Genetic diversity of Shepherd dog using pedigree analysis

ABSTRACT: The German Shepherd dog breed is the most popular breed globally and in Brazil. The study of the population structure through pedigree information is an essential tool to understand the history of the Brazilian German Shepherd dog breed. This study evaluated the status of genetic diversity and population structure of the Brazilian German Shepherd dog breed. The pedigree included a total of 77,938 animals born between 1970 and 2014. The average generation interval in this population was 3.91 years. Considering the reference population, 2,183 founders were identified. Approximately 3% of the genetic diversity of the current population (2010-2014) was lost, most of which was due to genetic drift. The effective population size was relatively small, and the pedigree showed bottlenecks indicating a loss of genetic diversity in this breed. These results indicated the need to adopt measures against the excessive increase in inbreeding and monitor effective population size to minimize genetic diversity loss.


INTRODUCTION
The German Shepherd dog breed is the most popular breed globally and in Brazil. This breed is widely used in the police work, military working, border guard working, shepherding animals, agility, personal and residential guard, due to its intelligence, faithful, alertness, and learning ability (KANIA-GIERDZIEWICZ et al., 2013). The German Shepherd is widely used in police and military service in Brazil. Despite the numerous international studies on the pedigree analysis of dog breeds, including German Shepherd (KASARDA et al. 2007;KALINOWSKA et al. 2010;KANIA-GIERDZIEWICZ et al. 2011;MELLANBY et al., 2013), unfortunately, until today, nothing is known about the population structure and genetic diversity of Brazilian dog populations.
Pedigrees are a valuable data source for monitoring the genetic diversity and the structure of a given population of dogs (LEROY et al., 2006;LEROY, 2011). This genetic information is particularly useful in dogs, as repeated matings and numerous matings between close relatives also negatively influence genetic variability (GŁAŻEWSKA, 2008). Consequently, there are severe losses of genetic diversity and an increased incidence of inherited diseases (LEROY, 2011). According to JANUTTA et al. (2008), increased inbreeding coefficients were associated with a higher genetic disposition for hip dysplasia in German Shepherd dogs. Furthermore, pedigree information would enable different kennel clubs to manage the rate of inbreeding and maintain the genetic diversity of highly specialized dog breeds. Bignardi & Santana The study of population structure through pedigree information is an essential tool for understanding the history of Brazilian German Shepherd dogs. It can provide useful information for breed selection, conservation, and expansion programs. Thus, the objectives of the present study were to evaluate the status of genetic diversity and population structure of the German Shepherd dogs using pedigree analysis.

Data
The pedigree of Brazilian German Shepherd dogs used in this study was obtained from the "Sociedade Brasileira de Cães Pastores Alemães" (SBCPA). The pedigree includes 77,938 animals born between 1970 and 2014. All population parameters were computed using pedigree analysis, with the entire and reference population defined as animals born between 2010 and 2014 (n = 11,299). This definition is equivalent to that of animals born in the last generation interval.

Analysis of pedigree
Pedigree analyses were performed using the PEDIG (BOICHARD, 2002), and CFC (SARGOLZAEI et al., 2006) software. The generation interval was defined as the average age of parents at the birth of their progeny, which was subsequently maintained for reproduction. Pedigree completeness was assessed by the number of equivalent complete generations (ECG) traced, computed as the sum of all known ancestors of the terms (1/2 d ), where d is the ancestor's generation number, which is equal to one for the parents, two for the grandparents, etc. (MAIGNEL et al., 1996). F-statistics (WRIGHT, 1969) were computed to characterize the genetic structure of the population of Brazilian German Shepherd dogs. The average inbreeding coefficient of dogs was denoted F IT , and the average inbreeding coefficient under random mating was denoted F ST . F ST was computed from a hypothetical population produced by randomly matching males and females. The F IS statistic was calculated as follows: . Effective population sizes were estimated based on the individual rates of inbreeding ΔF i (GONZÁLEZ-RECIO et al., 2007, GUTIERREZ et al., 2009) and coancestry ΔC ij (CERVANTES et al., 2011), considering: and , where F i is the inbreeding coefficient of the individual i, C ij is the coancestry coefficient between individuals i and j, and t i and t j are their respective ECG. The following formulas were used: and . The number of equivalent subpopulations was computed as suggested by Cervantes et al. (2008Cervantes et al. ( , 2011: . The principal ancestors (founders or not) of the reference population were identified as described by BOICHARD et al. (1997). The marginal contribution of each main ancestor was computed as the expected genetic contribution independent of the contributions of the other ancestors. The genetic contribution of a founder was calculated as the probability of selecting a gene randomly within the reference population that came from the founder (JAMES, 1972). The number of founders (f e ) and ancestors (ƒ a ) was obtained to evaluate the concentration of the origin of both animals and genes. The parameter f e is defined as the number of equally contributing founders that would be expected to produce the same genetic diversity as observed in the population under study (Lacy, 1989). This parameter was calculated as , where q k is the probability of gene origin of founder k. Parameter f a is the minimum number of ancestors, not necessarily founders, explaining the complete genetic diversity of a population. This parameter was calculated as , where q j is the marginal contribution of ancestor j, which is the genetic contribution made by an ancestor that was not previously explained by other ancestors chosen before. The founder genome equivalent (f g ) can be defined as the number of founders that would be expected to produce the same genetic diversity as observed in the population under study if the founders were equally represented and no loss of alleles occurred (BALLOU & LACY, 1995). Following CABALLERO & TORO (2000), the parameter f g was obtained by the inverse of twice the average coancestry of the individuals included in a pre-defined reference population.
Genetic diversity (GD) in the reference population was computed as: (LACY, 1989 and1995). In domestic populations, bottlenecks and genetic drift frequently occurred, leading to a loss of genetic diversity. The genetic diversity lost in the population since the generation of the founder can be estimated by 1 -GD. The loss of genetic diversity due to unequal contributions of founders was estimated by 1 -GD * , where (CABALLERO & TORO, 2000). The difference between GD and GD * indicates the loss of genetic diversity due to genetic drift that has accumulated since the founding of the population (LACY, 1995). Thus, this difference can be expressed as: , where f n is the effective number of non-founders. This parameter was computed using the following expression proposed by CABALLERO & TORO (2000): .

