0.86 (VHL20 in Hucul). In the case of HTG10 (both breeds) and HMS3 (Polish primitive horse) markers, significant differences between observed and expected heterozygosities were noticed (Table 2). Those deviations might have resulted from the occurrence of null alleles in HTG10 and HMS3 microsatellite markers, and thus the observed number of heterozygote genotypes was putatively underestimated. A similar situation was reported by Rendo et al. (2012) for the Pottoka pony breed. Therefore, it seems reasonable to consider the exclusion of both markers from the future analyses. On the other hand, both microsatellite markers (HTG10 and HMS3) are still recommended by ISAG for the horse parentage control and are broadly used in genetic diversity and phylogenetic studies, e.g. Van de Goor et al. (2011). Mean observed heterozygosity (0.68) calculated for Polish primitive horse does not differ from the results published by Gralak et al. (2001). Mean observed heterozygosity estimated for Hucul horses (0.71) corresponds with the results recently published by Kusza et al. (2013) for Hungarian, Slovakian and Austrian Hucul horses.
Mean homozygosity by locus (Hl) for Polish primitive horse and Hucul individuals born between 1980 and 2008 in Poland.
Results of the 12 microsatellite markers data analysis.
Mean expected heterozygosity was slightly higher in the Hucul horses (0.72) in comparison with the Polish primitive horse (0.70). Hucul breed also had a higher mean allelic richness (AR) than the Polish primitive horse, equal to 9.41 and 8.66, respectively. The number of observed alleles varied between 8 and 12 in the Polish primitive horse and between 7 and 12 in the Hucul breed (Table 2). In both breeds and for almost all of the genotyped microsatellite markers, the observed number of alleles was greater than reported in previous studies (e.g. Gralak et al., 2001; Georgescu et al., 2008; Van de Goor et al., 2010, 2011), although our investigation had spanned significantly larger animal populations. Taking into consideration that in the case of both breeds the parentage control based on microsatellite markers in Poland is mandatory, we decided to use available molecular data in our genetic diversity studies. However, as it was reported by Fernández et al. (2005) and Toro et al. (2009), the utility of molecular marker information in conservation programs is rather limited, especially if high-quality pedigrees are available. This situation might have changed if we replaced the limited information received from genotyping of just several STR markers by molecular data from genome-wide studies, including a large number of SNP genotypes. As it was shown by De Cara et al. (2011) such an approach can be very useful in maintaining genetic diversity on a high level. Application of genome-wide SNP information in phylogenetic and genetic diversity analyses of the domestic horse is developing dynamically, and results of such an investigations are published every year (e.g. Binns et al., 2012; Petersen et al., 2013).
Assessment of genetic diversity in the Polish populations of Polish primitive horse and Hucul based on pedigree and molecular data revealed an increased inbreeding level in both breeds during the analysed period of time (1980–2011) which is characteristic for small, closed animal populations, originating from a limited number of founders. Parameters related to genetic diversity (like effective population size or founder genome equivalent) are generally higher in Hucul horses whereas in the population of Polish primitive horse they can be considered as much stable. Overall, both sources of information (pedigree and genetic markers) indicate that genetic diversity of both breeds has declined over time which is particularly more apparent in the case of the Polish primitive horse. Conservation programs of both breeds should be revised and clear strategies for future populations management should be proposed, including optimized mating strategies and successive control of genetic diversity parameters.
Polish Horse Breeders Association for providing pedigree data. Horse Genetic Markers Laboratory for providing molecular data. Edited by: A.-E. Freifrau von Tiele-Winckler Reviewed by: three anonymous referees