Genome-wide diversity and runs of homozygosity in the “Braque Français, type Pyrénées” dog breed
Mastrangelo et al. BMC Res Notes
Genome-wide diversity and runs of homozygosity in the “Braque Français, type Pyrénées” dog breed
Salvatore Mastrangelo 2
Filippo Biscarini 0
Marco Ragatzu 1
Andrea Spaterna 3 4
Roberta Ciampolini 3
0 CNR-IBBA , Via Bassini 15, 20133 Milano , Italy
1 Club Italiano Braque Français Type Pyrénées , Capalbio, GR , Italy
2 Dipartimento di Scienze Agrarie e Forestali, Università di Palermo , Palermo , Italy
3 Centro Interuniversitario
4 Scuola di Scienze Mediche Veterinarie, University of Camerino , Matelica, MC , Italy
Objective: Braque Français, type Pyrénées is a French hunting-dog breed whose origin is traced back to old pointing gun-dogs used to assist hunters in finding and retrieving game. This breed is popular in France, but seldom seen elsewhere. Despite the ancient background, the literature on its genetic characterization is surprisingly scarce. A recent study looked into the demography and inbreeding using pedigree records, but there is yet no report on the use of molecular markers in this breed. The aim of this work was to genotype a population of Braque Français, type Pyrénées dogs with the high-density SNP array to study the genomic diversity of the breed. Results: The average observed (HO) and expected (HE) heterozygosity were 0.371 (± 0.142) and 0.359 (± 0.124). Effective population size (Ne) was 27.5635 runs of homozygosity (ROH) were identified with average length of 2.16 MB. A ROH shared by 75% of the dogs was detected at the beginning of chromosome 22. Inbreeding coefficients from marker genotypes were in the range FIS = [− 0.127, 0.172]. Inbreeding estimated from ROH (FROH) had mean 0.112 (± 0.023), with range [0.0526, 0.225]. These results show that the Braque Français, type Pyrénées breed is a relatively inbred population, but with still sufficient genetic variability for conservation and genetic improvement.
Dog; Braque Français; type Pyrénées; SNP; Genetic diversity; Molecular markers; Inbreeding; Runs of homozygosity; Heterozygosity
Genetic variability and structure in domestic animals
largely depend on breeders’ decisions and practices. In
selection, breeding within a closed population is
common practice, since it allows to fix the desired
characteristics and traits of the best representatives of the breed.
However, this mating practice can lead to high rates of
inbreeding and associated risks (higher frequency of
recessive disorders, inbreeding depression), which are
a serious threat especially to small populations and to
populations originating from a small number of ancestors
]. Furthermore, intense directional selection for
specialized animal types may result in a reduced genetic
basis available to the populations, which leads to a
dramatic loss of genetic variability, especially in dogs where
mating between close relatives is frequently used [
Concerns about the potential effects of inbreeding and
reduced diversity on health, functionality and welfare of
animals within dog breeds, have led to a call for improved
genetic management practices. Hence, managing genetic
diversity has become a major focus for dog breeders,
herd books and authorities [
]. Studies have shown that
there is a loss of the total amount of genetic diversity in
modern dog populations [
]. In particular, purebred
dogs have been intensely selected by resorting, in some
lines, to close-breeding where popular dominant sires
were repeatedly used for mating, resulting in a
reduction of genetic diversity. Traditionally, genealogical data
has been used to assess genetic diversity in dogs [
]. However, the use of genealogical data is limited by
the incomplete or inaccurate available pedigree records.