RESULTS AND DISCUSSION
The pedigree included 77,938 animals that were born between 1970 and 2014 ( Figure  1). There were 53.8% females and 46.1% males. During 1970During -1985, only 862 dogs were registered. Starting from 1995, the lowest value was recorded in 2008, with 1,243 dogs, and the highest in 2002, with 4,001 dogs.
The percentage of the animals used for breeding was lower in males than in females ( Figure 2). On the average, 19% of the males born in the 2000s were used for breeding; the proportion was 9% in females. Since 1995, the percentages of both males and females used for breeding have decreased. The average ratio of males to breeding females in the reference population was approximately 1:2.3. In contrast, this ratio was 1:2.43 between 1994 and 2009. This number reflects breeders' adoption of the reproduction system via polygamy.
Popular sires (defined as >100 recorded offspring) and popular dams (> 40 offspring) were identified. Popular sires were 102 (1.83% of the sires), while popular dams were 39 (0.30% of the dams). The most popular sires and dams in this study were Willy von der Ehrenfeste (556 progeny records) and Cheer Aus Agrigento (55 progeny records). CALBOLI et al. (2008) performed a study on German Shepherd dogs from the United Kingdom and reported 4% popular sires and 1% popular dams. The popular sire effect is one of the most significant contributors to the reduction in genetic diversity, inbreeding depression, inbreeding purging, and inherited disorders (WELLMANN & PFEIFFER, 2009;LEROY & BAUMUNG, 2011). Welfare concerns and effective population size should limit the use of popular sires. As a result, many more dogs are given the opportunity to breed, increasing the number of breeding dogs. Thus, more balanced use of breeding animals can be guaranteed.
The average generation interval for this population was 3.91 years. LEROY et al. (2009), MÄKI et al. (2010), and SHARIFLOU et al. (2011 reported an average generation interval similar in the German Shepherd. The generation intervals for the four different gametic pathways were similar and close to four years (Table 1). The long generational intervals could be attributed to the low replacement rate of breeders, as the most popular males and females have continued to contribute to their offspring for generations today (NAVAS et al., 2020). On average, breeding males were older than breeding females. This difference may be due to improved reproductive management. The intensive use of young females explains their shorter generation intervals as dams.
Until the 1990s, the average inbreeding coefficient (F IT ) was below 2%, probably towing to pedigree information changes (Figure 3).  BOICHARD et al. (1997), incomplete pedigree information can underestimate inbreeding levels. The ECG in this study was higher than the ones reported in other data sets of German Shepherd dogs (MÄKI et al., 2001;COLE et al., 2004;LEROY et al., 2009). Almost all the animals in the reference population were inbred ( Table 2). As dog mating between close relatives is a practice, dogs with high inbreeding are, in general, the products of full-sibs or parent-offspring mating.
As a result of the regular recording of genealogical information, animals in the reference population had nine mean equivalent generations (Table 2). This result indicated that the pedigree is deep enough to provide a comprehensive picture of the evolution of genetic diversity in the Brazilian German Shepherd dog breed. LEROY et al. (2009) studied 61 dog breeds and reported mean equivalent generations of 9.1 and 9.2 for breeds such as Beauceron and Epagneul Breton, respectively.  Twenty-four breeds had a complete generation equivalent value higher than 6. The high inbreeding values, connected to a lower inbreeding effective population size (Table 2), may increase the proportion of individuals affected by monogenic recessive genetic disorders concerning their allele frequency (LEROY & BAUMUNG 2011). SHARIFLOU et al. (2011 reported that the effective population size estimated from Australian German Shepherd dogs was more significant (250) than that in this study. According to LEROY et al. (2013), some measures should be recommended to limit close inbreeding practices, at least for breeds with effective inbreeding population sizes lower than 100, a very close scenario for the population of the Brazilian German Shepherd dog.
