Commercial pig producers generally use a terminal crossbreeding system with three breeds. Many pig breeding organisations have started to use genomic selection for which genetic evaluation is often done by applying single-step methods for which the pedigree-based additive genetic relationship matrix is replaced by a combined relationship matrix based on both marker genotypes and pedigree. Genomic selection is implemented for purebreds, but it also offers opportunities for incorporating information from crossbreds and selecting for crossbred performance. However, models for genetic evaluation for the three-way crossbreeding system have not been developed.Four-variate models for three-way terminal crossbreeding are presented in which the first three variables contain the records for the three pure breeds and the fourth variable contains the records for the three-way crossbreds. For purebred animals, the models provide breeding values for both purebred and crossbred performances. Heterogeneity of genetic architecture between breeds and genotype by environment interactions are modelled through genetic correlations between these breeding values. Specification of the additive genetic relationships is essential for these models and can be defined either within populations or across populations. Based on these two types of additive genetic relationships, both pedigree-based, marker-based and combined relationships based on both pedigree and marker information are presented. All these models for three-way crossbreeding can be formulated using Kronecker matrix products and therefore fitted using Henderson’s mixed model equations and standard animal breeding software.Models for genetic evaluation in the three-way crossbreeding system are presented. They provide estimated breeding values for both purebred and crossbred performances, and can use pedigree-based or marker-based relationships, or combined relationships based on both pedigree and marker information. This provides a framework that allows information from three-way crossbred animals to be incorporated into a genetic evaluation system. Commercial pig producers generally use a terminal crossbreeding system with three breeds. In this system, F1 sows from two maternal breeds are mated to purebred boars from a breed that has high-level production traits  to produce pigs for slaughter. Commonly, boar lines in Europe are Duroc and Pietrain and sows are crosses between Large White and Landrace. Genetic evaluation is usually done within each of these breeds based on recorded phenotypes on purebred animals. However, ideally genetic evaluation of purebreds should incorporate phenotypes of interest recorded on crossbreds, and breeding values for performance in the three-way cross should be estimated.Many pig breeding organisations have started to use genomic selection , for whiFor two-way terminal crossbreeding  and m(i), respectively, and b and breed i and i.Lo et al.  derived b specific partial relationship matrix and matrix b and breed b specific partial genetic vector, and b and breed García-Cortés and Toro  showed trix (see ). In thiLegarra et al.  proposedFirst, partial genetic and common genetic approaches for constructing pedigree-based relationships are presented, then the corresponding two different ways of constructing marker-based relationships are presented, and finally the genetic variances and covariances in model  are showFor the three-way crossbreeding system, the decomposition of the additive genetic effects by García-Cortés and Toro  is as foIn this way, a breed-specific partial genetic effect is defined for all animals containing the specific breed, and a breed-segregation partial genetic effect is defined for crossbred b proportion, and the breed-segregation partial relationship matrix is defined by the recursive formulas:The variance-covariance matrices of the partial genetic effects become (García-Cortés and Toro ):\docume in Eqs.  and 4)..\documenTo illustrate the different partial relationship matrices, we analysed the small pedigree in Table i, where f(i) is not inbred and since mother m(i) is not inbred, the variance is constant. In this way, the Mendelian sampling error term can be included into the residual error term Wei and van der Werf  presenteentclass1pt{miniman et al. , such a Note that model  with relb, andb, i.e. base animals are inbred with coefficient b and In the previous subsection, base animals were assumed to be unrelated. An alternative proposed by Legarra et al.  is to asmentclass2pt{minimLegarra et al.  suggestea et al.  showed tenderson  and submlgorithm  are as ub animals, and therefore the genetic variances for crossbred The parameter 2 in Eq.  does nota et al.  explaineb animals, matrices Marker-based partial relationship matrices are constructed by tracing breed of origin of alleles and defining relationships according to breed of origin. Assume that breed of origin of alleles can be determined for all animals and define breed-specific allele content matrices as: matrix software .i, j) equal to i inherited an Marker-based breed-specific partial relationship matrices are constructed as follows (details can be found in the “n is the number of markers. Note that diagonal elements of The breed-segregation partial relationship matrix is defined as:Relationship matrices that combine pedigree and marker information , 4 can tlgorithm ; see Chrlgorithm .The breed-specific partial marker-based relationship matrices above require estimates of breed-specific allele frequencies. Such estimates can be obtained from marker genotypes of purebred animals and breed-specific marker alleles for crossbred animals. Furthermore, there is a need to adjust these matrices to be compatible with partial pedigree relationship matrices similar to Christensen et al. , 19, i.eFinally, to incorporate the fact that marker genotypes only capture a fraction of the genetic effects, the partial marker-based relationship matrices enotypes .s is scaling parameter. As in Christensen [s such that the pedigree-based and marker-based relationship matrices are compatible. Parameters in matrix s can be estimated by matching s and can therefore be solved directly to obtain estimates.The marker-based relationship matrix is constructed as usual across all genotyped animals:istensen  and Legaistensen , we chosa et al. . For exaThe relationship matrix that combines pedigree and marker information becomesIn the previous sections, partial genetic and common genetic models for additive genetic effects for crossbred entclass1pt{minimaFor the partial genetic case, the vector of genetic effects on crossbred entclass1pt{minimaFor the common genetic case, all individuals are related, and breeding values for crossbred entclass1pt{minimaIn the common genetic case, there are three parameters For three-way crossbreeding, we presented models based on both pedigree-based, marker-based and combined relationships. Using combined relationship matrices results in a model for genetic evaluation where both pedigree and marker genotypes are used simultaneously for genetic evaluation, i.e. a single-step method for genomic evaluation. This paper provides the models and mathematical formulas, but a numerical implementation is needed before the methods are ready for use in practice. Such methods make it possible to incorporate phenotypes and genotypes on crossbreds into an existing genetic evaluation system, assuming that such a system is based on a single-step method.The models for three-way crossbreeding investigated in this paper were four-variate models where each variable was measured in a specific population, Two types of approaches for constructing additive relationships were presented, based on different assumptions about allele substitution effects of causal loci or SNPs. In the partial genetic approach, allele substitution effects of SNPs were assumed independent between breeds, whereas in the common genetic approach, they were assumed to be the same in different breeds. The partial genetic approach requires that alleles are traced according to breed of origin, which is feasible in some scenarios but may be difficult with sufficient accuracy in others. In particular, when crossbred Other terminal crossbreeding systems are of interest in pig production. Models for two-way crossbreeding are relevant for sow-traits measured on animals from breed n et al.  using paMany papers have reported genetic correlations between purebred and crossbred performances –26. The Models for genetic evaluation in the three-way crossbreeding system are presented. These models provide estimated breeding values for both purebred and crossbred performances, and can use pedigree-based or marker-based relationships, or combined relationships based on both pedigree and marker information. This provides a framework that allows information from three-way crossbred animals to be incorporated into a genetic evaluation system.