Genomics offer novel applications that have great
potential to increase our understanding of the genome of
domestic animals and to improve the efficiency of
conservation and selection programs [
(shorttandem repeats) molecular markers have been used
initially to estimate genetic diversity in the absence of
pedigree records [
9, 10, 14, 15
]. More recently, the
availability of high density single nucleotide polymorphism
(SNP) arrays, has increased the accuracy, the throughput
and the cost-effectiveness of genomic analyses for
conservation genetics [
]. Indeed, the large numbers of
SNPs throughout the genome makes these markers
particularly suitable for the detection of genomic regions
where a reduction in heterozygosity occurred and offers
new opportunities to improve the accuracy of genetic
Braques Françaises are hunting dogs, originating from
a very old type of gun-dog used for pointing the location
of game birds to hunters. There are two breeds of Braque
Français, both from the south of France: the Braque
Français, type Gascogne (larger size) and the Braque Français,
type Pyrénées (smaller size). They are popular
hunting dogs in France, but are seldom seen elsewhere. The
original Braque Français type of pointing dog has existed
since the eighteenth century. The first breed club was
formed in 1850, and the standards for both breeds were
written in 1880. The demography and inbreeding levels of
the Braque Français, type Pyrénées breed were estimated
using pedigree records [
]. There is yet no report of the
genetic characterization of this breed using molecular
markers. The aim of this work was to study the genomic
diversity of Braque Français, type Pyrénées dogs using
data high-density SNP data. Available data, methods and
results are hereby presented.
DNA sampling, genotyping and quality control
Blood samples were collected from 48 Braque Français,
type Pyrénées unrelated individual dogs (27 females, 21
males), to capture a representative sample of the
withinbreed genetic diversity. Genomic DNA (gDNA) was
extracted from blood samples through a standard
ethanol fractionation with concentrated sodium chloride (6 M
NaCl) and sodium dodecyl sulphate (10% SDS). The
concentration of DNA was adjusted to 50 ng/μL per sample.
All dogs were genotyped with the Illumina CanineHD
BeadChip, containing 173,662 SNPs. Genotyping was
carried out in the laboratories of “Dipartimento di Scienze
Agrarie, Alimentari e Forestali”, University of Palermo
(Italy). SNP data were filtered to exclude unmapped loci
not assigned to any chromosomes and loci on the sex
chromosomes: only SNPs located on the 38 autosomes
were considered. Additionally, SNPs with call-rate < 95%
and minor allele frequency (MAF) < 5% were removed
from the dataset, as well as animals with over 10% missing
Genetic diversity parameters, effective population size
and runs of homozygosity (ROH)
Basic genetic diversity indices were estimated,
including: (i) observed heterozygosity (HO = L1 lL=1 nNAB ); (ii)
expected heterozygosity (HE = L1 lL=1 1 − pl2 − ql2 );
(iii) the inbreeding coefficient based on the difference
between observed and expected homozygous genotypes
(FIS = L1 lL=1 1 − HHOEll , [
]); and (iv) minor allele
frequency (MAFl = 2nNBll). Here: L is the n. of SNP loci; nAB is
the number of heterozygous genotypes at each locus l; N
is the number of individuals (sample size); p and q are the
frequencies of, respectively, the A (major) and B (minor)
The contemporary effective population size (Ne) was
estimated based on linkage disequilibrium (LD; [
reduce the impact of SNP ascertainment bias from
linkage between loci, unlinked SNPs were selected based
on variance inflation factor (VIF, a measure of
multicollinearity in multiple regression) below 2 in 50-SNP
sliding window with 5-SNP step. A VIF threshold of 2 is
recommended for small sample sizes to avoid removing
too many SNPs
Runs of homozygosity (ROH: segments of
continuous homozygous genome, [
]) were detected in each
individual dog using the sliding-window based method
described in [
]. A 50-SNP long sliding window was
used to scan the genome; the proportion of overlapping
homozygous windows to call a ROH was 0.05; maximum
two missing and one heterozygous SNP were allowed in a
run; the minimum number of SNPs to call a ROH (s) was
calculated as proposed by Lencz et al.  to minimize
the number of false positive ROH:
ln(1 − HO)
where α is the tolerated proportion of false positive ROH
(0.05 in the present study); N and L are the sample size
and number of SNP loci; HO is the average observed
heterozygosity across individuals and SNPs. The minimum
length of a ROH was set to 1 MB to exclude short ROH
arising as a consequence of linkage disequilibrium (LD)
]. Additionally, a minimum density of one SNP every
50 kB and a maximum 100 kB gap between consecutive
SNPs were required to define a ROH.
