Segregation patterns and inheritance rate of copy number variations regions assessed in a Gochu Asturcelta pig pedigree.

Copy Number Variation Regions (CNVR) were subjected to pedigree analysis to contribute to the understanding of their segregation patterns. Up to 492 Gochu Asturcelta pig individuals forming 478 different parents-offspring trios (61 different families) were genotyped using the Axiom_PigHDv1 Array (658,692 SNPs). CNVR calling, performed using two different platforms (PennCNV and QuantiSNP), allowed to identify a total of 344 candidate CNVR on the 18 porcine autosomes covering about 106.8 Mb of the pig genome. Sixty-nine CNVR were identified, to some extent, in both the parents and the offspring and were classified as segregating CNVR. The other candidate CNVR were called in one or more progeny but in neither parent and classified either as singleton or recurrent de novo CNVR. Segregating CNVR were, on average, larger and more frequent than the recurrent de novo CNVR (444.8 kb vs 287.9 kb long and 34 vs 5 individuals, respectively). In any case, segregating CNVR did not conform to strict Mendelian inheritance patterns: estimates of average paternal and maternal transmission rates ranged from 11.0 % to 13.4 % and mean inheritance rate was below 21 %. This issue should be carefully considered when interpreting the results of CNV studies. Segregating CNVR, present across generations, are unlikely to be artifacts or false positives and can be hypothesized to be important at the population level.

Understanding Mendelian errors in SNP arrays data using a Gochu Asturcelta pig pedigree: genomic alterations, family size and calling errors.

Up to 478 Gochu Asturcelta pig parents-offspring trios (61 different families) were genotyped using the Axiom_PigHDv1 Array to identify the causes of Mendelian errors (ME). Up to 545,364 SNPs were retained. Up to 40,540 SNPs gathering 292,297 allelic mismatches were identified and were overlapped with SINEs and LINEs (Sscrofa genome 11.1). Copy number variations (CNV) were called using PennCNV. ME were classified into eight different classes according to the trio member ("Trio" meaning no assignment) and the allele on which ME was identified: TrioA/B, FatherA/B, MotherA/B, OffspringA/B. Most ME occurred due to systematic causes: (a) those assigned to the Father, Mother or Offspring occurred by null or partial null alleles characterized by heterozygote deficiency, varied with family size, involved a low number of loci (6506), and gathered most mismatches (228,145); (b) TrioB errors varied with family size, covaried with SINEs, LINEs and CNV, and involved most ME loci (33,483) and mismatches (65,682); and (c) TrioA errors were non-systematic ME with no sampling bias involving 1.2% of mismatches only and a low number of loci (1939). The influence of TrioB errors on the overall genotyping quality may be low and, since CNV vary among populations, their removal should be considered in each particular dataset. ME assignable to the Father, Mother or Offspring may be consistent within technological platforms and may bias severely linkage or association studies. Most ME caused by null or partial null alleles can be removed using heterozygote deficiency without affecting the size of the datasets.

Identification and Characterization of Copy Number Variations Regions in West African Taurine Cattle.

A total of 106 West African taurine cattle belonging to the Lagunaire breed of Benin (33), the N'Dama population of Burkina Faso (48), and N'Dama cattle sampled in Congo (25) were analyzed for Copy Number Variations (CNVs) using the BovineHDBeadChip of Illumina and two different CNV calling programs: PennCNV and QuantiSNP. Furthermore, 89 West African zebu samples (Bororo cattle of Mali and Zebu Peul sampled in Benin and Burkina Faso) were used as an outgroup to ensure that analyses reflect the taurine cattle genomic background. Analyses identified 307 taurine-specific CNV regions (CNVRs), covering about 56 Mb on all bovine autosomes. Gene annotation enrichment analysis identified a total of 840 candidate genes on 168 taurine-specific CNVRs. Three different statistically significant functional term annotation clusters (from ACt1 to ACt3) involved in the immune function were identified: ACt1 includes genes encoding lipocalins, proteins involved in the modulation of immune response and allergy; ACt2 includes genes encoding coding B-box-type zinc finger proteins and butyrophilins, involved in innate immune processes; and Act3 includes genes encoding lectin receptors, involved in the inflammatory responses to pathogens and B- and T-cell differentiation. The overlap between taurine-specific CNVRs and QTL regions associated with trypanotolerant response and tick-resistance was relatively low, suggesting that the mechanisms underlying such traits may not be determined by CNV alterations. However, four taurine-specific CNVRs overlapped with QTL regions associated with both traits on BTA23, therefore suggesting that CNV alterations in major histocompatibility complex (MHC) genes can partially explain the existence of genetic mechanisms shared between trypanotolerance and tick resistance in cattle. This research contributes to the understanding of the genomic features of West African taurine cattle.