Squalius cephalus). This fish species is not stocked and copes well with altered habitats, enabling an assessment of the effects of fragmentation per se. Using microsatellites, we genotyped 2133 chub from 47 sites within the catchment fragmented by 37 hydroelectric power stations, two weirs and the Rhine Falls. The shallow genetic population structure reflected drainage topology and was affected significantly by barriers to migration. The effect of power stations equipped with fishpasses on genetic differentiation was detectable, albeit weaker than that of man‐made barriers without fishpasses. The Rhine Falls as the only long‐standing natural obstacle (formed 14 000 to 17 000 years ago) also had a strong effect. Man‐made barriers also exacerbated the upstream decrease in allelic diversity in the catchment, particularly when lacking fishpasses. Thus, existing fishpasses do have the desired effect of mitigating fragmentation, but barriers still reduce population connectivity in a fish that traverses fishpasses better than many other species. Less mobile species are likely to be affected more severely.The Rhine catchment in Switzerland has been transformed by a chain of hydroelectric power stations. We addressed the impact of fragmentation on the genetic structure of fish populations by focusing on the European chub ( The ongoing landscape modification by humans leads to massive destruction or alterations of pristine ecosystems by a combination of fragmentation, habitat loss and degradation      (Hanfling and Weetman Gasterosteus aculeatus Linnaeus)   , gudgeon (Gobio gobio) and minnow (Phoxinus phoxinus) by Blanchet et al.  as our model species, a European cyprinid species that is very common in the Swiss midlands, where it lives in high number in rivers of the Barbel and Grayling Region [categorization according to Huet  of dams enable migration through fishpasses. We selected the common chub  to Huet ] as well to Huet . Chub re to Huet . Due to  to Huet . They re to Huet . Chub ar to Huet  or even  to Huet . Chub ar to Huet . The ups to Huet . Female  to Huet  and can  to Huet . BecauseIn this study, we describe the genetic population structure of the chub in the main Swiss lowland rivers of the Rhine catchment and assess the effects of barriers such as hydroelectric power plants on population connectivity and genetic diversity. In particular, we ask whether fishpasses that are present at most  barriers do have the desired effect of mitigating fragmentation.2, discharge 1037 m3/s). In addition to the Rhine, the Rhine catchment includes the river Aar  with its two tributaries Reuss  and Limmat   were air dried in 8‐strip microtubes before adding 300 μL of TNES buffer  and 5 μL of 10 mg/mL proteinase K . After incubation for 60 min in an incubation shaker  at 300 rpm, the proteins were precipitated by adding 85 μL of 5 m NaCl and shaking the tubes for 10 s. Proteins were pelleted in a centrifuge  at 4700 rpm for 10 min. The supernatants were carefully transferred into a 96‐deep‐well block. Afterwards, the DNA was precipitated by adding 400 μL of ice‐cold 100% ethanol and gentle mixing with a pipette. The DNA was pelleted by centrifugation for 10 min at 4700 rpm, washed with 70% ethanol, centrifuged again for 10 min at 4700 rpm and air dried. Finally, the DNA was resuspended in 100 μL of TE buffer  and stored at −20°C.DNA was extracted following the salting‐out protocol of Sunnucks and Hales  adapted μL PCR contained 5 μL Qiagen Multiplex PCR Master Mix , 3 μL of ultrapure water, 1 μL of DNA template and 1 μL of primer mix. The forward primers for the two multiplex reactions were labelled with fluorescent dyes , such that loci with the same dye had nonoverlapping allele size ranges. To balance peak heights, labelled forward primers of strongly amplifying loci were partially supplemented with unlabelled primer. Multiplex 1 contained the following primers : N7K4 0.4 nm labelled (YYE) and 14.5 nm unlabelled, N7G5 0.9 nm labelled (AT550) and 30.5 nm unlabelled, LC128 4.7 nm (AT550), LC 27 1.8 nm (FAM), LC 290 1.2 nm (YYE). Multiplex 2: LceA149 0.5 nm labelled (FAM) and 10 nm unlabelled, LC32 0.8 nm labelled (AT565) and 13.7 nm unlabelled, LceC1 1.6 nm (AT550), LceCb 6.8 nm (FAM), LC93 1.9 nm (AT565).Ten microsatellite loci amplified in two multiplex PCR were used to genotype the individuals: LC128, LC27, LC290, LC32, LC93  or 57°C (multiplex 2) for 90 s, extension at 72°C for 90 s, followed by a final extension at 72°C for 10 min .PCR thermal conditions were as follows: initial μL of PCR product was mixed with 8 μL Hi‐Di Formamide and 1 μL size standard diluted 1: 7 .PCR products were analyzed on an ABI 3730 capillary sequencer . For loading, 1 The genotyping accuracy was tested by extracting and genotyping a subset of samples twice. Locus LC290 showed a dropout of large alleles, due to its wide allele size range . To avoid genotyping errors resulting from this problem, all homozygotes with low peak heights were reamplified with more cycles and scored again. All other loci had scoring errors below 1%. Nevertheless, the scoring was performed three times to minimize mistakes. For the final data set, only individuals that were genotyped successfully at nine of the ten loci at least were considered . That resulted in an average of 45 individuals per site. For the sample sizes per site, see Table HO), expected heterozygosity (HE), mean number of alleles and allelic richness (AR) standardized for smallest N were calculated using FSTAT 2.9.4  was calculated according to Weir and Cockerham  to one . The neighbouring system is carried out by Voroni tessellation  was tested with 20 runs, for Kmax = 2–9. The total number of sweeps per run was 300 000 after a burn in of 50 000 sweeps. The best‐supported number of genetic clusters was determined by plotting the mean Deviance Information Criterion (DIC) of the 20 runs over the respective Kmax . Best Kmax was run an additional 20 times. For averaging the results and taking into account ‘label switching’, the programme CLUMPP 1.1.2  with the matrix of pairwise waterway distances extracted with ArcMap10  between sampling sites and the matrix of the pairwise number of barriers (barrier count) between sites. Partial Mantel tests were carried out according to Smouse et al.  approach, correlating the matrix of pairwise e et al. , using te et al.  with 10 n = 33), and power stations without fishpass (n = 5). The last group comprised one barrier (the two adjacent weirs) that was not a power station but was included because it could be assumed to be equally obstructive to fish migration. The age of artificial barriers could also influence the differentiation among fish populations they separate. However, we found that the pairwise number of artificial barriers separating two sampling sites and their cumulative age were strongly correlated (r = 0.97). This collinearity precluded an independent assessment of the age of barriers, thus we restricted our analyses on the number of barriers of each type. We constructed six linear models for comparison that had pairwise FST as the dependent variable and the following predictors:Assessing the impact of different barrier types on gene flow is important for assessing the effectiveness of the fish ladders. To evaluate the impact of the different types of barriers, we split the 39 barriers in our system into different groups: the Rhine Falls as the only major natural barrier (14 000–17 000 years old), hydroelectric power stations with fishpass  + count of barriers with fishpassdistance + Rhine Fallsdistance + Rhine Falls + count of all other barriersdistance + Rhine Falls + count of barriers with fishpass + count of barriers without fishpassn in the calculation of AIC to account for nonindependence inherent in pairwise data. R 2.15.1 was used to perform the analyses  as the sum of genetic distances for the stream sections between them. The coefficient of determination R2 is used to assess the fit of the resulting model to the data. Because a canal between the Aare river and Lake Biel  was investigated with a similar model selection approach as used for genetic differentiation. Six linear models of AR were constructed with the same predictors as above, but here distance was calculated from an arbitrary reference point below the most downstream site, namely where the Rhine crosses the Swiss border, and the numbers of the different types of barriers were counted along the river(s) between this reference point and each site.α = 0.05, this is close to the approx. 21 significant tests expected by chance, and the deviations were spread erratically over loci and populations, including similar numbers of heterozygote excesses and deficits. No deviation was significant after Bonferroni correction. Accordingly, only four of 47 multilocus estimates of FIS differed significantly from 0 at α = 0.05, and none of these deviations was significant at a Bonferroni‐corrected α of 0.001 (0.05/47 sites) . Table Ne obtained with NeEstimator. These are generally very high with most values in the hundreds or thousands. For many samples, Ne was estimated as infinite and the upper confidence limit reached infinity in the majority of cases. The lowest estimates tended to come from the river Limmat, which was the only river for which estimates <100 were obtained .Sample sizes and genetic diversity statistics for all collection sites are summarized in Table The DIC plot indicated four as the most likely number of genetic clusters present in our chub samples. These clusters corresponded well to the populations in the four rivers we sampled from above their confluence Fig. .FST and distance with increasing scatter . This corresponds to a case I relationship as defined by Hutchison and Templeton  as well as with increasing numbers of barriers separating the sites . Despite our efforts to break the collinearity when choosing sampling sites, waterway distances and barrier counts between sites were positively correlated with R = 0.827. However, the partial Mantel tests indicated that the number of barriers between sites is a somewhat better predictor of genetic differentiation than distance per se .The IBD plot shows a monotonic positive relationship between pairwise empleton  and is iFST between samples  and the model not distinguishing between different types of barriers  received virtually no support from the data. These results provide strong evidence that barriers influence the genetic structure of chub in the large Swiss lowland rivers and that fishpasses appear to mitigate the negative effects of barriers on population connectivity. The parameter estimates from the best‐supported model iii allow gauging the effects of barriers with and without fishpass relative to free‐flowing stretches of river. According to this model, the effect of a barrier without fishpass is equivalent to approx. 102 km of uninterrupted river distance, while barriers with fishpass correspond to approx. 12 km. These values are in a similar range when calculated from the second‐ranked model vi , which assigns an even stronger effect to the only long‐standing natural barrier, the Rhine Falls, corresponding to approx. 220 km of uninterrupted river. Unsurprisingly, commonality analysis of the two best‐supported models showed substantial contributions of all predictors via common effects, reflecting the multicollinearity of distance and the numbers of different barriers between sites, but it also showed that in both models the barriers without fishpasses had the largest unique effect on genetic differentiation (Table The best‐supported model predicting pairwise FST) from the most downstream site  was the most complex model vi that fitted separate effects for distance, the Rhine falls, barriers with fishpasses and barriers without fishpasses  than by distance , and this is to a large extent due to the samples from the river Limmat   positive Table . A possiWe show that the chub in the Swiss lowland rivers has a shallow population structure that is shaped by drainage topology, but also significantly affected by man‐made barriers to migration, most of which are hydroelectric power stations. From a management perspective, it is important to note that the fishpasses installed at many of these stations do indeed improve fish population connectivity across the barrier. Nevertheless, even barriers with fishpasses have a detectable effect on genetic differentiation, in a fish that is relatively large and mobile and copes equally well with lotic and lentic environments, that is a species that should be among those least susceptible to habitat fragmentation. The negative effects of river fragmentation are likely to be more severe for many other river‐dwelling fish species. Fortunately, improvements in the design of fishpasses show great promise in reducing these effects further, as seen for the Rheinfelden power station mentioned above. It is to be hoped that similar improvements will soon be realized for other barriers as well.http://dx.doi.org/10.5061/dryad.n41nk.Data available at Dryad Digital Repository: Figure S1. Map showing the location of all barriers included in Supplementary Table S1.Click here for additional data file.Figure S2. Neighbourhood Diagram used in the Bayesian clustering analysis (TESS 2.3.1 output).Click here for additional data file.Figure S3. Genetic cluster affiliations of individual fish estimated at Kmax = 4 in TESS 2.3.1 and visualized with DiSTRUCT.Click here for additional data file.Figure S4. Isolation‐by‐distance and ‘isolation‐by‐barriers’ of chub in the Swiss Lowland rivers depicted as linear regression plots of pairwise FST against waterway distance (A) and the number of barriers (B) between sampling sites.Click here for additional data file.Table S1. Table reporting ages of the barriers and their fishpasses.Click here for additional data file.


