Background

Small ruminants,like sheep(Ovis aries)and goats(Capra aegagrus hircus)were the first animals domesticated by human for food supply,being the most important group of ruminants raised in temperate and tropical regions[1].Sheep are multi-purpose animals,raised for meat,milk,wool,hides and skins,having a huge socioeconomic importance worldwide.However,there are several productive drawbacks associated with gastrointestinal parasites infection in small ruminants,since it represents the type of disease with the highest impact on animal health and productivity[2].Thus,the losses caused by gastrointestinal parasites and the costs due to excessive use of anthelmintic drugs are a struggling problem that restricts the sheep production in many regions of the world.

The main gastrointestinal nematodes of small ruminants belong to the Trichostrongylidae family.These parasites occur in the gastrointestinal tract and are seen as the main obstacle in the sheep industry,since they lead to significant economic losses due to high mortality and productivity losses.Within the Trichostrongylidae family,the Haemonchus genera has great pathogenic action and is the most prevalent parasite affecting small ruminants,mainly in tropical regions,where environmental conditions are characterized by high temperature and humidity,and abundant rainfall during summer[3-6].

To reduce the economic losses caused by nematode infections there are several management alternatives to minimize the damage,such as raise breeds or animals that are more resistant to these infections[5].In this regard,Santa Inês,an American hair breed,showed higher resistance to gastrointestinal nematode infections when compared to European sheep breeds[5,7-9].Several studies have shown that selection for gastrointestinal parasite resistance is possible in sheep,since genetic progress in research and commercial herds[10-16].

The identification of genetic markers associated with gastrointestinal parasites resistance could increase the genetic response for this trait by marker-assisted selection[15].Furthermore,the identification of genomic regions associated with resistance or susceptibility to gastrointestinal parasites will help to deeper understand the biological and physiological processes underlying this trait[17].Several studies reported genetic markers associated with gastrointestinal parasites resistance close to the Major Histocompatibility Complex(MHC)[18-20]and IFN-gama genes[18-22].Recently,genome-wide association studies have identified genetic variants for gastrointestinal parasite resistance in some sheep breeds[23-26].The identification of genomic regions that play a role in gastrointestinal parasite resistance may become an important tool to improve the resistance of Santa Inês or other sheep breeds.Therefore,the aim of this study was to estimate variance components and to identify genomic regions and pathways associated with resistance to gastrointestinal parasites in a Santa Inês population adapted to tropical conditions.

Methods

Data

The phenotypic records were collected from 700 naturally infected animals of Santa Inês breed belonging to four flocks located in the Minas Gerais and São Paulo southeast states and Sergipe northeast state of Brazil.The samples collection and phenotypes evaluations were performed from October to November of 2011.Phenotypes evaluations were achieved to verify resistance to gastrointestinal parasites,and were divided into two categories:i)farm phenotypes,assessing body condition score(BCS),degree of anemia assessed by the famacha card(FAM),fur score(FS)and feces consistency(FC);and ii)lab phenotypes,comprising blood analyses for hematocrit(HCT),white blood cell count(WBC),red blood cell count(RBC),hemoglobin(HGB),platelets(PLT)and the egg counts per gram of feces(EPGlog).The EPG values were transformed to log10(n+1)=EPGlogto meet the basic requirements of normality and homogeneity in an attempt to stabilize the variance prior to analyses,where n is the number of EPGlogper sample.

Blood samples were collected by puncture of the jugular vein using vacuum tubes with anticoagulant EDTA and clot activator.Subsequently,samples were submitted to the Veterinary hematology analyzer Sysmex PocH-100iV Diff to perform a complete blood cell count for HCT,WBC,RBC,HGB and PLT.Fecal samples used for EPG evaluation were taken directly from the rectum of the animals and sent to the laboratory for analyses.The EPG count was assessed using the modified McMaster technique[27]and the parasites’genera were identified using the morphometric key by Van Wyk et al.[28].The feces were classified through visual inspection according to its consistency appearance(FC)developed by Gordon et al.[29]and modified by the authors considering three out of the original five-value scale:1 for normal stool,2 for pasty stool,and 3 for diarrheal feces.