The inbreeding coefficient based on ROH (FROH) for
each animal was estimated as the ratio between the sum
of the length of all ROH (LROH) and the total length of
the autosomal genome covered by SNPs on the array
(LAUTO = 2268.83 MB).
Quality control filtering of genotypic data, ROH
detection and the estimation of genetic parameters were
all performed using the PLINK software package [
Ne was estimated using NeEstimator v.2 [
unlinked SNP selected using PLINK. The R programming
environment for statistical computing v.3.2.3 [
used for data manipulation, summary statistics,
preparation of tables and figures. The specific PLINK command
lines are detailed in Additional file 1.
After filtering for quality (call-rate, genome assembly),
the final number of SNPs retained for the analysis was
94,065. All 48 dogs had high quality genotyping and were
included in the analysis. The average observed (HO) and
expected (HE) heterozygosities were 0.371 (± 0.142) and
0.359 (± 0.124), respectively; the average MAF was 0.269
(± 0.132). The HO and HE values reported here are
comparable with previous studies that examined the genetic
diversity in dogs: Pollinger et al. [
] and Pilot et al. [
estimated average HO between 0.2 and 0.3 in modern dog
]. Pertoldi et al.  reported HE estimates
in five Danish dog breeds similar to the expected
heterozygosity found here in Braque Français, type Pyrénées
dogs. The reported MAF values also were consistent with
the range found in literature [
]. Recently, Stronen
et al.  reported lower values of genetic diversity in
the endangered Norwegian Lundehund (e.g. HO = 0.038)
compared to three reference breeds. In the same breed, a
very low MAF (0.033) has been estimated [
comparison, the genetic diversity levels reported here for Braque
Français, type Pyrénées dogs seem to indicate that this
is not an endangered breed. The 5560 SNPs on the sex
chromosomes were not used in this study, because of
potentially ambiguous heterozygous SNP calls in males.
As an indication, we report basic SNP descriptive
statistics in males and females. Monomorphic SNPs on the sex
chromosomes were 41.2 and 37.3% in males and females
respectively, with MAF 0.145 and 0.151, and missing-rate
0.0301 and 0.0293.
The estimated contemporary effective population size
(Ne) in Braque Français, type Pyrénées dogs was 27; this
value indicates a potential risk of inbreeding and
reduction in genetic diversity. In a recent a study on Bullmastiff
dogs, a similar Ne value (29.1) was estimated using the
same method [
]. The authors reported that a small Ne
is a reflection of population size, unequal use of breeding
animals and unequal founder contributions.
A total of 5635 ROH were identified with an
average of 117.38 ROH per dog (range between 52 and 230
ROH per animal). Table 1 reports the number of ROH
and their average length per length class (0–2, 2–4, 4–8,
8–16 MB). The average number of ROH in
Mediterranean dog breeds ranged from 12 (Mastino Abruzzese) to
114 (Saint Bernard) [
]: this positions the Braque
Français, type Pyrénées in the higher part of the range. The
longest ROH (12.5 MB) was found on chromosome 16.
Figure 1 shows the proportion of times (dogs) each SNP
falls inside a ROH, plotted against the SNP position along
the dog genome. Especially on chromosome 22 there are
a number of SNP loci which appear to fall relatively often
within a ROH in the Braque Français, type Pyrénées
dog breed. Indeed, at the beginning of chromosome 22
(5–10 MB) there is a ROH shard by most of the dogs in
the analysed sample (Fig. 2).
Inbreeding coefficients estimated from SNP marker
loci as FIS were in the range [− 0.127, 0.172]: some
negative values were obtained, which corresponded to dogs
with lower than average homozygosity. Inbreeding
estimated from runs of homozygosity (FROH) had a mean of
0.112 (± 0.023), with a range between 0.0526 to 0.225.