Achillea millefolium aggregate is one of the most diverse polyploid complexes of the Northern hemisphere and has its western Eurasian boundary in the Iberian Peninsula. Four ploidy levels have been detected in A. millefolium, three of which have already been found in Iberia , and a fourth (tetraploid) reported during the preparation of this paper. We collected a sample from 26 Iberian populations comprising all ploidy levels, and we used microsatellite markers analyzed as dominant in view of the high ploidy levels. Our goals were to quantify the genetic diversity of A. millefolium in the Iberian Peninsula, to elucidate its genetic structure, to investigate the differences in ploidy levels, and to analyse the dispersal of the species. The lack of spatial genetic structure recovered is linked to both high levels of gene flow between populations and to the fact that most genetic variability occurs within populations. This in turn suggests the existence of a huge panmictic yarrow population in the Iberian Peninsula. This is consistent with the assumption that recent colonization and rapid expansion occurred throughout this area. Likewise, the low levels of genetic variability recovered suggest that bottlenecks and/or founder events may have been involved in this process, and clonal reproduction may have played an important role in maintaining this genetic impoverishment. Indeed, the ecological and phenologic uniformity present in the A. millefolium agg. in Iberia compared to Eurasia and North America may be responsible for the low number of representatives of this complex of species present in the Iberian Peninsula. The low levels of genetic differentiation between ploidy levels recovered in our work suggest the absence of barriers between them.The  Achillea millefolium agg. is one of the most diverse polyploid complexes of the Northern hemisphere in terms of morphological, genetic and ecological features  ,12 x, 4xferences ). The diribution . In the The lack of genetic studies on Iberian yarrows motivated us to investigate this species aggregate at population level using SSR markers (microsatellites). Microsatellites are widely used to investigate genetic variation, structure and dynamics both at population and species level e. g., ,18). The. The18])A. millefolium in the Iberian Peninsula; (ii) to elucidate its genetic structure and gene flow; (iii) to investigate the putative genetic differentiation between ploidy levels; and (iv) to evaluate alternative dispersal methods for this species within the reported area.The aims of the present work are: (i) to quantify the genetic diversity of A. millefolium in the Iberian Peninsula , measurements were carried out on five individuals from desiccated leaves of the same individuals used for microsatellite analysis. For the rest of populations, one to three individuals were used. Foliar segments were excised from the middle part of the leaf, softened in NaOH 0.5 M for 24 h, and bleached in a saturated solution of chloral hydrate for 6–12 h. Epidermis fragments were excised from the adaxial side of the distal half of the segments, semipermanently mounted in glycerol sealed with nail varnish, and measured with a micrometric ocular mounted in an Olympus CH-2 optic microscope at 400x. Two measures, long axis (stoma length or SL) and short axis (stoma width or SW), were made on an ideal ellipse adapted to the stoma. Measurement of SL equals the length of the guard cell. For each mounted slide, measurements were made on different zones of the epidermis of the segment, discarding stomata on the margins of the leaf. A total of 30–63 stomata were measured per population. For the five populations for which we lacked chromosome counts, 100–120 stomata were measured.A. millefolium from Iran [A. millefolium, plants were collected at least 1 meter apart to avoid duplication. For this reason, we obtained data for 12–16 random individuals per site, accounting for a total of 375 individuals belonging to 26 populations  and data was scored manually.Total genomic DNA was extracted from desiccated leaves using the CTAB method of , as modirom Iran  were tesrom Iran . Differeulations  and reprx, 4x, 6x and 8x), we followed the approach of [As mentioned in the introduction, polyploidy frustrates the statistical treatment of microsatellite data and prevents the performance of many analyses and calculations, especially when individuals with different ploidy levels co-exist in the same matrix, as in the present work –24,31–34roach of  and recoroach of .k), the mean number of alleles (Na), the number of effective alleles (Ne), the number of private alleles (PA) and the unbiased diversity (uh) were calculated. These parameters were also computed at ploidy level. The genetic relationship between pairwise populations and ploidy levels was estimated by means of Nei’s unbiased genetic distance (D) and ΦPT, a measure of population genetic differentiation analogous to FST (GenAlEx documentation). The significance level of ΦPT was computed through 99 permutations.All the genetic diversity parameters were computed using GenAlEx 6.5 ,42. At pA. millefolium in Iberia. Firstly, the Bayesian method implemented in the software Structure 2.2 [4 Markov MonteCarlo (MCMC) replications. This analysis did not yield any structure in the data. We carried out a second analysis to see whether we could improve the results, restricting K to a range varying from K = 1 to K = 15. Each K was estimated 10 times, the length of burn-in period comprising 105 iterations and 106 MCMC replications. The optimal number of clusters was determined using the ΔK statistical approach of [Structure assumes Hardy-Weinberg equilibrium within populations and linkage equilibrium between loci within populations [tructure, we also applied a non-model-based approach: nonhierarchical K-means clustering [Four different approaches were used in order to gain insights into the patterns of spatial genetic structure of ture 2.2  was usedroach of . Structuulations  and, givustering , as implustering . This apustering ). The anustering ), based ustering . We perfPT) and the matrix of geographical distances, by applying the Mantel test (1000 permutations).The remaining three methods were performed using GenAlEx 6.5. software. An analysis of molecular variance (AMOVA) was computed with respect to three different factors: 1) between and within populations; 2) between and within ploidy levels; and 3) between and within soil type . In addition, a Principal Co-ordinate Analysis (PCoA) was performed based on the pair-wise genetic distances of individuals and populations. Finally, isolation-by-distance between populations was investigated by computing the correlation between the matrix of pair-wise population genetic distance (Φuh) for A. millefolium was 0.077. At population level (k) ranged from 16 (Hu1) to 35 , the mean number of alleles (Na) ranged from 0.224 (Hu1) to 0.645 (B1) and the number of effective alleles (Ne) ranged from 1.032 (Hu1) to 1.169 (B1). Forty-five exclusive alleles were detected ranging from one  to six (HU3), all of them occurring at low frequencies (mean frequency 0.097). Only five of the populations surveyed lacked private alleles . The lowest value for unbiased diversity was found in population Hu1 (uh = 0.022), whereas the highest value was found in population B1 (uh = 0.112). Nei’s genetic distance (D) between pairs of populations ranged from 0.002 between L1 and Sa1 to 0.049 between Gi1 and Lo1 (see PT were significant (P < 0.05) and ranged from 0.044 between B1 and S2 to 0.334 between Lu1 and S1  ranged from 22 (2x) to 77 (8x), the mean number of alleles (Na) ranged from 0.383 (2x) to 0.567 (6x) and the number of effective alleles (Ne) ranged from 1.106 (8x) to 1.141 (4x). Fifty-five exclusive alleles were detected ranging from one (4x) to 28 (8x). The lowest value for unbiased diversity belonged to 2x and 8x (uh = 0.071) cytotypes, whereas the highest value belonged to 4x and 6x (uh = 0.088) cytotypes. Nei’s genetic distance (D) between ploidy levels ranged from 0.002 between 6x and 8x to 0.030 between 2x and 4x (see PT between ploidy levels were significant (P < 0.05) and ranged from 0.005 between 6x and 8x to 0.105 between 2x and 4x cytotypes . Similarly, when the partition was based on the ploidy level, most of the variation was again detected within groups (97%), while only 3% of the variation was detected between ploidy levels . Finally, the same pattern was detected when considering soil type, since 99% of the variation was detected within groups, while only 1% of the variation was detected between the two soil types . With regard to the PCoA analysis, the first three components explain 43.27% and 64.55% of total variance of data when considering individuals and populations, respectively. At individual level, in the scatter-plot for the two first components (35.70% of the variance), individuals are distributed across the graph without any grouping pattern, regardless of the population to which they belong ,,Structury belong . Similary belong  in the sy belong . Accordiy belong , no corrA. millefolium in Iberia grows only in ruderal and nitrified environments, and this ecological uniformity may, in turn, have introduced similar adaptive pressures to the entire colonized area and may, in part, have been responsible for the genetic uniformity of this species. Moreover, a study investigating the colonization of North America [A. millefolium var. gigantea or A. millefolium var. puberula) and phenological differences , the few taxa comprising Iberian A. millefolium agg. may be due to their uniform ecological preferences .Our results for Bayesian clustering Figs  and 6, n America , where y America . Given tA. millefolium to the Iberian Peninsula has not had sufficient time to produce new variations and to develop a clear genetic structure [A. millefolium agg. phylogeny based on both nuclear and plastid genomes found low levels of genetic variation and limited resolution [A. millefolium, and thus, the Iberian Peninsula should be considered both in evolutionary and geographical terms, a dead-end for this species. However, given its therapeutic properties and ornamental value, several independent, human-mediated introductions  cannot be ruled out, and this may