Poorer Resolution of Low-Density SNP Vs. STR Markers in Reconstructing Genetic Relationships Among Seven Italian Sheep Breeds

Ciani_imp:ok 12-02-2014 13:23 Pagina 236

236 Poorer resolution of low-density SNP vs. STR markers in reconstructing genetic relationships among seven Italian sheep breeds

E. CIANI1, F. CECCHI2, E. CASTELLANA1, M. D’ANDREA3, C. INCORONATO4, F. D’ANGELO5, M. ALBENZIO5, F. PILLA3, D. MATASSINO4, D. CIANCI1, R. CIAMPOLINI2 1 Dipartimento di Bioscienze, Biotecnologie e Biofarmaceutica, Università degli Studi di Bari, 70126 Bari, Italy 2 Dipartimento di Scienze Veterinarie, Università degli Studi di Pisa, 56124 Pisa, Italy 3 Dipartimento di Scienze Animali Vegetali e dell’Ambiente, Università degli Studi del Molise, 86100 Campobasso, Italy 4 Consorzio per la Sperimentazione, Divulgazione e Applicazione di Biotecniche Innovative (ConSDABI) Sub National Focal Point italiano FAO Biodiversità Mediterranea, 82100 Benevento, Italy 5 Dipartimento di Scienze delle Produzioni e dell’Innovazione nei Sistemi Agro-alimentari Mediterranei, Università degli Studi di Foggia, 71100 Foggia, Italy

SUMMARY Single Nucleotide Polymorphisms (SNPs) are increasingly used in livestock to infer genetic variability and relationship within and among breeds. Since displaying different mutational properties, bi-allelic markers could reconstruct dissimilar scenarios with respect to long-established Short Tandem Repeats (STRs). Here we present the results from the genetic characterization of seven Italian breeds using 111 SNPs, compared to those previously obtained from 19 STR loci typed on the same population sample (Sarda: 97; Comisana: 67; Altamurana: 83; Leccese: 86; Gentile di Puglia: 74; Bagnolese: 33; Laticauda: 30). All the 111 SNP loci resulted to be polymorphic in the total sample, with only six loci being monomorphic in at least one breed. Af- ter pruning of loci displaying MAF<0.1, a total of 103 SNPs were left in the overall sample dataset. The overall SNP gene diversity was 0.40 (±0.09). A remarkably low proportion of SNPs (on average 2.2%) displayed significant (P<0.01) deviations from the Hardy–Weinberg equilibrium within the seven breeds. The Leccese breed displayed not only the highest levels of

Hardy-Weinberg and gametic disequilibrium (4.9% of SNPs), but also the highest inbreeding coefficient (FIS=0.05) and the highest within-breed allele-sharing distance (D=0.31). These results are consistent with the evidence of sub-structuring within the Leccese breed, as suggested by both the STRUCTURE analysis and the NJ tree based on the inter-individual allele-sharing distance. In addition, the analysis among-breeds highlighted a marked differentiation of Sarda, likely consistent with its insular nature. A more complex scenario was observed when looking at the ability of SNP in reconstructing genetic connections among Italian sheep breeds in comparison to what obtained previously with STR. SNP generally provided poorer resolution in reconstructing the genetic relationships among the seven Italian sheep breeds, likely due to their limited number. Intere- stingly, the study produced also a first clue of overdominance as shaping the pattern of variability at olfactory receptor genes in the ovine species.

KEY WORDS Genetic variability, Microsatellite, Single Nucleotide Polymorphisms.