The BCS was assessed after evaluating the prominences of the spinous and transverse bones of the spine,fat coverage,and muscle development between the last rib and the ileum wing,as described by Russel et al.[30].Coloration of the ocular mucosa was measured by trained people by observing the medial part of the lower conjunctiva and comparing it with the famacha chart(FAM;FAMACHA©System)considering a five-value scale:1 for red robust,2 for rosy red,3 for pink,4 for pale pink,and 5 for white[31].The descriptive statistics for the analyzed traits are presented in Table 1.

Genotyping of animals

The extraction of the genomic DNA from each animal was performed from blood samples collected with EDTA.For this,red blood cells were lysed using 1 mL of whole blood and 500 μL of lysis solution(0.32 mol/L Sucrose,12 mmol/L Tris-HCl pH 7.5,5 mmol/L MgCl2,1%Triton X-100)followed by centrifugation at 13,000 r/min.The supernatant was discarded to reach the leucocytes.The pellets were slowly washed adding 1 mL of ultrapure water and then the microtube was poured out of the water and held for a few minutes on absorbent paper to completely dry the pellet.This washing step was repeated approximately three times until a clean white pellet was obtained.The white pellet was then kept frozen(-20°C)until sending to the DEOXI laboratory in Araçatuba-SP,where the DNA was completely extracted.The DNA of each sample was quantified and the degree of purity(ratio of optical densities between 260 and 280 nm)(Table 1)was evaluated.After these processes,the samples were stored at-20°C until genotyping was performed.

Table 1　Descriptive statistics for eggs per gram of feces(EPGlog),red blood cells(RBC),famacha(FAM),platelet(PLT),white blood cells(WBC),hematocrit(HCT),and hemoglobin(HGB)

aSD:Standard deviation;bCV:Coefficient of variation

Trait　n　Mean　SDa　CVb,%　Min　Max EPGlog　517　2.57　0.53　20.6　1.71　4.04 RBC,106/μL　513　9.97　2.12　21.2　4.39　19.25 FAM　518　3.08　0.97　31.16　1.0　5.0 PLT,103/μL　514　383.8　241.6　62.9　8.0　2101.0 WBC,103/μL　514　10.96　3.73　34.0　3.60　44.40 HCT,%　514　29.05　6.79　23.4　10.90　58.00 HGB,g/dL　514　8.43　1.94　23.0　3.30　17.30

A total of 576 animals were genotyped with the Ovine SNP12k BeadChip(Illumina,Inc.),that contained 12,785 biallelic SNP markers.Quality control consisted of excluding markers with unknown genomic position,locatedonsexchromosomes,monomorphic,with minor allele frequency(MAF)lower than 0.05,call rate lower than 90%,and with excess heterozygosity.After quality control,there were 11,602 SNPs and 574 samples left for analyses.

Quantitative genetic analyses

The SNPs windows regions which accounted for more than 1%of the genetic additive variance were used to search for candidate genes(CG),which are described in Additional file 1.A total of 22,21,23,20,26,25 and 23 windows for EPGlog,FAM,WBC,RBC,PLT,HCT and HGB traits were identified,respectively.Among the associated windows,10 are common for HCT and HGB traits,located on OAR1,OAR2,OAR3,OAR5,OAR8 and OAR15(Additional file 1:Table S9 and S10-Figs.3 and 4).

where A22is a numerator relationship matrix for genotyped animals and G is the genomic relationship matrix created as described by[34]:

where Z is the gene matrix containing allele frequency adjustment;D is the matrix that have the SNP weight(initially D=I);and,q is a weighting/standardization factor.According to[35],such factors can be obtained by ensuring that the G average diagonal is next to A22.The pedigree file has a total of 1196 animals and the last three generations of animals with records were considered.A linear model was used to analyze HCT,WBC,RBC,HGB,PLT,and EPGlog.The model can be represented by the following matrix equation:

where y is the observations vector;β is the vector of fixed effects;a is the additive direct vector;X is the incidence matrix;Z is the incidence genetic random effects additive direct matrix(the β vector associated with the y vector);e is the residual effect vector.The priori distributions of vectors y,a and e were given by:

where H is the relationship coefficients matrix among animals obtained from the single-step analyses(singlestep);R is the residual variance matrix;I is the identity matrix;G is genetic additive variance matrix and⊗is the Kronecker product.An inverted chi-square distribution was used for the prior values of the direct and residual genetic variances.A uniform distribution was used a priori for the fixed effects.