explain both the low levels of genetic diversity and the presence of exclusive alleles found in most of the populations studied.Similarly, the low levels of genetic variability recovered suggest that bottlenecks and/or founder events may have been involved in this process ,52. Whentructure . Furthertructure . Previousolution . Accordisolution , the gensolution ,55. Convsolution ,56–60. ORubus alceifolius displays least genetic diversity in areas where it has become a serious weed [Achillea millefolium is considered such a weed and meets the parameters attributed to invasive plants [A. millefolium during the colonization of the Iberian Peninsula may be considered as invasive.Another factor that may affect genetic variability is invasiveness. For example, ous weed , and it e plants , namely,A. millefolium, it is expected to be greater for polyploids. Polyploidy allows more recombination events to occur during meiosis and therefore results in increased diversity of the genome, thus allowing adaptive plasticity [x and 6x ecotypes of A. millefolium agg. in America [A. millefolium agg. This may have masked the separation between sympatric populations and highlights the lack of barriers between polyploids, even those of different species. Consequently, previously published karyological data [A. millefolium agg. are quite similar, show limited structural differentiation and lack obvious obstacles to hybridization.With reference to the correlation between ploidy level and genetic diversity in asticity ,62 and rasticity . Accordiasticity  found th America  and this America  highlighcal data –65, provAchillea millefolium aggregate, and this involved a limited number of individuals. The species behaves in Iberia as an invasive plant colonizing ruderal and disturbed habitats. The low genetic variability of populations and lack of differentiation between the different ploidy levels all indicate that the Iberian Peninsula constitutes a geographical cul-de-sac for the expansion of the aggregate and a genetic and evolutionary dead-end for the Iberian populations.All of our results indicate a very recent colonization of the Iberian Peninsula by the S1 Dataset0 = absence; 1 = presence. See (TXT)Click here for additional data file.S1 FigSL = Stomata length; SW = Stomata width.(EPS)Click here for additional data file.S2 FigSee (EPS)Click here for additional data file.S1 TableSee (DOCX)Click here for additional data file.S2 TableSee (DOCX)Click here for additional data file.S3 TableSee (DOCX)Click here for additional data file.


More than 14 million  live in the Bangkok Metropolitan Region, located in Central Thailand (http://worldpopulationreview.com/countries/thailand-population/) . Subsequent generations of Chinese in Thailand have Thai names, speak Thai, consider themselves Thai, and have married original Thai. Therefore, it is now very difficult to distinguish the original Thai from Chinese Thai. Although not strictly prohibited, consanguineous marriages are strongly discouraged in both original Thai and Thai–Chinese cultures.Thailand's history goes back more than 700 years, and since 1782, Bangkok has been the capital. The population of Thailand in 2013 was 69.52 million, ranked 20th in the world (n/) Fig. . Seventyhttp://data.worldbank.org/country/thailand). The politics of Thailand are a constitutional monarchy, whereby the Prime Minister is the head of government and a hereditary monarch is head of state, and the judiciary branch is independent of the executive and the legislative branches. Since 2001, populist policies have been implemented including a universal healthcare scheme, which has tremendously impacted the medical system and genetic service in Thailand.Thailand's gross domestic product (GDP) was 366 billion U.S. dollars (USD), ranked 21st in the world, and is grouped in the upper middle income category by the World Bank. (http://bps.ops.moph.go.th/).Thailand has had a successful history of health improvement with a life expectancy of 71 years for males and 78 years for females, and with a reduction in infant mortality from 68 per 1000 in 1970 to 13 per 1000 today  with 125,866 beds were available to provide in-patients services. Both the private and public sector healthcare workers receive training funding from the human resource development effort of the MOPH  or the United States (U.S.A.) established clinical genetic services in Thailand in both hematology and pediatric departments. In the 1980s, cytogenetic services began in Thai university hospitals as laboratory specialist received training abroad, and since the 1980s, governmental cytogenetic laboratories have become available throughout the country. On a much smaller scale, private sector laboratories also provide genetic services.Molecular genetic testing for thalassemia became available 30 years ago and was the first available genetic test excluding karyotypes. With its abundance of thalassemia, Thailand has always been one of the world leaders for advancing research in hemoglobinopathies  have been made available over the past 20 years with test panels now comparable to most developed countries. Noninvasive prenatal fetal trisomy (NIFTY) testing as well as rapid invasive diagnostic methods for common aneuploidy are available as send out tests to out of country laboratories. Two university-based laboratories and one private laboratory are currently providing PGD services for a few Mendelian diseases including thalassemia  technologies for research have been available in Thailand for a few years with at least five institutes in Thailand possessing NGS technology. NGS services are currently limited by a deficiency in well-trained bioinformaticians.With all the progress in genetic services over the last 40 years, there are still only 22 clinical geneticists available to cover 69 million people, and much of their time is spent as genetic laboratory supervising. There are no genetic counselors in Thailand, adding to the manpower shortage. A local postgraduate fellowship training program for medical (pediatric) geneticist is currently approved for 2014 with the hope of supplementing the next generation of graduates to work in this field.Neonatal screening programs are administered by two organizations, the national neonatal screening operation center of MOPH that operates in all provinces and metabolic genetic center of Mahidol University located in Bangkok. Since 1996, these centers have provided coverage for 95% of all newborns for congenital hypothyroidism  and pilot testing for HLA-B*1502 has been launched in Bangkok through supports of NHSO's Bangkok Office.Pharmacogenetics testing has been implemented nationwide and in 2013, university- based medical centers including Chulalongkorn University, Mahidol University, KhonKaen University, and Prince of Songkla University established pharmacogenetics testing services. Recently, HLA-B*1502 testing used for prevention of severe cutaneous adverse reactions (SCAR) from carbamazepine was proven to be cost-effective in Thailand which is the reason this makes up most of the pharmacogenetic testing .The Thailand Birth Defects Registry which provides preventive measures and an educational program was developed in 2008 to decrease the incidence of birth defects and to provide care for children with birth defects in a holistic manner. Care maps of five birth defects – Down syndrome, neural tube defects, cleft lip/palate, limb defects, and Duchenne muscular dystrophy – were established  can prevent many congenital anomalies. In 2003, a survey of 383 pregnant Thai women showed 23% knew that FA helped to prevent birth defects, 3% knew that FA should be taken before pregnancy, and only 0.3% reported taking FA before pregnancy ; however, the direct-to-consumer genetic tests are regulated by consumer law and such testing is currently not included under the jurisdiction of the national FDA. It will be interesting to see how Thai society will cope with the influx of personalized medicine as this type of testing becomes more available. There is also currently no law to prevent discrimination based on genetic status such as the Genetic Information Nondiscrimination Act (GINA) in the United States.Medical school curriculum has included medical genetics as one of the required courses for more than 20 years. However, due to the shortage of medical genetic specialists, these courses are often not given by geneticists. Biologists, medical technologists, and clinical pathologists in some universities have been responsible for such teaching. With different types of curriculum, medical genetics is generally offered either as a week- or month-long program during preclinical rotations.Clinical genetics has not been recognized as a specialty by the Medical Association of Thailand. However, a formal training program for pediatricians to become medical geneticists will be launched in the academic year of 2014, under the Royal College of Pediatricians. Also as noted above, there are no training programs for genetic counselors.Molecular genetics is a well-established field in Thailand. There are hundreds of scientists using molecular techniques for research. However, cytogeneticists and biochemical geneticists are extremely insufficient.Thailand harbors rare genetic diseases, unique to Thailand, which have contributed to the international knowledge on diseases such as benign adult familial myoclonic epilepsy , a type of neural tube defect, characterized by a congenital bone defect of the anterior cranium between the frontal and ethmoidal bones resulting in herniation of meninges and brain tissues Fig. , has a up63 which causes an isolated cleft lip  (Leoyklang et al. Oral clefts are common in Thailand and genetic studies have demonstrated many genetic susceptibility loci and new mutations (Shotelersuk et al. Genetic diseases with a higher prevalence in Thailand compared to other parts of the world are a compelling reason to conduct research here. Unfortunately, there are limitations including inadequate budget, insufficient advanced technologies and knowledgeable personnel in bioinformatics, and inefficient supporting systems. However, we are optimistic that collaborations between local and foreign researchers will lead to better care of patients both in Thailand and globally.


