ENGLISH VERSION

ZESZYT 348

Brygida Ślaska
Structural genomics of the raccoon dog 
(Nyctereutes procyonoides procyonoides)

The racoon dog (Nyctereutes procyonoides procyonoides) is one of the fur-bearing animal species, the breeding of which aims at pelt production. The contemporary knowledge of the raccoon dog genome is scarce. 
Aim of work. The aim of the studies presented in the dissertation was to extend the knowledge of the raccoon dog genome in terms of structural genomics, in cognitive as well as application aspects, by: determination of the level of genetic conservatism between the dog and the raccoon dog; estimation of biodiversity of raccoon dogs bred in the national fur-bearing animal farms and wild-living raccoon dog; development of a reliable system of origin control including verification of the thesis of presence of half-sibling originating from more than one male in the same litter; determination of the consequences of and solutions to the promiscuity phenomenon in the raccoon dog; construction of a genetic map of the raccoon dog, and genome mapping in order to detect QTL. 
Material and methods. The studies involved raccoon dogs bred in three farms situated in various region of Poland in the years 2005–2009 and wild-living raccoon dogs. A total of 376 individuals were investigated. Genetic variability at the molecular level was analyzed with the use of 20 microsatellite sequences. The amplification primers were taken from literature data; they were developed for the genome of the domestic dog, which together with the raccoon dog belongs to the family Canidae. The microsatellite sequences analysed were subjected to sequencing. A comparative in silico analysis of microsatellite sequences in the raccoon dog and domestic dog was performed. The study also included characterisation of the genetic structure and phylogenetic analysis of three groups of farm and wild-living raccoon dogs. An analysis of the origin was also performed using the data of co-dominant markers in order to develop a reliable system of checking the origin, to verify the thesis of presence of half-siblings originating from more than one male in the same litter and to determine the consequences of and solutions to the promiscuity phenomenon in the raccoon dog. Linkage analysis between the polymorphic lociof interest was performed. Lineage and phenotypic data provided by farm documentation as well as animal genotypes in the polymorphic microsatellite loci were used to detect the relationship between usability and functional traits in the raccoon dog. The material for QTL identification was a reference family – a three-generation population of 290 individuals. Two single-trait mixed animal models were used for the QTL detection tests. Genetic correlations were estimated on the basis of the results of multi-trait model analysis. Estimators of the epistatic effects were also de-termined.
Results and discussion. By using information about the sequence of the dog genome, amplification products were obtained in all the raccoon dog microsatellite markers analysed. In the sequencing reaction of the microsatellite loci, it was observed that the repetitive motifs in the case of 80% of the markers were identically arranged in the genomes of both the raccoon dog and the domestic dog. A high level of similarity of the repetitive motifs was observed in the case of 10% of the loci. A total of 130 alleles were identified in the 20 loci analysed. The mean number of alleles in all the loci in various raccoon dog groups ranged from 4.45 to 6.15. Both conservative and specific alleles were noted in the particular raccoon dog groups. 80% of the analysed markers were characterised by a high polymorphism index. The genetic distance among the four raccoon dog groups displayed low values, ranging between 0.083 and 0.143. Its highest value was observed between the raccoon dogs from population A and wild-living individuals. When only three microsatellite loci with the highest polymorphism index were used, the total probability of exclusion was 0.95, whereas it exceeded the value of 0.9999 when 11 most informative markers were used. The probability of detecting two individuals with the same genotype in all the twenty loci concurrently was 5.33e-18. Based on the genetic analysis of the origin control in the raccoon dog, half-siblings in the same litter were observed in a predominant number of the litters. 66.7% of the litters comprised offspring from different fathers in the case of a female mating during one oestrus with more than one male. The linkage analysis demonstrated two linkage groups, each containing 9 microsatellite loci. The total length of the map was 419.5 cM, with distances between the markers ranging between 9.0 to 39.3 cM. The average distance between the loci was 26.2. A varied recombination rate was observed between the sexes. The total length of the female map was 493.6 cM, whereas the male map was 410.8 cM long. The raccoon dog markers were classified into linkage groups, likewise in the case of the domestic dog. Group LGN1in the raccoon dog contained markers which formed a linkage group in dog chromosome CFA4, whereas the microsatellite loci from LGN2 were present in dog CFA15. The sequence of arrangement of four markers in linkage group LGN1and five in LGN2 was consistent with that in the domestic dog. Differences in the arrangement sequence of the other microsatellite loci were observed between the species. The values of heritability coefficients of the conformation traits showed a relatively high level (0.35–0.48), which indicated that the variability of usability traits observed in the population was to a high degree conditioned by the additive effect of the individual’s genotype, and the values of the estimations of reproductive trait heritability (0.12–0.20) indicated a low share of genetic variability in the phenotypic variability. On the basis of the results of QTL mapping and the magnitude of additive and dominant effects, presence of hypothetical QTL regions with a statistically confirmed effect on body weight and purity of the raccoon dog coat colour was noted. The highest values of the test statistics were obtained for purity of the coat colour and body weight in LGN2 (respectively: LR = 14.2 (p ≤ 0.05) and LR = 14.1 (p ≤ 0.05)). A slightly lower value of the likelihood test, also statistically confirmed, was noted for purity of the coat colour in LGN2. The mean effects of addictiveness and dominance estimators of potential QTL were negative in most cases. A probable position of the QTL with the highest value of the test statistics for body weight is a fragment which comprises IGF-I gene on the dog map and on the raccoon dog map. The scan analysis of LGN1also implied possible presence of QTL determining body weight and purity of the raccoon dog coat; one of the regions in the dog genome mentioned, and probably in the raccoon dog genome, contains the growth hormone receptor gene (GHR). Since there are QTL pairs related to raccoon dog conformation traits in the particular linkage groups, epistatic interactions within the particular loci pairs were estimated. Epistatic interaction between loci related to raccoon dog body weight was observed both in LGN1 and in LGN2. The results clearly indicate a model with epistatic interactions between the QTL in each linkage group. 
conclusions. The use of genome marker maps elaborated for the domestic dog is one of the possibilities contributing to development of knowledge about the raccoon dog genome. DNA sequence orthology in the raccoon dog and the domestic dog corroborates genome conservatism in both Canidae species. Phylogenetic analyses based on microsatellite sequences demonstrated genetic separateness of farm and wild-living raccoon dogs in Poland. A reliable system of origin verification has been elaborated, which should be put into practice in raccoon dog breeding in Poland, particularly in the case of individuals chosen for the selection stock. Offspring origin control should become an indispensable element in breeding practice and BLUP estimation. Presence of paternal half-siblings in the same raccoon dog litter has been confirmed and a hypothesis has been formulated predicting potential mechanisms of post-copulation selection of semen in females. The possibility of determination of biological fathers solely on the basis of breeding documents has been excluded in the case of promiscuity. Arrangement of markers in linkage groups has contributed to development of the raccoon dog genetic map. Segregation analysis of microsatellite sequences displayed advanced conservatism of the linkage arrangements in the genomes of the raccoon dog and domestic dog. The magnitudes of additive, dominant and epistatic effects of the identified loci of the quantitative traits have confirmed polygenic determination of the traits in question. The loci of the quantitative traits determined may be useful in genetic improvement of conformation traits and, consequently, in marker-aided selection. The IGF1 and GHR genes have been recognised to be candidate genes for production traits in the racoon dog. The molecular study results obtained may have an application character.