A threshold model was applied to analyze the FAM trait.The scores of FAM for each individual i,were defined by Ui in the underlying scale yi=(1)t0＜Ui＜t1;(2)t1＜Ui＜t2;(3)t2＜Ui＜t3;(4)t3＜Ui＜t4;i=1,…n,where n is the number of observations;t1to t4were the threshold values;and U is the unobservable continuous variable,in underlying scale,limited between two unobservable thresholds.After specifying the thresholds t0to t4it is necessary to adjust one of the thresholds(from t1to t4)into an arbitrary constant.In the present study,it was assumed that t1=0,in such a way that the vector of estimable thresholds was defined as t=t2,t3,t4.Afterdefining　themodelparameters,thelink between categorical and continuous scales could be established based on the contribution of the probability of an observation that fitted the first category,which is proportional to:

where yv is the response variable for the vth observation;t is the threshold value arbitrarily assigned as the true value is unobservable;Uv is the value of the underlying variable for the vth observation;(φ)is the cumulative distribution function of a standard normal variable;and w’v is the scale of the incidence matrix that linked θ to the vth observation.As the observations are conditionally independent,given θ,the likelihood function is defined by the product of contributions of each record.

The analyses were performed using the GIBBS2F90 and THRGIBBSF90 programs[33,36].The a posteriori variance component estimates were obtained using the POSTGIBBSF90 program[36].The analysis consisted of a single chain of 500,000 cycles discarding the first 20,000 cycles,taking a sample at every 100 iterations.Thus,48,000 samples were used to obtain the parameters.The data convergence was verified with the graphical evaluation of sampled values versus interactions according to the criteria proposed by several authors[37-39],using the Bayesian Output Analysis(BOA)of the R 2.9.0 software[40].

Linkage disequilibrium estimation

反应过程中苯酚的残余浓度随时间的变化情况如图3(a)所示。由该图可知，反应液中苯酚的浓度在反应开始后急剧下降，5 min后即可达到90%的去除率，30 min后反应达到平衡，说明制备的Fe3O4-C空心微球具有较高的催化活性。以ln(Ct/C0)对t作图，可得一条直线，见图3(b)，说明苯酚的催化氧化降解反应符合伪一级反应动力学[8]。

The CD80 and CD86 genes identified in almost all traits(RBC,PLT and HCT)probably participate effectively in the activation of T cells,which requires engagement of two separate T-cell receptors.The antigen-specific T-cell receptor(TCR)binds foreign peptide antigen-MHC complexes,and the CD28 receptor binds to the B7(CD80/CD86)costimulatory molecules　expressed　on　the　surface　ofantigenpresenting cells(APC).The simultaneous triggering of these T-cell surface receptors with their specific ligands results in the activation of this cell.Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells.Recently,functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice,and the authors concluded that CD86 could be more important for initiating T-cell responses,while CD80 could be more significant for maintaining these immune responses[73].Recently,[24]observed some important genes matching to important pathways involved in host immune response against parasites.The PDGFRA gene(OAR6:67,950,121-69,892,816)was also observed by Benavides et al.[24],whose findings have pointed out as a key gene in cytokine signaling.

where,

Atlija et al.[26]observed some significant chromosomewise QTL detected by linkage analysis and from the combined LD and linkage analysis.These authors reported some CG involved in immune response.Theirs findings support our results,since genes such as MBN,BTC,CXCL1,CXCL10,EREG,RASSF6,SCARB2,TMPRSS11D,AMBN,and AREG were observed in both studies,indicating that these genes can be used and deply studied as CG for resistance to gastrointestinal nematodes in sheep.Some of the genes cited in Additional file 1:Table S9 and S10 have functions related to immune response or to immunoglobulin development,and were associated with HCTand HBG traits,respectively.