INTRODUCTION (within populations, between populations within geographic regions, and between geographic regions) differs between Analysis of genetic variability is essential to preserve and ex- STRs and SNPs5. By comparing loci from the same genomic ploit biodiversity. At the molecular level, microsatellite poly- regions, Payseur and Jing4 revealed substantial differences in morphisms (Short Tandem Repeats, STRs) are by far the levels of structure in human populations also at linked STRs most employed loci to investigate biodiversity in livestock, and SNPs, thus attributing these differences primarily to va- including sheep, but more recently SNPs have also been in- riation in the mutational process of the two marker classes. troduced1,2,3. However STRs and SNPs have different muta- In addition, they highlighted the influence of population di- tional properties4 so, for a more effective characterization of vergence time on differences in population structure genetic variability, they can be used in a complementary way. between STRs and SNPs, suggesting that the informativeness Studies carried out by high-throughput SNP genotyping in gap between SNPs and STRs increases as divergence times human datasets previously genotyped at hundreds of STRs between taxonomic units decrease. highlighted how the fraction of diversity attributable to va- In a previous paper, the molecular characterization of seven rious hierarchical components of population structure Italian sheep breeds carried out using STR markers has been discussed6. Here we present the results from the genetic characterization of the same breed dataset using 111 SNPs, to- Autore per la corrispondenza: gether with a first evidence of overdominance at sheep olfac- Francesca Cecchi ([email protected]). tory receptor genes. Ciani_imp:ok 12-02-2014 13:23 Pagina 237

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MATERIAL AND METHODS In order to take into account more complex evolutionary relationships (reticulated evolution) among the studied Animals and breeds breeds, network analysis using Reynolds genetic distance was A total of 470 animals, representative of seven breeds from also carried out by the software Neighbor-net14. southern and insular Italy (Sarda, SAR: 97; Comisana, COM: Breed differentiation was also investigated using the Bayesian 67; Altamurana, ALT: 83; Leccese, LEC: 86; Gentile di Puglia, clustering algorithm implemented in the STRUCTURE GEN: 74; Bagnolese, BAG: 33; Laticauda, LAT: 30) were sam- software v. 2.215. We adopted the “admixture model”, without pled from different flocks trying to avoid closely related in- providing a priori information on population membership. dividuals. In particular, from 3 (Altamurana) to 6 (Leccese Ten independent runs were performed for each K value from and Gentile di Puglia) farms were considered for the small 2 to 10 (where K is the number of cluster to be tested), adop- local sheep populations while samples from the more repre- ting a burn-in period of 100,000 generations, followed by sented Sarda and Comisana were supplied by national bree- 100,000 iterations. The algorithm of Evanno et al.16 was ding stations to guarantee a wider territorial coverage. adopted in order to evaluate the most probable value of K. Sarda is a high milk-yielding breed indigenous of the Sardi- Only loci displaying MAF ≥ 0.1 were used in all the above nian island, thought to derive from the Sardinian mouflon. analyses. After a long-lasting period of very strict isolation, half a century ago it began to spread out over the Italian peninsula re- placing low-producing local sheep breeds; it counts at pre- RESULTS sent more than 3 million heads, mainly located in Sardinia, Tuscany and Latium. Whitin-breed diversity Comisana originated at the beginning of the XX century in All the 111 SNP loci resulted to be polymorphic in the total southeast Sicily by crossbreeding local ewes with rams from sample, with only six loci being monomorphic in at least one Malta and North Africa; it is nowadays spread throughout breed (DU179443, DU216500, DU222192, DU355225, the peninsula though being most concentrated in Southern DU446541, DU487538; data not shown). After pruning of Italy where the breed is preferred to Sarda for its rusticity and loci displaying MAF<0.1, a total of 103 SNPs were left in the excellent adaptation to harsh and semi-arid climate condi- overall sample dataset (data not shown). tions, associated to a valuable milk productivity also when The overall SNP gene diversity (0.40±0.09; data not shown) reared under extensive or semi-extensive systems. was appreciably lower than that estimated previously at STR Out of the three autochthonous Apulian breeds, two (Altamu- loci (0.79±0.08) by Ciani et (2013) 6 on the same samples, rana and Leccese) are considered as belonging to the Zackel likely due to the bi-allelic nature of SNPs. Within-breed gene group of sheep and display a prevailing attitude toward milk diversity ranged from 0.37 (ALT, BAG, LAT) to 0.40 (GEN) production while Gentile di Puglia, also known as Apulian and the average observed heterozygosity ranged from 0.36 Merino, is a fine-wool sheep whose true origin is still object of (LEC) to 0.41 (LAT) (Table 1). debate7. All these breeds have suffered a severe numerical re- A remarkably low proportion of SNPs (on average 2.2%) di- duction in the last fifty years, more pronounced for Altamura- splayed significant (P<0.01) deviations from the Hardy- na, whose population size dropped from 140.000 heads in Weinberg equilibrium within the seven breeds (data not 1960s to very few hundred heads at the present days. shown), with LEC exhibiting the highest number of loci Laticauda and Bagnolese, both autochthonous of Campania, (4.9% of SNPs) in significant departure from HWE. Intere- are usually reared by small family farms under semi-extensi- stingly, all the seven breeds displayed a significant (P<0.01) ve systems and are thought to derive by crossbreeding local departure from HWE due to excess of heterozygous genoty- sheep from the Apennines with fat-tailed North African pes at locus DU526865 (OAR15) (Table 2). sheep, likely imported under the Bourbons dynasty in the The squared allele-frequency correlation, r2, as a measure of XVIII century. Laticauda has a prevailing attitude toward gametic disequilibrium, was assessed for the 5.253 pair-wise meat production, associated to a good prolificacy8, while Ba- combinations possible with 103 bi-allelic loci. SNP r2 values gnolese is a dual-purpose breed, particularly valued for the in the total sample were generally higher than STR values6, excellent quality of dairy products derived from its milk. with a maximum value of 0.64 (data not shown). Within- breed percentages of locus pairs with significant P values we- SNP analysis re generally consistent with those observed at STR loci6, hi- Genomic DNA was extracted from peripheral blood samples ghlighting LEC as the breed displaying the highest values and following standard protocols. Animals were genotyped using BAG and LAT as the breeds displaying the lowest values (da- 111 ovine SNPs selected among 1.5K markers from the ISGC ta not shown).