In addition, three environmental parameters – depth and two depth-independent temperature indices – were collected on a 4 m grid across 85 locations throughout the reef. We use ordinary kriging to spatially interpolate our environmental data and estimate the three environmental parameters for each colony. Partial Mantel tests indicate a significant correlation between genetic relatedness and depth while controlling for space. These results are also supported by multi-model inference. Furthermore, spatial Principle Component Analysis indicates a statistically significant genetic cline along a depth gradient. Binning the genetic dataset based on size-class revealed that the correlation between genetic relatedness and depth was significant for new recruits and increased for larger size classes, suggesting a possible role of larval habitat selection as well as selective mortality in structuring intra-reef genetic diversity. That both pre- and post-recruitment processes may be involved points to the adaptive role of larval habitat selection in increasing adult survival. The conservation importance of uncovering intra-reef patterns of genetic diversity is discussed.Relative to terrestrial plants, and despite similarities in life history characteristics, the potential for corals to exhibit intra-reef local adaptation in the form of genetic differentiation along an environmental gradient has received little attention. The potential for natural selection to act on such small scales is likely increased by the ability of coral larval dispersal and settlement to be influenced by environmental cues. Here, we combine genetic, spatial, and environmental data for a single patch reef in Kāne‘ohe Bay, O‘ahu, Hawai‘i, USA in a landscape genetics framework to uncover environmental drivers of intra-reef genetic structuring. The genetic dataset consists of near-exhaustive sampling ( Local adaptation occurs when populations become fine-tuned to their environment through the process of natural selection. Locally adapted genotypes, however, can be swamped out by the arrival of immigrants from populations differently adapted. This is the dominant paradigm in terrestrial systems, where physical isolation is considered important, if not essential for populations to diverge . In contIn marine systems, indication of local adaptation comes from both experimental studies involving reciprocal transplant experiments e.g., , 9) as w as w9]) The term local adaptation implies the existence of patterns on a small spatial scale. This is often interpreted to mean adaptive differences between discreet demes existing in distinct habitats. It is important to note, however, that local adaptation also can refer to differences within continuous populations along a continuous environmental gradient . Small-sInvestigations that look at individual-level genetic variation across an environmental gradient are common in the terrestrial plant literature . CoralsPocillopora damicornis, within a single reef. The reef we chose was in Kāne‘ohe Bay, Hawai‘i, USA and was ∼40m in diameter with an abundance of Pocillopora damicornis colonies and a depth range of between 5 and <1 m. P. damicornis is distributed across the Indo-Pacific and can be quite common in some locations. In our genetic study )th space . We teasth space , here weth space –66 that p = 0.019) with regards to depth. In other words, genetic diversity exhibits a depth cline whereby individuals at similar depths tend to have more similar genotypes than individuals at different depths. When we restrict our partial Mantel tests to the smallest size class, we see that this is true even for likely recent coral recruits. Planulae of P. damicornis have the ability to remain in the plankton for more than 100 days after release [P. damicornis to disperse over long distances, it has also been shown that planulae are competent  as soon as 12 h after release [Based on the Monte Carlo randomization procedure of our sPCA, allele frequencies at our study site show positive autocorrelation . Put another way, since there are no genetic differences between newer and older coral recruits overall, the increasing correlation between genetic relatedness and depth for increasing size classes is being driven by differences in the depth distribution between older and younger corals, thus pointing to depth as a selective factor in post-recruitment processes. In particular, increased environmental dependence of genetic relatedness for larger size classes may be explained by selective mortality . In. InAMOVAy  and displaying the combined mixed colors.(TIF)Click here for additional data file.


Biogeography investigates spatial patterns of species distribution. Discontinuities in species distribution are identified as boundaries between biogeographic areas. Do these boundaries affect genetic connectivity? To address this question, a multifactorial hierarchical sampling design, across three of the major marine biogeographic boundaries in the central Mediterranean Sea  was carried out. Mitochondrial COI sequence polymorphism of seven species of Mediterranean benthic invertebrates was analysed. Two species showed significant genetic structure across the Tyrrhenian-Ionian boundary, as well as two other species across the Ionian Sea, a previously unknown phylogeographic barrier. The hypothesized barrier in the Ligurian-Tyrrhenian cannot be detected in the genetic structure of the investigated species. Connectivity patterns across species at distances up to 800 km apart confirmed that estimates of pelagic larval dispersal were poor predictors of the genetic structure. The detected genetic discontinuities seem more related to the effect of past historical events, though maintained by present day oceanographic processes. Multivariate statistical tools were used to test the consistency of the patterns across species, providing a conceptual framework for across-species barrier locations and strengths. Additional sequences retrieved from public databases supported our findings. Heterogeneity of phylogeographic patterns shown by the 7 investigated species is relevant to the understanding of the genetic diversity, and carry implications for conservation biology. Through the study of species distribution as well as patterns of genetic structuring, a link between biogeography and phylogeography can be explored, which aids in the identification of discontinuities in the spatial distribution and genetic diversity of species Major barriers to gene flow between oceanic basins have previously been identified . In each site 2 sampling areas about 25 km apart were randomly selected, and named counter-clockwise starting from Location 1 , Polyplacophora (1), Ascidiacea (1); Crustacea (1), and Porifera (1). Data retrieved on life history traits and larval ecology are summarized in In each area 30 individuals per species were collected, fixed in 80% ethanol and maintained at 4°C prior to processing. Only the sponge was sampled as pieces rather than as whole organism. To minimize the possibility of clonality, samples were collected at least 5 m apart http://www.fokker.wi.mit.edu/primer3/input.htm) in order to increase amplification success and the quality of sequences obtained . The COI gene was chosen for analysis due to its intermediate rate of variation that provides information on genetic structure across a wide range of spatial and temporal scales obtained .2 25 mM, 0.5 µl of dNTP mix 10 mM, 0.5 µl of each primer, 0.15 µl of GoTaq DNA polymerase (Promega) and 2.5 µl of template DNA (from a 1∶50 dilution of the extracted DNA). A hold at 94°C for 3 min was followed by 30 cycles of denaturation at 94°C for 45 s, annealing at a primer specific temperature for 45 s and extension at 72°C for 90 s, finishing with a final extension at 72°C for 5 min on a thermal cycler  , 4 µl of MgClmp 2700) . PCR proAll sequences were checked and edited manually on Chromas Pro . Sequence alignments were performed using Mega v. 5 Balanus perforatus, 1500 km apart, so decided not to analyse Location 2 samples.Preliminary analysis showed no differentiation in any of the investigated species between the two replicated areas of each site. As a result, individuals from the two areas were pooled and hereafter data have been analysed considering only the sites , haplotype and nucleotide diversity , were calculated for each species by site, location, and for the whole study area, using DnaSP v. 4.50.3 The fixation index (Fst) between sites across each PB was used to quantify the differentiation due to genetic structure. Large scale genetic structuring across the whole study area was also assessed, as the genetic differentiation between all pairs of sites. Pairwise Fst values were calculated in Arlequin v 3.1 Analysis of Molecular Variance (AMOVA) was performed to investigate sources of variability within and between the three locations studied. Significance was tested with 16000 permutations in Arlequin v 3.1 Isolation by distance across the whole study area was tested with a Mantel test as implemented in Genepop, following the Isolde procedure which compares a semi matrix of Fst/(1-Fst) with a semi matrix of the natural logarithm of the