可见，地下水评价不仅要对地下水动态进行深入的研究，对地下水资源量的动态变化也应该予以重视。对降水量系列资料应不断补充，充分考虑近期降水和其他补给项对地下水资源的影响。

Principal component analysis

The principal component analyses(PCAs)were obtained from the genomic relationship matrix through the preGS90 program.The preGS90 is an interface program to process genomic information for the BLUPF90 family[36].Efficient methods for the creation of the genomic relationship matrix,relationship based on genomic data,and their inverses are described by[41].The PCAs applied to genotype data can be used to calculate principal components(PCs)that explain differences among individual samples in the genetic data.The top PCs are viewed as continuous axes of variation that reflect genetic variation due to ancestry in the sample.Individuals with similar values for a particular principal component will have a similar ancestry for that axes.

三是加快中西部地区金融服务体系发展，推动地区之间的技术转移和扩散，促进区域经济的协调发展和产业结构的合理化。

Genome-wide association analysis

The genome-wide association analysis for each trait was performed using the single-step GWAS(ssGWAS)methodology[42].The same linear animal model for HCT,WBC,RBC,HGB,PLT and EPGlog,and the threshold model for FAM used to estimate the variance components were applied.The effects were decomposed in genotyped(ag)and non-genotyped(an)animals as describe by[42],considering the effect of genotyped animals as:

3.财务报表分析报告难读。上面提到，财务报表分析是财务人员的工作，是对财务数据的对比分析。财务是相对专业的知识，专业名词、术语较多，非财务人员对这些财务专业名词、术语比较陌生，不易理解，需要财务人员在撰写财务报表分析报告上，进行表述转述，将一些会计专业术语，转变为一些日常用语，这样有利于报告阅读者更好理解、使用财务报表分析报告。但在实际操作中，财务人员习惯于其专业表述方式，撰写的财务报表分析报告过于“专业”，影响了财务报表分析报告阅读、使用。

where Z is a matrix that relates genotypes of each locus and u is a vector of marker effects,and the variance of animal effects was assumed as:

where D is a diagonal matrix of weights for variances of markers(D=I for GBLUP),is the genetic additive variance captured by each SNP marker when no weights are present,and G*is the weighted genomic relationship matrix.The ratio of covariance of genetic effects(ag)and SNPs(u)is:

where λ is a variance ratio or a normalizing constant.According to[34],

where M is the number of SNP and piis the allele frequency of the second allele of the ithSNP.According to[43],the markers effect can be described by:

The estimated SNP effects can be used to estimate the variance of each individual SNP effect[44]and apply a different weighting for each marker,such as:

The following iterative process described by[42]was used considering D to estimate the SNP effects:

针对2017年发布《京杭运河通航管理办法(试行)》(重新发布)有关船舶通航尺度条款的修订内容，分析了大运河水运的宏观形势变化和市场需求导向，讨论了政策初衷有关疏堵问题已经基本解决，需将发展视角转向船舶主尺度突破标准的常态化与新兴运输的体量化。据此，基于主要等级航道通航技术现状，依据相关通航管理标准，从理论层面延拓修订内容，分析船舶总长、总宽、吃水及净高等尺度限值，从实践层面讨论尺度放宽的制约要素与合理区间，同时兼顾江海直达新兴业态的发展，最终明确通航尺度条款的修订内涵，为本《办法》修订背景作诠释，亦为船型标准研究、市场运力管控及行业政策制定作保障。

Search for genes

Poliovirus receptor-like proteins(PVRLs),such as PVRL4,are adhesion receptors of the immunoglobulin superfamily and function in cell-cell adhesion[66].The encoded protein contains two immunoglobulinlike(Ig-like)C2-type domains and one Ig-like V-type domain.It is involved in cell adhesion through transhomophilic　and　-heterophilic　interactions.Itisa single-pass type I membrane protein　[provided by RefSeq,Jan 2011].

The chromosome regions that explained more than 1.0%of the additive genetic variance were selected to explore and determine possible quantitative trait loci(QTL).The Map Viewer tool of ovine(Ovis aries)genome was used for identification of genes,available at“National Center for Biotechnology Information”(NCBI-http://www.ncbi.nlm.nih.gov)database,using the bank references of HuRef assembly,CHM1 1.0,CRA TCAGchr7v2 and Ensembl Genome Browser(http://www.ensembl.org/index.html).The classification of the genes according to its biological function,identification of metabolic pathways and gene enrichment was performed by DAVID tool v6.8[46]and GeneCards(http://www.genecards.org/).Gene ontology(GO;P＜0.01)and Kyoto Encyclopedia of Genes and Genomes(KEGG;P＜0.05)pathways were identified by DAVID tool v6.8[46].All annotated genes in the Ovis aries genome were used as background.