pilot sheep SNP array (http://www.sheephapmap.org/). The When estimating inbreeding, lower FIS values were observed full list of the selected 111 SNPs is available upon request to compared to STR loci6, the highest being observed in LEC the authors. (0.05, Table 1). The allele-sharing distance (D) between all Minor Allele Frequency (MAF) and pair-wise gametic dise- possible individual pairs within each breed highlighted LAT quilibrium were evaluated using PowerMarker v. 3.259. De- as the less differentiated breed (0.26), while the most diffe- viations from the Hardy-Weinberg equilibrium (HWE) were rentiated breeds resulted to be LEC and GEN (0.31, Table 1). evaluated using the ARLEQUIN package v. 3.1110. The genetic distance (D) between all pair-wise combinations Genetic relationships among breeds of individuals was calculated, using PLINK v 1.0111, as one The neighbour-net networks based on the Reynolds distance minus the average proportion of alleles shared identical by (Figure 1) highlighted a poor structure resolution (star-like state12 and then used to construct a NJ tree via MEGA v.4.113. topology with internal reticulations), with a slightly more Ciani_imp:ok 12-02-2014 13:23 Pagina 238

238 Poorer resolution of low-density SNP vs. STR markers in reconstructing genetic relationships among seven Italian

Table 1 - Within-breed genetic diversity parameters.

Breed N Region of origin Ho*He*FIS*D* Altamurana 83 Apulia 0.38 0.37 -0.01 0.29 Bagnolese 33 Campania 0.37 0.37 -0.01 0.28 Comisana 67 Sicily 0.38 0.38 0.01 0.30 Gentile di Puglia 74 Apulia 0.39 0.40 0.02 0.31 Laticauda 30 Campania 0.41 0.37 -0.11 0.26 Leccese 86 Apulia 0.36 0.38 0.05 0.31 Sarda 97 Sardinia 0.38 0.38 0.00 0.29

*Ho, observed heterozygosity; He, expected heterozygosity, FIS, inbreeding coefficient; D, allele sharing distance.

Table 2 - Observed (Ho) and expected (He) heterozygosity for the locus DU526865 (OAR15).