In German shepherd dogs, similar FROH values have
been reported [
]. FROH from 0.061 (Jack Russell
Terrier) to 0.151 (Bulldog) was estimated in a panel of canine
]. The Pearson linear correlation between the
two measures of molecular inbreeding (FIS and FROH was
0.91. Previously, from pedigree records an average
genealogical inbreeding coefficient (F, [
]) of 4.3% was
The results presented in this note constitute the first
report on the genomic characterization of the Braque
Français, type Pyrénées dog breed using molecular
markers. Previously, the demographics and diversity
of this breed had been analysed resorting to pedigree
data exclusively. The use of molecular data represents
% SNP in ROH in the dog breed Braque Français, type Pyrénées − BRQ
an important step towards a more accurate description
of the genome of the Braque Français, type Pyrénées
dogs. This improved genomic information will prove to
be very useful for the management of the breed, both in
the perspective of conservation and in that of breeding
and selection. However, this is but a preliminary study,
that aimed at providing essential initial information to be
used subsequently for a larger and more comprehensive
studies. Genomic data on a panel of relevant dog breeds
are currently being acquired, to be used for a large-scale
genome-wide characterization of dog breeds and the
genetic distances and phylogenetic relationships between
them. Additionally, phenotypes on the hunting ability
and morphology of Braque Français, type Pyrénées dogs
are being collected: together with the already available
genotypic data, these phenotypes will be used in
genomewide association studies (GWAS) and similar approaches
(e.g. see [
]) to detect SNP loci and regions of the
genome that play a role in relevant phenotypes for the
breed. As an illustration, the strong ROH signal detected
on chromosome 22 may be associated to hunting ability
or other phenotypic characteristics of the Braque
Français, type Pyrénées dog breed. Furthermore, genomic
data can be used to improve the accuracy of pedigree
records, thereby allowing for more meaningful
comparisons between genealogic and molecular inbreeding. All
together, the results presented here provide an
interesting example of the use of molecular markers to
understand the genetic background and history of small canine
breeds like the Braque Français, type Pyrénées.
Additional file 1. Plink command lines. File with the Plink command
lines used to: (i) edite the SNP data; (ii) select unlinked SNP loci for the
estimation of Ne; (iii) detect runs of homozygosity (ROH).
SNP: single nucleotide polymorphisms; ROH: runs of homozygosity.
FB, BA and SM performed all statistical analyses. FB wrote most of the
manuscript. RC, SM, MR and AS contributed largely to the writing of the paper. RC
sampled the animals and generated the SNP genotype data. All authors read
and approved the final manuscript.
We thank “ENCI” (Ente Nazionale Cinofilia Italiana) for contributing the data
used for this work. We acknowledge Mr. Antonino La Barbera (Vice-President
of “Club Italiano Bracco Francese”, Capalbio-GR) for fostering research on this
breed and for his endeavours in maintaining and collecting data and
information on the Braque Français, type Pyrénées dog breed.
The authors declare that they have no competing interests.
Availability of data and materials
The genotypic data used in this preliminary work will be publicly available
after publication of results from the main study including Braque Français,
type Pyrénées and all other breeds selected for comparison. In the meantime,
data may be requested directly to the corresponding author.
Consent for publication
Ethics approval and consent to participate
No experimental studies were conducted on the animals. Animal records
were provided by “ENCI” (Ente Nazionale Cinofilia Italiana), the institution that
officially manages data for all dog breeds registered in Italy. Blood samples
for genotyping were collected by official veterinarians. “Club Italiano Bracco
Francese”, whose president is coauthor of the paper, consented for this study
to access the blood samples.
This research work was funded by “ENCI” (Ente Nazionale Per la Cinofilia
Italiana—Italian Kennell Club) through the “Italian Club Bracco Francese”. The
Club has then commissioned this work out to the “Centro Interuniversitario di
Ricerca e di Consulenza sulla Genetica e la Clinica del Cane”, whose Director is
Prof. Andrea Spaterna.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Taberlet P , Valentini A , Rezaei H , Naderi S , Pompanon F , Negrini R , AjmoneMarsan P. Are cattle, sheep, and goats endangered species ? Mol Ecol . 2008 ; 17 ( 1 ): 275 - 84 .