日本政府不顾中方的坚决反对和严正交涉，不顾胡锦涛主席日前在APEC会场的郑重告诫，执意推进“购岛”进程，伤害了中国人民的民族感情，是二战结束以来对中国主权最为赤裸裸的挑战。野田政府对外宣称其“国有化”的一大理由是，“为继续平稳安定地维持管理”。真是可笑之极，钓鱼岛由谁来管理？管理什么？日本政府凭什么“管理”中国的神圣领土？实际上，日本政府企图借此强化对钓鱼岛的控制权，单方面背弃了40年前中日邦交正常化时“搁置争议”的共识。

Results and discussion

The predicted values of LD vs.linkage distance between genetic markers were presented in Fig.2.On the basis of this figure,it is possible to state that when considering a distance between markers lower than 1 Mb,the level of LD indicates that the Ovine SNP12k Bead-Chip may be a suitable tool for identifying genomic regions associated with traits related to gastrointestinal parasites resistance.Most tightly linked SNP pairs have the highest r2and average r2rapidly decreases as linkage distance increases(Fig.2).The overall LD mean between markers considering windows up to 10 Mb was 0.07.The LD mean between adjacent SNPs across autosomes ranged from 0.02 to 0.10(Table 2).Several authors have study the pattern of LD between markers in the genome of various sheep breeds[47-52].It is difficult to compare the level of LD obtained in different studies since different sample sizes,LD measures,marker types,marker densities and historical population demographics[51]were used.However,the level of LD obtained in the present study was similar to those reported in previous studies for wool sheep breeds using the Ovine SNP50 BeadChip[50-52].

The Fig.1 represents the first two principals(PC1 and PC2)component analysis(PC)based on the genomic relationship matrix.Despite the animals being originated from flocks located in different regions of Brazil,it is important to note that there was no subpopulation structure in this population.

精密量取以水为溶出介质的对照品溶液和供试品溶液，用0.45 μm的过滤膜过滤，弃去不同体积的初滤液，取续滤液测定溶液含量随初滤体积的变化，并与未过滤的工作对照品溶液或离心的样品溶液相比较，计算回收率。结果见表1。

Linkage disequilibrium and population structure

Genetic parameters estimates

The descriptive statistics for FAM indicates an incidence of animals with some degree of anemia(mainly levels 4 and 5)and animals that are not affected(levels 1 and 2)(Table 1).In the present study,animals were free of other sources that lead to anemia,i.e.fluke or minerals deficiencies such as copper,so,it is possible to asseverate that the presence of anemia in these animals was probably due to the prevalence of a Haemonchus population and the susceptibility or not of these ewes to infection.Similarly,the descriptive statistics for EPGlogindicates an infection by gastrointestinal parasites(Table 1).Most of the gastrointestinal parasites belonging to the Trichostrongylidae family have similar shape and eggs size,and unless the EPGlogis accompanied by a larvae cultivar,the eggs can belong to any specie.Moreover,the larva 4 and 5 of Haemonchus develops in the wall of the stomach with two particularities:(i)they feed with blood and(ii)do not lay eggs,which make FAM more precise than EPGlogto predict infection by Hemonchus contortus in all damaging stages.

Fig.1　First two principal components(pc1:3.16%;pc2:15%)of the genomic relationship matrix of genotyped animals

The variance components and heritability estimates for EPGlog,RBC,FAM,WBC,PLT,HCT,and HGB were described in Table 3.The parameter estimates convergence was verified by inspection of trace-plots[37,38]in which the convergence diagnosis indicates theconvergenceofthechain.Thus,theburn-in period used was considered enough to achieve the convergence of the estimates of all parameters.The marginal posterior distributions of heritability estimates for the traits showed accurate values,tending to a normal distribution,since the mean and the median were similar(Table 3).Symmetric distributions of central tendency statistics were an indicative that the analysis is reliable.