Breed Ho He P Altamurana 0.52 0.39 * Bagnolese 0.55 0.40 * Comisana 0.75 0.47 * Gentile di Puglia 0.59 0.42 * Laticauda 0.58 0.42 * Leccese 0.70 0.46 * Sarda 0.61 0.43 * *Hardy-Weinberg exact test P-value <0.01.

the total sample is indicated with K followed by the corresponding integer. In general, bi-allelic markers provided mo- Figure 1 - Neighbor-Net network constructed using Reynolds re noisy STRUCTURE patterns than STR markers6. Notwith- distance. standing, they confirmed the higher differentiation of Sarda, as highlighted both in Figure 2 (with Sarda clustering in a se- pronounced differentiation of the Sarda breed. A rough parate group already at K = 2) and in Table 3, where the pro- breed resolution was also observed when plotting the NJ portions of genomic ancestry (Q) obtained at each K value dendrogram based on the inter-individual allele-sharing di- for each breed are reported. As can be observed, at almost all stance (D) calculated by PLINK (data not shown), with the the tested K values, Sarda was the best discriminated breed, exception of the Sarda animals, well discriminated from the though lower Q values were observed with SNPs compared other subjects, and the Leccese animals, grouped into two to STRs6. The second best discriminated breed was Altamu- different sub-clusters. rana that displayed, at least at lower K values (≤4), Q values Breed differentiation was also investigated adopting a Baye- higher or nearly approaching those of Sarda. In addition, the sian clustering analysis carried out using the software STRUCTURE analysis (Figure 2) confirmed the presence of STRUCTURE v. 2.215. The plot of the results is presented in genetic sub-structuring within the Leccese breed sample pre- Figure 2, where the number of sub-populations assumed in viously observed by STR markers6.

Table 3 - Average proportion of membership to assumed STRUCTURE clusters for each breed.

Average Q values* Breed K = 2 K = 4 K = 6 K = 7 K = 8 K = 10 K = 12 Altamurana 0.95 0.87 0.77 0.72 0.53 0.49 0.46 Bagnolese 0.89 0.44 0.80 0.76 0.73 0.61 0.54 Comisana 0.88 0.86 0.49 0.72 0.70 0.44 0.29 Gentile di Puglia 0.86 0.78 0.80 0.75 0.70 0.65 0.37 Laticauda 0.87 0.74 0.52 0.46 0.42 0.30 0.38 Leccese 0.91 0.43 0.39 0.38 0.38 0.36 0.35 Sarda 0.92 0.88 0.86 0.84 0.82 0.79 0.76 *For each K value, only data for the cluster showing the highest average Q value is reported. Ciani_imp:ok 12-02-2014 13:23 Pagina 239

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Figure 2 Plot of genomic components estimated using the software STRUCTURE v. 2.2 (Falush et al., 2007).