Ancient autozygous segments subject to positive selection suggest adaptive immune responses in West African cattle.

Small-sized and trypanotolerant West African taurine (Bos taurus) cattle are a unique case of human-mediated process of adaptation to a challenging environment. Extensive gene flow with Sahelian zebu (B. indicus), bigger and with some resistance to tick attack, occurred for centuries and allowed the apparition of stable crossbred populations (sanga) having intermediate characteristics. Up to 237 individuals belonging to 10 different taurine, zebu and sanga cattle populations sampled in Benin, Burkina Faso and Niger were typed using the BovineHD BeadChip of Illumina to identify signatures of selection, assessed using three different Extended-Haplotype-Homozygosity-based statistics, overlapping with ancient, originated 1024 or 2048 generations ago, Homozygosity-By-Descent segments in the cattle genome. Candidate genomic regions were defined ensuring their importance within cattle type and using zebu as reference. Functional annotation analysis identified four statistically significant Annotation Clusters in taurine cattle (from ACt1 to ACt4), one (ACs1) in sanga, and another (ACz1) in zebu cattle, fitting well with expectations. ACt1 included genes primarily associated with innate immunity; ACt2 involved bitter taste receptor genes of importance to adaptation to changing environments; ACt3 included 68 genes coding ATP-binding proteins, some of them located on trypanotolerance-related QTL regions, that can partially underlie immune response and the additive mechanism of trypanotolerance; ACt4 was associated with growth and small size (NPPC gene); ACs1 included genes involved in immune response; and ACz1 is related with ectoparasite resistance. Our results provide a new set of genomic areas and candidate genes giving new insights on the genomic impact of adaptation in West African cattle.

Functional characterization of Copy Number Variations regions in Djallonké sheep.

A total of 184 Djallonké (West African Dwarf) sheep of Burkina Faso were analysed for Copy Number Variations (CNV) using Ovine 50 K SNP BeadChip genotyping data and two different CNV calling platforms: PennCNV and QuantiSNP. Analyses allowed to identify a total of 63 candidate Copy Number Variations Regions (CNVR) on 11 different ovine chromosomes covering about 82.5 Mb of the sheep genome. Gene-annotation enrichment analysis allowed to identify a total of 751 potential candidate ovine genes located in the candidate CNVR bounds. Functional annotation allowed to identify five statistically significant Functional Clusters (FC; enrichment factor > 1.3) involving 61 candidate genes. All genes forming significantly enriched FC were located on ovine chromosome (OAR) 21. FC1 (22 genes including PAG4 and PAG6) and FC5 (three genes: CTSC, CTSW and CTSF), coding proteases (peptidases and cathepsins, respectively), were involved in reproductive performance and modulation of gestation. Both FC3 and FC4 were involved in inflammatory and immunologic response through coding serum amyloid A and B-box-type zinc finger proteins, respectively. Finally, FC2 consisted of 27 genes (including OR10G6 and OR8B8) involved in olfactory receptor activity, key for animals adapting to new food resources. CNVR identified on at least 15% of individuals were considered CNVR hotspots and further overlapped with previously reported quantitative trait loci (QTL). CNVR hotspots spanning genes putatively involved with lipid metabolism (SKP1, TCF7, JADE2, UBE2B and SAR1B) and differential expression in mammary gland (SEC24A and CDKN2AIPNL) on OAR5 and dairy traits (CCDC198 and SLC35F4) on OAR7 overlapped with QTL associated with lipid metabolism, milk protein yield and milk fat percentage. Information obtained from local sheep populations naturally adapted to harsh environments contributes to increase our understanding of the genomic importance of CNV.

Population Structure Assessed Using Microsatellite and SNP Data: An Empirical Comparison in West African Cattle.