shortest oceanic distance in km.Migration rates between areas were estimated with the software MIGRATE-n v. 3.3.1 COI sequences of the target species available in Genbank  were aliThe history of effective population size (Ne) was assessed by testing the fit of the mismatch distributions to the theoretical distribution in an expansion scenario For those sites that were not significantly different, and did not deviate from the expansion model, the coalescence time τ (i.e. time since the start of a population expansion) was estimated following the formula T = 2υτ For the same undifferentiated sites, past changes in effective population sizes and estimates of expansion time were also inferred by generating Bayesian Skyline Plots (BSPs) using the software BEAST v. 1.7.5 To test for consistency of the phylogeographic patterns across species, two types of multivariate analysis were used.Site and Location as crossed factors in order to test for the effect of each of the PB. A Bray Curtis similarity matrix between all sites was represented graphically using a Multidimensional Scaling plot (MDS). Apparent groupings across the whole study area were also statistically tested with ANOSIM.The multivariate Analysis of Similarity (ANOSIM), as implemented in Primer v.6 Discriminant analysis of principal components (DAPC), available in the Adegenet package Chondrosia reniformis and 48 in Osilinus turbinatus. Patella caerulea and Balanus perforatus presented 36 and 43 haplotypes, respectively. Hexaplex trunculus, Chiton olivaceus, and Halocynthia papillosa showed lower number of haplotypes  and southeastern sites . P. caerulea migration rate estimates were higher from the southeastern towards the northwestern sites , whereas H. trunculus migration rate estimates were higher from northwestern towards southeastern sites . The Mantel test on the geographic and genetic distances matrix was not significant (p = 0.135 for P. caerulea and p = 0.082 for H. trunculus).Fst values failed to detect significant genetic differentiation across the putative barriers PB1 and PB3 for any of the seven species studied . Signifirunculus . PairwisOsilinus turbinatus and Chondrosia reniformis significant pairwise Fst values were detected between the E and F sites (Location 3) and most other sites . Mantel tests were not significant in either species .In er sites , except niformis . MigratiChiton olivaceus showed significant genetic differentiation between sites A, B, C and site F. Site E was also significantly different from site A .m site A . A signiHalocynthia papillosa showed significant genetic differentiation between several pairs of sites , or even across sites located more than 1000 km apart  through time, coinciding with estimates of times since expansion  largely preceding the Last Glacial Maximum . The more recent lineages showed a slightly growing population size in the BSP , and coincided with the LGM for O. turbinatus . Expansion estimates for C. olivaceous dated around 32,600 ya, before the LGM, in contrast with the BSP, which showed increasing population size after the LGM.Bayesian skyline plots (BSP) showed large confidence intervals in most cases , yet the the BSP , but stiBalanus perforatus) across sampling areas was generated. The analysis of similarity (ANOSIM) indicated no significant effect of the factor site crossed by location, i.e. no multispecies significant differences across any of the three putative barriers. The MDS of the Bray-Curtis matrix of distances between all six sites . The resulting DAPC showed a distribution of the samples (sites) similar to the MDS plot: sites A, B and C grouped together. Site D was separated from all the others, and sites E and F clustered together , corresponding to all polymorphic sites across all species Click here for additional data file.Figure S2Bayesian Skyline Plots for the species showing signs of expansion according to mismatch distribution or neutrality tests.Patella caerulea and Hexaplex trunculus plots are calculated for each haplogroup. When shown, the black square indicates the date of the Last Glacial Maximum (LGM). The most likely evolutionary model selected by jModelTest is indicated in each plot.(TIF)Click here for additional data file.Table S1Specific primers. Names, sequence, amplified fragment length, and optimal annealing temperature for each of the seven pairs of primers used.(DOCX)Click here for additional data file.Table S2Previously published sequences. Accession numbers, geographical origin and identification with the haplotypes found in this study, as named in (DOCX)Click here for additional data file.Table S3Analyses of Molecular Variance. Results of the Amova on the groups corresponding to the three Locations.(DOCX)Click here for additional data file.Table S4Demographic history analyses. Fit to sudden expansion model and neutrality tests.(DOCX)Click here for additional data file.


Due to its geo-climatic conditions, Algeria represents a biodiversity hotspot, with sheep breeds well adapted to a patchwork of extremely heterogeneous harsh habitats. The importance of this peculiar genetic reservoir increases as climate change drives the demand for new adaptations. However, the expansion of a single breed  which occurred in the last decades has generated a critical situation for the other breeds; some of them are being subjected to uncontrolled cross-breeding with the favored breed and/or to marginalization (effective size contraction). This study investigated genetic diversity within and among six of the nine Algerian breeds, by use of 30 microsatellite markers. Our results showed that, in spite of the census contraction experienced by most of the considered breeds, genetic diversity is still substantial (average gene diversity ranging 0.68 to 0.76) and inbreeding was not identified as a problem. However, two breeds (Rembi and Taâdmit) appeared to have lost most of their genetic originality because of intensive cross-breeding with Ouled-Djellal. Based on the above evidence, we suggest Hamra, Sidaoun, and D’man as breeds deserving the highest priority for conservation in Algeria. The Near East and North Africa (NENA) region is recognized as the reservoir of genetically unique breeds  playing The blood used for all of the analyses was collected by veterinarians during routine blood sampling on commercial farm animals . Those animals were not linked to any experimental design and blood sampling was not performed specifically for this study, therefore no ethical authorization was required. All the samples and data processed in our study were obtained with the breeders and breeding organizations' consent.th century would have been the basis for the development of the Taâdmit sheep  IC95%.The observed heterozygosity (H = 0.16) . Distribrom zero . The meaWe failed to detect significant linkage disequilibrium between pairs of loci in each considered breed after False Discovery Rate correction .ST was 0.038 [0.029–0.047] (IC95%). Pair-wise FST values between breeds were particularly low between the Ouled-Djellal/Rembi, Ouled-Djellal/Taâdmit, Rembi/Taâdmit pairs  and among the group formed by Sidaoun/D’man/Hamra: mean FST were respectively of 0.011 and 0.077 and were found significantly different .Considering all the breeds, the mean Fit pairs . FSTAT [ST values were also calculated among seven Italian sheep breeds  from a previously published dataset [ST value  was found for the Italian breeds compared to the Algerian ones . Pair-wise FST values between Rembi/Taâdmit, Ouled-Djellal/Rembi and Ouled-Djellal/Taâdmit were significantly lower than all the pair-wise FST values observed between Italian breeds; the only exception concerned the FST values between Ouled-Djellal/Taâdmit and Bagnolese/Laticauda which showed overlapped confidence intervals. Bagnolese and Laticauda are known to be genetically very close, as they share a common origin and the same breeding area [As a general reference, mean and pair-wise F dataset , using aing area .The Bayesian analyses for cluster assignment suggested K = 2 as the most likely number of clusters using the ΔK criterion by Evanno et al. , with ΔKIn the DAPC analysis, 80 PCs of the PCA were retained as input to DA, accounting for approximately 89% of the total genetic variability. The scatterplot of the first two components of the DA  showed tThe current study investigated the genetic diversity of six main Algerian breeds, enabling the acquisition of original information concerning the level of variability, both within and among breeds.www.dagris.ilri.cgiar.org; FAO DAD-IS database: www.fao.org/dad-is). If reductions in flock size experienced by breeds are correlated with reductions in effective population size low genetic diversity could be found for these breeds.Some of the breeds studied have been largely marginalized over time, implying reduced flock size, mostly because of farmers' preference for the Ouled-Djellal. Hamra, Sidaoun, Taâdmit and D’man show flock contraction, and contribute currently to a very low proportion of the Algerian sheep livestock  than that observed in Italian breeds . Hence, in spite of flock size reductions experienced by Hamra, Sidaoun, D’man and Taâdmit, the