Fig.2　Linkage disquilibrium(r2)between markers considering windows up yo 10 Mb

Table 2　Summary of SNP markers analyzed and average linkage disequilibrium(LD;r2)between synthetic adjacent markers obtained for each autosome(OAR)

aSD:Standard deviation

OAR　n　Mean LD　SDaLD　Mean Distance,Mb SDaDistance Mb,1　707　0.09　0.17　0.75　1.5 2　683　0.09　0.15　1.01　1.9 3　222　0.04　0.04　2.60　3.0 4　297　0.05　0.09　2.64　3.1 5　369　0.10　0.19　0.72　1.5 6　112　0.03　0.10　2.7　2.5 7　79　0.02　0.04　3.4　2.8 8　288　0.06　0.13　1.9　2.8 9　313　0.07　0.14　1.3　2.3 10　108　0.04　0.10　3.7　2.8 11　171　0.06　0.11　1.5　2.5 12　224　0.06　0.12　1.9　2.7 13　228　0.07　0.14　2.1　2.6 14　131　0.07　0.14　3.0　3.1 15　253　0.08　0.16　1.5　2.5 16　257　0.08　0.16　1.7　2.4 17　152　0.07　0.16　2.6　3.1 18　190　0.07　0.12　1.7　2.5 19　87　0.03　0.09　2.6　2.9 20　137　0.03　0.10　3.4　2.9 21　168　0.06　0.16　2.1　2.8 22　137　0.03　0.06　2.2　2.7 23　201　0.08　0.12　0.9　1.7 24　125　0.09　0.17　1.6　2.1 25　84　0.02　0.11　3.2　2.6 26　175　0.07　0.13　0.93　1.7

Heritability estimates were low for EPGlog,moderate for RBC,PLT,HCT,HGB and WBC,and high for FAM.Studies involving Santa Inês breed are scarce in the literature and most of them refers to genetic parameters for traits related to parasites resistance performed in wool sheep breeds.Bisset et al.[53]reported heritability estimate for resistance and resilience of sheep to Haemonchus contortus in a South African Merino flock and observed high heritability for EPG,FAM and HCT,with values ranging from 0.47 to 0.55.Contrasting with our results,moderate heritability estimates for EPG among different studies were reported.Bishop et al.[54]studying Texel lambs observed a weighted average heritability of 0.26 and 0.38 for Strongyle and Nematodirus nematode resistance,respectively.Pickering et al.[25]observed an estimate of 0.25 in New Zealand sheep,and more recently,Benavides et al.[55]reported a heritability estimate of 0.36 in Australian Merino flock.Riley et al.[56]found a low heritability estimate for FAM in a Merino flock in South Africa,with values ranging from 0.06 to 0.24.The heritability estimate obtained for FAM pointed out that genetic progress for this trait is feasible,and this trait should be included in Santa Inês breeding programs.Moreover,the FAM has low cost and it is easily measured.In general,all traits showed genetic variability,therefore,it is important to investigate the presence of genomic regions or genes affecting these traits so as to elucidate and better understand their genetic architecture,especially for Haemonchus contortus,since it is one of the most predominant,highly pathogenic and economically important gastrointestinal parasite in sheep 5.

Table 3　Estimates of additive genetic variance(Va),residual variance(Vr),and heritability(h2)for log10of eggs per gram of feces(EPGlog),red blood cells(RBC),famacha(FAM),platelets(PLT),white blood cells(WBC),hematocrit(HCT),and hemoglobin(HGB)

aSD:heritability standard deviation;HPDi:lower limit of credibility interval(95%)for heritability posterior distribution;HPDs:upper limit of credibility interval(95%)for heritability posterior distribution

Trait　Va　Vr　h2　SDa　Median　HPDi　HPDs EPGlog　0.13　1.03　0.11　0.08　0.09　0.001　0.28 RBC　0.45　2.01　0.18　0.10　0.17　0.004　0.37 FAM　0.03　0.06　0.35　0.11　0.35　0.14　0.58 PLT　6.14　28.77　0.17　0.09　0.17　0.02　0.35 WBC　2.71　9.27　0.22　0.10　0.22　0.03　0.41 HCT　4.93　19.04　0.20　0.11　0.19　0.001　0.41 HGB　0.29　1.44　0.16　0.10　0.15　0.001　0.36