A sample of 185 West African cattle belonging to nine different taurine, sanga, and zebu populations was typed using a set of 33 microsatellites and the BovineHD BeadChip of Illumina. The information provided by each type of marker was summarized via clustering methods and principal component analyses (PCA). The aim was to assess differences in performance between both marker types for the identification of population structure and the projection of genetic variability on geographical maps. In general, both microsatellites and Single Nucleotide Polymorphism (SNP) allowed us to differentiate taurine cattle from zebu and sanga cattle, which, in turn, would form a single population. Pearson and Spearman correlation coefficients computed among the admixture coefficients (fitting K = 2) and the eigenvectors corresponding to the first two factors identified using PCA on both microsatellite and SNP data were statistically significant (most of them having p < 0.0001) and high. However, SNP data allowed for a better fine-scale identification of population structure within taurine cattle: Lagunaire cattle from Benin were separated from two different N'Dama cattle samples. Furthermore, when clustering analyses assumed the existence of two parental populations only (K = 2), the SNPs could differentiate a different genetic background in Lagunaire and N'Dama cattle. Although the two N'Dama cattle populations had very different breeding histories, the microsatellite set could not separate the two N'Dama cattle populations. Classic bidimensional dispersion plots constructed using factors identified via PCA gave different shapes for microsatellites and SNPs: plots constructed using microsatellite polymorphism would suggest the existence of weakly differentiated, highly intermingled, subpopulations. However, the projection of the factors identified on synthetic maps gave comparable images. This would suggest that results on population structuring must be interpreted with caution. The geographic projection of genetic variation on synthetic maps avoids interpretations that go beyond the results obtained, particularly when previous information on the analyzed populations is scant. Factors influencing the performance of the projection of genetic parameters on geographic maps, together with restrictions that may affect the election of a given type of markers, are discussed.

Ancient Homozygosity Segments in West African Djallonké Sheep Inform on the Genomic Impact of Livestock Adaptation to the Environment.

A sample of Burkina Faso Djallonké (West African Dwarf) sheep was analyzed to identify stretches of homozygous segments (runs of homozygosity; ROH) overlapping with ancient homozygosity-by-descent (HBD) segments. HBD segments were considered ancient if they were likely to be inherited from ancestors living from 1024 to 2048 generations ago, roughly coinciding with the time in which sheep entered into West Africa. It is hypothesized that such homozygous segments can inform on the effect of the sheep genome of human-mediated selection for adaptation to this harsh environment. PLINK analyses allowed to identify a total of 510 ROH segments in 127 different individuals that could be summarized into 124 different ROH. A total of 32,968 HBD segments were identified on 119 individuals using the software ZooRoH. HBD segments inherited from ancestors living 1024 and 2048 generations ago were identified on 61 individuals. The overlap between consensus ROH identified using PLINK and HBD fragments putatively assigned to generations 1024 and 2048 gave 108 genomic areas located on 17 different ovine chromosomes which were considered candidate regions for gene-annotation enrichment analyses. Functional annotation allowed to identify six statistically significant functional clusters involving 50 candidate genes. Cluster 1 was involved in homeostasis and coagulation; functional clusters 2, 3, and 6 were associated to innate immunity, defense against infections, and white blood cells proliferation and migration, respectively; cluster 4 was involved in parasite resistance; and functional cluster 5, formed by 20 genes, was involved in response to stress. The current analysis confirms the importance of genomic areas associated to immunity, disease resistance, and response to stress for adaptation of sheep to the challenging environment of humid Sub-Saharan West Africa.

Genomic scan of selective sweeps in Djallonké (West African Dwarf) sheep shed light on adaptation to harsh environments.

The Djallonké (West African Dwarf) sheep is a small-sized haired sheep resulting from a costly evolutionary process of natural adaptation to the harsh environment of West Africa including trypanosome challenge. However, genomic studies carried out in this sheep are scant. In this research, genomic data of 184 Djallonké sheep (and 12 Burkina-Sahel sheep as an outgroup) generated using medium-density SNP Chips were analyzed. Three different statistics (iHS, XP-EHH and nSL) were applied to identify candidate selection sweep regions spanning genes putatively associated with adaptation of sheep to the West African environment. A total of 207 candidate selection sweep regions were defined. Gene-annotation enrichment and functional annotation analyses allowed to identify three statistically significant functional clusters involving 12 candidate genes. Genes included in Functional Clusters associated to selection signatures were mainly related to metabolic response to stress, including regulation of oxidative and metabolic stress and thermotolerance. The bovine chromosomal areas carrying QTLs for cattle trypanotolerance were compared with the regions on which the orthologous functional candidate cattle genes were located. The importance of cattle BTA4 for trypanotolerant response might have been conserved between species. The current research provides new insights on the genomic basis for adaptation and highlights the importance of obtaining information from non-cosmopolite livestock populations managed in harsh environments.