DISCUSSION the 103 SNPs were polymorphic in all the seven breeds, with Sarda and Altamurana displaying a fairly lower proportion Neither STR nor SNP used here were specifically selected of polymorphic loci (98 and 97%, respectively, data not for information content on the Italian populations under shown). On the contrary, Sarda was among the breeds di- study. An SNP/STR ratio of about six was adopted, based splaying the highest proportion of polymorphic SNPs on the main literature contrasting the two marker ty- (90%) in the work of Kijas et al.1 where contrasted with pes4,17,18,19,20,21. Rosenberg et al.22 and Liu et al.21 showed that, world-wide sheep breeds. The observed difference (90% vs. in general, randomly chosen set of microsatellites provided 98%) may be explained considering that we used a sub-set greater informativeness to discriminate between human of SNPs selected from the 1.5K SNP chip adopting a crite- populations (respectively, 2.8 to 4.3 and 2.5 to 6.3 times) rion of heterozygosity maximization, therefore removing than a random chosen set of SNPs. Narum et al.19 found from our panel loci less likely to be polymorphic in our mean ratios of random SNPs to microsatellites in Chinook breeds. In fact, a higher proportion of loci (56%) displayed salmon (from 3.9 to 4.1) to be within the range of those a high degree of polymorphism (MAF >0.30 in the total previously shown in humans. Adopting a SNP to microsa- sample) compared to the proportion (45%) observed by tellite ratio of 5.5, we observed a better discrimination abi- Kijas et al. (2009)1. For a similar reason, when the 50K SNP lity of microsatellite markers versus SNP loci. The selection chip was adopted on a set of 2,819 sheep, belonging to 74 of highly diagnostic SNPs from larger panels has been pro- breeds sampled from around the world (Kijas et al., 2012)3 ven to dramatically increase population differentiation, as the within-breed proportion of polymorphic loci never ex- demonstrated by Glover et al.18 in Atlantic salmon and by ceeded 96%, with generally higher values in Southern and Lao et al.23 in a large panel from humans where the best 10 Western European breeds. loci allowed to clearly assign all the individuals to the cor- The average (1-IBS) distances between all possible individual rect continental region of sampling. A similar scenario may pairs within each of the seven breeds (data not shown) were be assumed for sheep, where the current availability of me- higher than observed by Kijas et al.1 in 22 sheep populations dium- to high-density SNP chips will allow to identify the typed at a 1.5K SNP chip, thus suggesting a more pronoun- most discriminating SNPs. ced allelic variability within Italian breeds. To date, only a very restricted number of studies have re- However, average distance values at our 103 SNP panel reported levels of genetic diversity in sheep using biallelic sulted to be higher than those observed at the 1.5K SNP pa- markers. Hoda et al.2 investigated three Albanian sheep nel, also when considering a breed (Sarda) represented in breeds using 36 SNP markers originally developed by Pari- both studies (0.294 vs. 0.267, respectively). This may be due set et al.24 in known sheep genes; Kijas et al.1 performed a ge- to a markedly larger sample size of the breeds considered he- nome-wide survey of SNP variation in sheep breeds sam- re compared to population samples of far less than 40 ani- pled at a global scale using a 1.5K pilot SNP chip and Kijas mals per breed adopted in the study of Kijas et al.1. Moreo- et al.3 investigated the history of domestic sheep through a ver, values observed in our survey may have been inflated by medium density SNP chip. In our study, on average, 99% of the SNP selection process, as already specified above. Ciani_imp:ok 12-02-2014 13:23 Pagina 240

240 Poorer resolution of low-density SNP vs. STR markers in reconstructing genetic relationships among seven Italian

Analysis of Hardy-Weinberg proportions confirmed SAR patterns of genetic structure inferred by using the Bayesian and GEN, respectively, as one of the less and the most va- approach implemented in the popular software package riable breed. Interestingly, a significant excess of heterozy- STRUCTURE resulted to be roughly consistent between the gous genotypes was shared among the seven breeds at the two classes of markers, with STR showing only slightly bet- locus DU526865 (OAR15). Heterozygote excess may be due ter discrimination ability than SNP loci. to several factors, like balancing selection (overdominan- The branching pattern of the NJ dendrogram based on the ce), negative assortative mating, population mixing, CNVs, inter-individual allele-sharing distance (D, data not shown), segmental duplications, pseudogenes. Out of all the loci di- well differentiated Sarda animals from the others and highli- splaying a significant (P<0.01) departure from HWE in at ghted sub-clustering of the Leccese animals in two groups, least one breed (n = 8), only a minor proportion (25%; n = grossly corresponding to animals belonging, respectively, to 2) displayed heterozygote excess, therefore suggesting that the old-type and to the improved Leccese breed. These re- this may not be due to factors acting on a genome-scale like sults would support the oral historical knowledge that mo- negative assortative mating or population mixing. It must dern-day improved Leccese animals were obtained during be pointed out that the SNP DU526865 is harboured in a the second half of the last century by crossing local Leccese region homologous to the human OR5W2 gene, known to ewes with rams from different breeds, mainly Bergamasca, a be a functional olfactory receptor gene. Interestingly, by in- sheep breed from Northern Italy (also known as the Giant of vestigating the contribution of overdominance to the Bergamo, due to its body size) widely used in the past as maintenance of polymorphism in the human genome crossing breed for several Alpine and Apenninic breeds. using the HapMap genotypic data, Alonso et al.25 found Breed sub-structuring within the Leccese population was al- that olfactory receptor activity is a molecular function en- so evident from the Bayesian assignment test performed on riched in genes with higher heterozygosity than expected, the whole population sample. Moreover, when the analysis also when accounting for pseudogenes or functional genes was repeated uniquely on the Leccese sample an incredible not significantly expressed in olfactory epithelium. The fine and precise dissection of genetic variability was obtai- maintenance of high variability in olfactory receptors has ned, with animals from different flocks falling each in a cor- been observed so far also in primates, mouse and pig and responding cluster (data not shown). This result cannot be would be related to the ability to discriminate among clo- explained only by the above-cited crossbreeding events (un- sely related structural odorants. Considering all the above less assuming that rams from different breeds were used in evidences, the results presented in this work may be inter- each flock) and seems more likely due to a very close repro- preted as a first clue to a role of overdominance in shaping ductive isolation among flocks, that would also justify the hi- the pattern of variability at olfactory receptor genes in the gh levels of inbreeding and gametic disequilibrium observed ovine species. in this study. Unfortunately, literature on inference of genetic relationships among sheep breeds by SNP markers is still very poor. 1 Kijas et al. observed FST values comparable to those reported CONCLUSION in the present study among breeds of European origin such