2. Kristensen TN , Sørensen AC . Inbreeding-lessons from animal breeding, evolutionary biology and conservation genetics . Anim Sci . 2005 ; 80 ( 2 ): 121 - 33 .
3. Leroy G , Rognon X , Varlet A , Joffrin C , Verrier E. Genetic variability in french dog breeds assessed by pedigree data . J Anim Breed Genet . 2006 ; 123 ( 1 ): 1 - 9 .
4. Cecchi F , Paci G , Spaterna A , Ciampolini R . Genetic variability in bracco italiano dog breed assessed by pedigree data . Ital J Anim Sci . 2013 ; 12 ( 3 ): 54 .
5. Mortlock S-A , Khatkar MS , Williamson P. Comparative analysis of genome diversity in bullmastiff dogs . PLoS ONE . 2016 ; 11 ( 1 ): 0147941 .
6. Pertoldi C , Kristensen TN , Loeschcke V , Berg P , Praebel A , Stronen AV , Proschowsky HF , Fredholm M. Characterization of the genetic profile of five danish dog breeds . J Anim Sci . 2013 ; 91 ( 11 ): 5122 - 7 .
7. Pfahler S , Distl O. A massive reduction of the genetic diversity in the lundehund . Anim Genet . 2014 ; 45 ( 1 ): 154 .
8. Mortlock S-A , Booth R , Mazrier H , Khatkar MS , Williamson P. Visualization of genome diversity in german shepherd dogs . Bioinform Biol Insights . 2015 ; 9 ( Suppl 2 ): 37 .
9. Leroy G , Verrier E , Meriaux J , Rognon X . Genetic diversity of dog breeds: within-breed diversity comparing genealogical and molecular data . Anim Genet . 2009 ; 40 ( 3 ): 323 - 32 .
10. Ciampolini R , Cecchi F , Paci G , Policardo C , Spaterna A. Investigation on the genetic variability of the american pit bull terrier dogs belonging to an italian breeder using microsatellite markers and genealogical data . Cytol Genet . 2013 ; 47 ( 4 ): 217 - 21 .
11. Vaysse A , Ratnakumar A , Derrien T , Axelsson E , Pielberg GR , Sigurdsson S , Fall T , Seppälä EH , Hansen MS , Lawley CT , et al. Identification of genomic regions associated with phenotypic variation between dog breeds using selection mapping . PLoS Genet . 2011 ; 7 ( 10 ): 1002316 .
12. Pilot M , Malewski T , Moura AE , Grzybowski T , Oleński K , Kamiński S , Fadel FR , Alagaili AN , Mohammed OB , Bogdanowicz W. Diversifying selection between pure-breed and free-breeding dogs inferred from genomewide snp analysis . Genes Genomes Genet . 2016 ; 6 ( 8 ): 2285 - 98 .
13. Dreger DL , Rimbault M , Davis BW , Bhatnagar A , Parker HG , Ostrander EA . Whole genome sequence, snp chips and pedigree structure: building demographic profiles in domestic dog breeds to optimize genetic trait mapping . Dis Models Mech . 2016 . https://doi.org/10.1242/dmm.027037.
14. Ciampolini R , Cecchi F , Bramante A , Casetti F , Presciuttini S. Genetic variability of the bracco italiano dog breed based on microsatellite polimorphism . Ital J Anim Sci . 2011 ; 10 ( 4 ): 59 .
15. Bigi D , Marelli S , Randi E , Polli M. Genetic characterization of four native italian shepherd dog breeds and analysis of their relationship to cosmopolitan dog breeds using microsatellite markers . Animal . 2015 ; 9 ( 12 ): 1921 - 8 .
16. Allendorf FW , Hohenlohe PA , Luikart G . Genomics and the future of conservation genetics . Nat Rev Genet . 2010 ; 11 ( 10 ): 697 .
17. Bruford MW , Ginja C , Hoffmann I , Joost S , Orozco-terWengel P , Alberto FJ , Amaral AJ , Barbato M , Biscarini F , Colli L , et al. Prospects and challenges for the conservation of farm animal genomic resources, 2015 - 2025 . Front Genet . 2015 ; 6 : 314 .