Identification of genomic regions and candidate genes of functional importance for gastrointestinal parasite resistance traits in Djallonké sheep of Burkina Faso.

A total of 184 Djallonké lambs from Burkina Faso with phenotypes for packed-cell volume (PCV), log-transformed fecal egg count (lnFEC), and FAffa MAlan CHArt (FAMACHA©) eye scores were typed with the OvineSNP50 BeadChip of Illumina to contribute to the knowledge of the genetic basis of gastrointestinal (GIN) parasite resistance in sheep. Association analysis identified a total of 22 single-nucleotide polymorphisms (SNPs) related with PCV (6 SNPs), lnFEC (7), and FAMACHA scores (9) distributed among 14 Ovis aries chromosomes (OAR). The identified SNPs accounted for 18.76 % of the phenotypic variance for PCV, 21.24 % for lnFEC, and 34.38 % for FAMACHA scores. Analyses pointed out the importance of OAR2 for PCV, OAR3 for FAMACHA scores, and OAR6 for lnFEC. The 125 kb regions surrounding the identified SNPs overlapped with seven previously reported quantitative trait loci (QTLs) for the traits analyzed in the current work. The only chromosome harboring markers associated with the three traits studied was OAR2. In agreement with the literature, two different chromosomal areas on OAR2 can play a major role in the traits studied. Gene-annotation enrichment analysis allowed us to identify a total of 34 potential candidate genes for PCV (6 genes), lnFEC (4), and FAMACHA scores (24). Annotation analysis allowed us to identify one functional term cluster with a significant enrichment score (1.302). The cluster included five genes (TRIB3, CDK4, CSNK2A1, MARK1, and SPATA5) involved in immunity-related and cell-proliferation processes. Furthermore, this research suggests that the MBL2 gene can underlie a previously reported QTL for immunoglobulin A levels on OAR22 and confirms the importance of genes involved in growth and size (such as the ADAMTS17 gene on OAR18) for GIN resistance traits. Since association studies for the ascertainment of the genetic basis of GIN resistance may be affected by genotype-environment interactions, obtaining information from local sheep populations managed in harsh environments contributes to the identification of novel genomic areas of functional importance for GIN resistance for that trait.

Assessing performance of single-sample molecular genetic methods to estimate effective population size: empirical evidence from the endangered Gochu Asturcelta pig breed.

Estimating effective population size (N e ) using linkage disequilibrium (LD) information (N e( LD ) ) has the operational advantage of using a single sample. However, N e( LD ) estimates assume discrete generations and its performance are constrained by demographic issues. However, such concerns have received little empirical attention so far. The pedigree of the endangered Gochu Asturcelta pig breed includes individuals classified into discrete filial generations and individuals with generations overlap. Up to 780 individuals were typed with a set of 17 microsatellites. Performance of N e( LD ) was compared with N e estimates obtained using genealogical information, molecular coancestry (N e(M) ) and a temporal (two-sample) method (N e( JR ) ). Molecular-based estimates of N e exceeded those obtained using pedigree data. Estimates of N e( LD ) for filial generations F3 and F4 (17.0 and 17.3, respectively) were lower and steadier than those obtained using yearly or biannual samplings. N e( LD ) estimated for samples including generations overlap could only be compared with those obtained for the discrete filial generations when sampling span approached a generation interval and demographic correction for bias was applied. Single-sample N e(M) estimates were lower than their N e( LD ) counterparts. N e(M) estimates are likely to partially reflect the number of founders rather than population size. In any case, estimates of LD and molecular coancestry tend to covary and, therefore, N e(M) and N e( LD ) can hardly be considered independent. Demographically adjusted estimates of N e( JR ) and N e( LD ) took comparable values when: (1) the two samples used for the former were separated by one equivalent to discrete generations in the pedigree and (2) sampling span used for the latter approached a generation interval. Overall, the empirical evidence given in this study suggested that the advantage of using single-sample methods to obtain molecular-based estimates of N e is not clear in operational terms. Estimates of N e obtained using methods based in molecular information should be interpreted with caution.