genetic diversity appeared moderately high for these breeds and in any case not significantly lower than the genetic diversity of Ouled-Djellal. This conclusion must be considered critically, as blood collection goes back to the period 1998–2004. Over recent years the situation has gradually deteriorated [Consideration of the six breeds showed moderately high genetic diversity, with homogenous levels of expected heterozygosity, allelic richness and private allelic richness among the breeds. When comparing Algerian breeds with Italian breeds, using a set of fifteen common microsatellites, gene diversity in Algerian breeds was significantly , but to a moderate extent, lower  some alleles or allele combinations can be lost from the population; (ii) the limited animal numbers in a breed can imply an increased inbreeding level. The mean Fexample, –41). Forexample,  for an aIn sum and according to our sampling, inbreeding did not appear as an immediate problem for the breeds considered.Cross-breeding between low and high productive breeds aims to improve breeds faster than through selection schemes, but such practices do not always achieve the desired results . This prST values were calculated. Very low FST values (ranging 0.007 to 0.015) were observed between breeds belonging to the "white breeds" group while pair-wise FST values between the remaining breeds were significantly higher. Pair-wise FST values were also calculated between seven Italian breeds, using a common set of 15 microsatellites, in order to provide a general reference term to interpret pair-wise FST values between Algerian breeds. The comparison clearly showed poor genetic differentiation among the “white breeds”, with pair-wise FST values significantly lower than those observed for Italian breeds known to be genetically differentiated. Considering the known origin of Taâdmit and the hypothesized origin of Rembi, both as Ouled-Djellal-derived breeds, limited FST values were expected in this group; however such very low values of FST are likely the consequence of cross-breeding phenomena largely recorded between Ouled-Djellal and the two other breeds [In order to evaluate the level of genetic dilution in Algerian breeds due to uncontrolled cross-breeding with the pervasive Ouled-Djellal breed, pair-wise Fr breeds .The Bayesian analysis showed signals of admixture between the “white breeds” whereas Hamra, Sidaoun and D’man proved to be well differentiated. In spite of this observed levels of admixture, the model-based clustering algorithm implemented in the STRUCTURE software was able to distinguish Ouled-Djellal, Rembi and Taâdmit when they were analyzed together in a reduced dataset. Results of DAPC were more telling, highlighting such a low differentiation level between the three “white breeds” that they appeared almost completely overlapping; while, Hamra, Sidaoun and D’man clearly differentiated from each other and from the central cluster formed by the “white breeds”.Hence, we can suppose that cross-breeding has spread to such a degree in Algeria that Rembi and Taâdmit prove to be genetically very close to Ouled-Djellal. For Rembi, practices of cross-breeding with the popular Ouled-Djellal led to the disappearance of the initial specimen’s phenotype , replacThis study investigates the genetic diversity within and among Algerian breeds. Previous studies on Algerian sheep used very limited number of microsatellites and/or focused only on phylogenetic purposes –47. Our S1 Table(TIF)Click here for additional data file.S2 Tablee, effective number of alleles.Microsatellites are grouped  according to amplification and fragment analysis conditions (see details in (PDF)Click here for additional data file.S3 TableThe data file has individuals organized in rows, with the name of the population in the first column. The subsequent columns correspond to microsatellite data (two columns per locus). Missing data are coded by zeros.(CSV)Click here for additional data file.S1 FigHamra, lattitude: 34° 45' 34.06"N longitude: 0° 8' 44.51"E; Rembi, lattitude: 35° 13' 12.84"N longitude: 2° 19' 2.04"E; Ouled-Djellal, latitude: 36°17'N longitude: 6°37'E; Sidaoun, latitude: 22°47'N longitude: 5°31'E; Taâdmit, latitude: 34° 43'N longitude: 3° 13'E; D’man, latitude: 31°37'N longitude: 2°14' W.(TIF)Click here for additional data file.S2 FigOn the top, the three “white breeds” ; on the bottom, from the left to the right: Sidaoun—D’Man—Hamra.(TIF)Click here for additional data file.


Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-β1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in −/−Matn2 relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in −/−Matn2 fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration  Skeletal muscle regeneration following injury is a multistep process that restores the tissue architecture by the sequential activation of multiple signaling pathways . The earMyog) requires the replacement of TFIID with the TRF3–TAF3 complex , cytokines and various stimuli from the extracellular matrix (ECM) . TGF-β fThe ECM has a variety of direct and indirect effects on muscle differentiation and regeneration. Importantly, the inhibition of ECM assembly interferes with myogenic differentiation independently of MRF expression . During Matn2)  . Matn2 iMatn2) . MatriliMatn2) . Matn2 hMatn2) . Recent Matn2) , and tha healing .Matn2 upregulation during notexin-induced muscle regeneration in vivo and in differentiating myoblast cultures. We report delayed myogenic differentiation from Matn2-silenced myoblast cultures, and a mild dystrophy with delayed muscle regeneration in −/−Matn2 mice. We also establish that TGF-β can inhibit C2 myoblast differentiation by decreasing Matn2 expression, and lack of Matn2 delays the onset of Trf3, Nfix and MRF gene expression. Overall, our data implicate Matn2 in a signaling network controlling the onset of the myogenic program, thereby linking ECM–cell interactions to tissue differentiation and repair.Here, we reveal transient Matn2 upregulation until 4 days after treatment, followed by a decline in its expression , a common integrin target . Unlike the weak Matn2 staining of the endomysium relative to nerves and blood vessels in the untreated muscle, an intense Matn2 signal was observed at the periphery of necrotic myofibers and in the interstitial tissue at day 1 post-injury . This further implied a proteolytic release of Matn2 epitopes from oligomers and ECM linkages in the old endomysium, thereby resulting in better recognition by the antibody. Using myogenic, basement membrane and inflammatory cell markers, we observed Matn2 deposition around MyoD-, Myog- and Lama2-positive proliferating and differentiating myoblasts on day 2, but not around granulocytes and macrophages . Matn2 colocalized with laminin in the basement membrane surrounding desmin-positive fusing myoblasts, myotubes and myoblasts merging with newly formed myofibers .To reveal which cell types might express neration , which wyofibers , implyinyofibers  but stilyofibers . Matn2 eyofibers . The MatWhile keeping the muscle nerves intact, notexin treatment destroys their motor endplates. Unlike myoblast proliferation, myofiber maturation depends on nerve signals . Matn2 dMatn2 expression in C2 myoblasts cultured in differentiation medium, as this cell line model was reported to faithfully mimic fetal muscle differentiation  or the control vector (line Ctrl) were established and tested for differentiation and the expression of marker genes. Matn2 mRNA levels were successfully reduced in lines sh7, sh4 and sh3, with expression levels ranging from 52.2% to 17.6% of that of the control line, as determined by QRT-PCR . Prior to differentiation, the Matn2-silenced cells were morphologically identical to control myoblasts (data not shown) and proliferated at similar rates, as based on their mitochondrial activity . The primary myotube frequency, fusion index and differentiation index decreased in correlation with the reduction in Matn2 immunofluorescence and mRNA expression of the cultures . Thus, silencing Matn2 expression delayed both the differentiation and fusion of myoblasts.To address whether Matn2 might be needed for myogenic differentiation, we silenced its expression in C2 myoblasts using an efficient transposon-based short-hairpin (sh)RNA expression system . Indepen QRT-PCR ; expressactivity  and on csupplementary material Fig. S2C; data not shown). Thus, Matn2 silencing interfered with the transient activation of MyoD and Myog, as well as with the progressive increase in the p21 mRNA level observed in control myoblasts, readily explaining the delayed commitment, cell cycle exit and differentiation of the silenced cell lines. Matn2 silencing also hampered the activation of Fak, a gene encoding a modulator of cell–ECM contacts, and the transient rise in the expression of ECM genes , the expression of which paralleled that of Matn2 in the control line. As no marked alteration was seen for the unrelated control p53 and Col1a1 mRNAs, this indicated that Matn2 silencing exerts a specific effect on the expression of MRF and differentiation-related genes.Delayed myoblast differentiation was further supported by