Based on the reference population, 2,183 founders were identified. These founders showed unbalanced genetic contributions because the effective number of founding dogs was only 103.70. The founder genome equivalents were 21.41 for the entire population and 17.13 for the reference population. According to NAVAS et al. (2020), a small number of founders leads to a decrease in genetic diversity. Only 20 ancestors explained more than 50% of the genetic contribution in the reference population, 92 contributed 75%, and 234 contributed 90% of the genetic contribution. It must be noted that these values indicated how much inbreeding is caused by the abusive use of specific studs.
According to BOICHARD et al. (1997), genetic diversity within populations after a small number of generations can be measured using parameters derived from gene origin probabilities. The estimated parameters derived from the probability of the gene origin are listed in table 2. The estimates of f e , f a , and f g were relatively low. This was probably due to the intensive use of few dogs as breeding animals. The f a parameter complements the effective amount of founder information by accounting for the genetic variability losses caused by the unbalanced use of reproductive individuals, which produces population bottlenecks. According to the definition of LACY (1989), considering the reference population, only 17 unrelated founders would be necessary to generate the genetic diversity for that period.
The lowest fa/fe ratio indicated bottlenecks in the pedigree of the German Shepherd dog in Brazil. CECCHI et al. (2009) observed a similar pattern in the fa/fe ratio of an Italian population of German Shepherd dogs. Bottlenecks in pedigrees are a common problem in domestic populations and contributed to a reduction in genetic diversity (HUBY et al., 2003). The fa/fe ratio was lower, reflecting a higher impact of random genetic drift on the loss of genetic diversity in the population. The small number of founder animals shows the need to control inbreeding levels, maintain desirable allele founders, search for control tools, and even introduce animals not directly related to the principal ancestors in the population. Approximately 3% of the genetic diversity of the current population (2010-2014) has been lost. SHARIFLOU et al. (2011) reported that 1% of the genetic diversity of Australian German Shepherds was lost. These results suggest that genetic drift is a common cause of genetic diversity loss in Shepherd dogs.
The loss of genetic diversity in Brazilian German Shepherd dogs is shown in table 2. Approximately 2.34% of genetic diversity was lost 1 -GD = genetic diversity lost in the population since the founder generation; 1 -GD * = loss of genetic diversity due to the unequal contributions of founders; GD * -GD = loss of diversity by genetic drift accumulated over non founder generations.
from the first generation of founders until 2014.
Considering the entire population, approximately 19% of the loss was due to the unequal contribution of founders, and the remaining 81% to genetic drift over generations of non-founders. These results suggested that genetic drift is a common cause of genetic diversity loss in dogs.
The current average inbreeding of the population (FIT) was greater than that expected under random mating (FST). The FIS value, which measures the deviation of causality in mating, was higher than zero, indicating that the inbreeding exceeded the expected level of random mating. This implies that resources were involved among the dogs most closely related to the average, and consequently, the population was distributed into subpopulations. The ratio between NeCi and NeFi allows for the estimation of the equivalent number of subpopulations (S), which was 1.43 in the reference population. Hardy-Weinberg equilibrium deviation in dog breeds can be related to either population subdivision or inbreeding practice, that is, mating between close relatives.
The monitoring of population structure, inbreeding, and other related parameters has excellent potential to prevent major losses of genetic diversity in populations of German Shepherd dogs. The excessive use of ten highly favored ancestors resulted in genetic contributions of 40% to the reference population. Thus, it is recommended that breeders use a more significant number of studs, especially those less genetically related to each other, to at least minimize the loss of genetic diversity and control the level of inbreeding.

CONCLUSION
Evaluation of the data registration histories of German Shepherd dogs showed inbreeding trends over time. The effective population size was relatively small, and the pedigree showed bottlenecks, indicating a loss of genetic diversity in the breed. These results indicated the need to adopt measures against the excessive increase in inbreeding and monitor the effective population size to minimize genetic diversity loss.