as in the comparisons Sarda vs. Merino (FST = 0.053), vs. This paper presents the results from the genetic characteriza- German Mountain Brown (FST = 0.070) and vs. Comisana tion of seven Italian sheep breeds through a low-density SNP (FST = 0.078) while obviously observing much higher FST va- panel. Results are compared to those previously observed at lues (>0.25) when comparing breed pairs sampled from dif- STR loci for the same population data-set. The two class of ferent continents. markers generally displayed low consistency both in measu- Interestingly, although SNPs generally depicted less defined ring within-breed genetic variability and in reconstructing genetic relationships among breeds than microsatellites6, genetic relationship among breeds, likely due to a poor infor- they constituted the majority among the most informative mativeness of bi-allelic loci as a consequence of the limited

markers ranked on the basis of FST (data not shown). This re- number. However, STR and SNP loci coherently highlighted sult is in agreement with those observed by Rosenberg et some striking evidences, notably the greater differentiation al.22, who found some SNP loci outperforming the average of Sarda from all the other breeds and the presence of gene- SNPs by a large margin. We hypothesize that this phenome- tic sub-structuring within the Leccese population sample. non may be due to the fact that the stronger “selection in- Advancing fast and cheap whole genome sequencing and ge- tensity” usually applied on the choice of SNP markers than nome-wide SNP genotyping techniques are now providing microsatellites due to the availability of far larger panels of very large panels of bi-allelic markers, validated over many bi-allelic markers would facilitate detection of highly infor- world-wide sheep breeds, that will facilitate the detection of mative SNPs showing breed-specific allele frequency distri- highly informative SNPs displaying breed-specific allele fre- butions. In addition, homoplasy may contribute to keep quency distributions. informativeness of the best-discriminating STR loci at lower levels than that of the best SNPs. As anticipated, SNP produced poor network resolution com- AKNOWLEDGEMENTS pared to STR6, with patterns generally inconsistent with any previous knowledge about breed inter-relationships. These Authors would like to thank the Ministero delle Politiche results do not confirm those of Smith et al.26, Ryynänen et Agricole, Alimentari e Forestali (MIPAAF) for funding the al.20, Narum et al.19 and Glover et al.18 who observed general- research, Associazione Nazionale della Pastorizia, Istituto ly comparable topologies adopting the two classes of Zooprofilattico della Sicilia, Azienda Sperimentale Cavone di markers in various salmon populations. On the contrary, Spinazzola and farmers for assistance with sample collection. Ciani_imp:ok 12-02-2014 13:23 Pagina 241

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