comparative QRT-PCR, which revealed the most robust changes in myogenic gene expression between the control line and the sh4 and sh3 silenced cell lines in repeated differentiation experiments  for further analyses . Smad4 expression was not altered, but large differences were seen in the expression of p21 , it is likely to modulate several signaling pathways. Thus, the decreased expression of Matn2 hampered the induction of p21 and overt differentiation, despite the increased expression of the TGF-β signaling intermediate Smad4, and this effect could be reversed by restoring Matn2 expression and its deposition in the ECM. QRT-PCR confirmed the restoration of muscle-specific gene expression in differentiating Matn2-rescued myoblasts  mRNAs.Next, we tested whether rescuing Matn2 synthesis in a silenced cell line can restore the proper timing of myogenic differentiation. To this end, we mutated the sequence targeted by the shRNA in the mouse get site . Then, wanalyses . The Matanalyses . As expeanalyses , and theanalyses , yieldedn of p21 . Unlike yoblasts , and theMatn2 expression. Nfi proteins have been implicated in the control of adult stem cells and tissue regeneration in connection with TGF-β signaling , implying that Matn2 and Nfix might be part of a genetic switch driving an all-or-none differentiation response.The finding that Matn2 expression might regulate myoblast differentiation prompted us to assess the regulation of transcription factors that are possibly involved in the onset of ignaling . Nfix hale genes . When moed cells . The NfiMatn2 has been shown to be regulated by Smad signaling in other cell types  or downstream promoter (Pd) can direct the transient gene activation during muscle regeneration and myoblast differentiation . To further establish the function of Matn2 in myogenesis in vivo, we next compared the regeneration of tibialis anterior muscle following notexin-induced injury in Matn2 knockout (−/−Matn2) and wild-type mice . Consistently, decreased desmin staining and an increased number of Ki67+ and Ki67+MyoD+ cells were observed in Matn2-deficient mice at days 3 and 5 of regeneration, respectively . Central myonuclei and myofiber splitting also occurred more frequently, whereas fibrosis was not more pronounced in older −/−Matn2 than in wild-type animals .Similarly to rat muscles , high Maype mice . Signifiype mice . The aveype mice . The incectively . Immunoft-injury . At lateTrf3 expression, followed by robust downregulation in the expression of Nfix, Nfia, Bmp7 and late differentiation markers  in −/−Matn2 fetal limbs at embryonic day 16.5 (E16.5)  . Matn2 dnd E17.5 , along wnd E17.5 , our datTrf3, Taf3, Bmp7, MyoD, Fak, Nfic, Nfix and Itga5 were all decreased in −/−Matn2 versus +/+Matn2 cultured myoblasts .By contrast, in the absence of Matn2, the expression of  induced . Thus, Mignaling . p42 , p21 and muscle-specific genes (Myh1). In addition to identifying Matn2 as an upstream global modulator of myogenic differentiation, these data further support the proposal that the Trf3–Taf3 complex plays a pivotal role in terminal muscle differentiation by specifically activating a subset of myogenic marker genes while inhibiting many others  and fetal muscle genes (Myh7) by forced Nfix expression in embryonic myotubes and in differentiating C2 cells . Our results provide evidence for a molecular mechanism whereby TGF-β1-mediated regulation of Matn2 might concomitantly control NFI expression.By controlling the expression of regulatory and ECM target genes, Nfix can increase MyoD-dependent muscle-specific gene expression . Whetherromoters , and witromoters . Our intromoters . Our conromoters  and of Bromoters  to contrMatn2, MyoD and Myog during myogenic differentiation. As MyoD expression is turned on in response to increased expression of Matn2 and possibly also of Nfix, a negative-feedback loop can be established whereby MyoD, overcoming the effect of Nfix, will block Matn2 expression at late stages of differentiation . Muscle necrosis was induced by intramuscular injection of 20 µg of notexin, as described previously . The ani−/−Matn2 mice has been reported previously (−/−Matn2 mice and wild-type littermates by injecting 0.3 µg (60 µl) of notexin (Sigma-Aldrich) into the tibialis anterior muscle. All animal treatments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and under the approval of the Animal Health Care and Control Institute, Csongrád County, Hungary.The generation of eviously . Muscle C2 myoblast cell line clone 7  and primsupplementary material Table S1. Nonspecific binding of the antibodies was blocked with 10% proper normal serum. Nuclei were stained with DAPI. NMJs were detected with tetramethylrhodamine α-bungarotoxin . The specimens were mounted with fluorescent mounting medium (Dako), viewed with a Nikon Eclipse E600 microscope equipped with epifluorescence and photographed with a Spot RT Slider camera. The images were processed using SPOT software . Figures were created with Adobe Photoshop 8.0 and CorelDraw X4 softwares.The average CSA and fiber-size distributions were determined using the Digimizer software . Immunohistochemistry was performed as described previously . Primaryg for 10 min at 4°C, supernatant proteins were separated by SDS-PAGE, blotted and developed as described previously . Experiments were repeated at least three times.The myoblast cell layer was extracted in ice-cold buffer containing 20 mM Tris-HCl pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 and protease inhibitors. After centrifugation at 10,000 eviously . Primaryn = 3) and used for northern analysis as described previously . For QRT-PCR, pooled total RNA was isolated from three cultures of differentiating C2 cell lines, three primary myoblast cultures grown either in growth medium or differentiating ex vivo in differentiation medium and also from E16.5 fetal limbs. QRT-PCR was performed with the SYBR green protocol, as described previously  values were normalized either to the average Ct values of three internal control genes  (SYBR green) or to the Ct values of Hprt (TaqMan probe). Relative gene expression levels are presented as log2 ratios or fold values.Pooled total RNA was isolated from rat soleus muscles . At 24 h after plating, the cells were seeded into 10-cm dishes for a 2-week selection in growth medium containing 500 µg/ml G418 (Cambrex), and stably transfected Matn2-silenced cell lines were established from G418-resistant single colonies.We designed shRNA targeting Matn2 using the program RNAi explorer . OligonuMatn2 silencing, we modified the cDNA encoding full-length mouse Matn2 by mutating the sequence targeted by the shRNA but preserving its protein coding capacity (Matn2. Clones that were resistant to 1 µg/ml puromycin were pooled. Independent control (Ctrl), silenced  cell lines and rescued (sh3-res2) cultures were tested for myogenic differentiation and marker gene expression in at least three independent experiments.To rescue capacity . The modThe MTS assay for mitochondrial function was performed with the CellTiter96 AQueous One solution cell proliferation kit (Promega), according to the manufacturer's protocol. For the evaluation of differentiation, myoblast cultures were stained for sarcomeric α-actinin and nuclei (DAPI). In randomly chosen microscope fields containing a total of 3000–4000 nuclei, α-actinin-positive myoblasts were counted. Primary myotube frequency was calculated as the number of myotubes containing <3 nuclei divided by the total number of cells in a given microscope field. The fusion index was defined as the number of myotubes harboring ≧3 nuclei divided by the total number of cells. The differentiation index was defined as the number of myotube nuclei divided by the total number of nuclei. Data represent the mean±s.e.m. from three to five independent experiments.1-µl aliquots of total RNA samples treated with RNase-free DNase  were subjected to oligo(dT)-primed first-strand cDNA synthesis, followed by PCR using promoter-specific primer pairs , as descThe program Jaspar was used to search for putative NFI and MyoD recognition sequences in the Matn2 promoter and to synthesize double-stranded oligonucleotides . ConstruPd(−342)Luc was constructed by inserting the −342/+60 Pd fragment into the PvuII/PstI site of the pGL3-Basic Vector (Promega). Derivatives carrying point mutations in the MyoD motif near the start site  in the NFI motif (Nfm1) or in both the NFI and MyoD consensus sequences (NfMdm1) of the Nfi/MyoD composite element Pd−135/−107 were made using the PCR based QuikChangeTM Site-Directed Mutagenesis kit (Stratagene).Pd(−342)Luc. Results are the mean±s.e.m. from at least three independent experiments.C2 myoblasts (cultured in growth medium) and COS-7 cells were transfected with 3–5 µg of wild-type and mutant reporters in six-well plates using ExGene 500 (Fermentas) or the calcium phosphate coprecipitation method, respectively, and luciferase activities were measured, as described previously . Forced P<0.05, **P<0.01, ***P<0.001.Data are presented as the mean±s.e.m. Statistical significance was determined using a one-way analysis of variance (ANOVA) with KyPlot version 2.0 beta 15; *