18. Cecchi F , Paci G , Spaterna A , Ragatzu M , Ciampolini R . Demographic approach on the study of genetic parameters in the dog Braque Français type Pyrénées italian population . Ital J Anim Sci . 2016 ; 15 ( 1 ): 30 - 6 .
19. Wright S. The interpretation of population structure by f-statistics with special regard to systems of mating . Evolution . 1965 ; 19 ( 3 ): 395 - 420 .
20. Jones A , Ovenden J , Wang Y . Improved confidence intervals for the linkage disequilibrium method for estimating effective population size . Heredity . 2016 ; 117 ( 4 ): 217 - 23 .
21. McQuillan R , Leutenegger A-L , Abdel-Rahman R , Franklin CS , Pericic M , Barac-Lauc L , Smolej-Narancic N , Janicijevic B , Polasek O , Tenesa A , et al. Runs of homozygosity in European populations . Am J Hum Genet . 2008 ; 83 ( 3 ): 359 - 72 .
22. Purcell S , Neale B , Todd-Brown K , Thomas L , Ferreira MA , Bender D , Maller J , Sklar P , De Bakker PI , Daly MJ , et al. Plink: a tool set for whole-genome association and population-based linkage analyses . Am J Hum Genet . 2007 ; 81 ( 3 ): 559 - 75 .
23. Bjelland D , Weigel K , Vukasinovic N , Nkrumah J . Evaluation of inbreeding depression in holstein cattle using whole-genome snp markers and alternative measures of genomic inbreeding . J Dairy Sci . 2013 ; 96 ( 7 ): 4697 - 706 .
24. Lencz T , Lambert C , DeRosse P , Burdick KE , Morgan TV , Kane JM , Kucherlapati R , Malhotra AK . Runs of homozygosity reveal highly penetrant recessive loci in schizophrenia . Proc Natl Acad Sci . 2007 ; 104 ( 50 ): 19942 - 7 .
25. Do C , Waples RS , Peel D , Macbeth G , Tillett BJ , Ovenden JR . Neestimator v2: re-implementation of software for the estimation of contemporary effective population size (ne) from genetic data . Mol Ecol Resour . 2014 ; 14 ( 1 ): 209 - 14 .
26. R Core Team. R: a language and environment for statistical computing . Vienna: R Foundation for Statistical Computing; 2017 . https://www.Rproject.org/.
27. Pollinger JP , Lohmueller KE , Han E , Parker HG , Quignon P , Degenhardt JD , Boyko AR , Earl DA , Auton A , Reynolds A , et al. Genome-wide snp and haplotype analyses reveal a rich history underlying dog domestication . Nature . 2010 ; 464 ( 7290 ): 898 .
28. Parker HG , Dreger DL , Rimbault M , Davis BW , Mullen AB , CarpinteroRamirez G , Ostrander EA . Genomic analyses reveal the influence of geographic origin, migration, and hybridization on modern dog breed development . Cell Rep . 2017 ; 19 ( 4 ): 697 - 708 .
29. Stronen AV , Salmela E , Baldursdóttir BK , Berg P , Espelien IS , Järvi K , Jensen H , Kristensen TN , Melis C , Manenti T , et al. Genetic rescue of an endangered domestic animal through outcrossing with closely related breeds: a case study of the norwegian lundehund . PLoS ONE . 2017 ; 12 ( 6 ): 0177429 .
30. Dreger DL , Davis BW , Cocco R , Sechi S , Di Cerbo A , Parker HG , Polli M , Marelli SP , Crepaldi P , Ostrander EA . Studies of the Fonni's dogs from sardinia show commonalities between development of pure breeds and population isolates . Genetics . 2016 ; 204 : 737 - 55 .
31. Wright S. Coefficients of inbreeding and relationship . Am Nat . 1922 ; 56 ( 645 ): 330 - 8 .
32. Biscarini F , Biffani S , Stella A . Más allá del gwas: alternativas para localizar qtls . arXiv preprint arXiv:1504.03802 . 2015 .