GWAS,genomic regions and enrichment analysis

The variance components were estimated using a single animaltraitmodelby　singlestep　genomicBLUP(ssGBLUP)procedure,under Bayesian inference[32].For all studied traits the fixed effects considered in the model were contemporary groups(farm and management group),month of sample collection,sex,covariable body condition(linear effect),and age at the collection(linear and quadratic effect).All phenotypes were tested for data consistency and contemporary groups with less than three animals and records out of three standard deviations from the contemporary group mean were discarded,remaining 500 animals with phenotypes records.The ssGBLUP is a modified version of the animal model(BLUP)with additive relationship matrix A-1replaced by H-1[33]:

Fig.3　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for hematocrit(HCT)trait

The genomic regions associated with EPGlogare detailed in Additional file 1:Table S4 and illustrated in Fig.5.Genes such as CYP11A1 and CYP1A1 located on chromosome 18,and CYP19 and SFXN1 located on chromosome 7 have functions associated with transportation or construction of iron molecules,and its absence in the blood can be an indicator of anemia in animals.The B2M,SFXN1,IL25,BMP4,TSHR,CCL28,PIK3R1,FGF10,IL15,IL2,TP-1,BPMG,BCL10,HSPD1,and MALT1 genes are described to have functions related to the body’s immune and defense response.Indeed,these genes participate in metabolic pathways related to the development of the immune system and in the regulation of the immune process effects.Atlija et al.[26]also observed that the IL25 gene is involved in EPG trait in adult sheep,with functions also linked to immune response.The enrichment analysis revealed the PI3K-Ark signaling pathway,which controls several cellular responses and has important functions in the immune system by regulating many key events in the inflammatory response related to damage and infection[57].The genomic regions associated with FAM are described in Additional file 1:Table S5 and illustrated in Fig.6.The identified CGs have functions related to the development of the immune system and the body’s defense response.Genes,such as ADAM10,IL6ST,TNFRF13B,SIVA1,JUN,PAX1,PIK3R1,SIT1,and AKT1 are related to the differentiation of T cells,a group of white blood cells(leukocytes)responsible for defending the body against antigens.The GUCY1A2 gene,which interacts selectively and noncovalently with iron Fe and the SIVA1 gene,both are expressed in different subpopulations of T and B cells and provide co-stimulatory signals for the proliferation of Tand B cells and immunoglobulin production by B cells[58].The GUCY1A2 gene operates in the process of regulating the proliferation and elimination of T cells,maintaining its number stable in the absence of external stimulus.

Fig.4　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for hemoglobin(HGB)trait

Fig.5　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for egg count per gram of feces(EPGlog)trait

The results of the enriched genes and functional grouping analyses showed that genes associated with FAM are involved in functions related to body’s immune response.The biological processes(Additional file 2-Table S11 and S12)showed that these genes are significantly(P＜0.05)involved in lymphocytes and leukocytes homeostasis.

The CCL28 gene located on OAR16 identified both for EPG and FAM acts as a chemotactic for CD4 and CD8 inactive T cells[59].The metabolic pathways associated with the CCL28 gene and with others associated with EPGlogand FAM traits,whose functions were found related to immunoglobulin synthesis in the intestine,are presented in Fig.7.

实话实说，本期针锋相对是我们结论下得最晚的一次。当你越是了解两台机器的特性，就越难分辨孰好孰坏。对于拍摄体育或者野生动物这类“硬核”需求，尼康D500的优势明显。尽管两者纸面数据非常接近，但在极限条件下，D500的光学取景器具备先天优势，表现超强的对焦系统保证了更高的拍摄成功率，再加上超大的缓存容量以及更快速的存储介质支持，D500在这一领域上优势明显。

The genomic regions associated with WBC are described in Additional file 1:Table S6 and illustrated in Fig.8.The genes found in the SNP windows encode proteins that are involved in many immune system processes,responding to any potential internal or invasive threat.The vertebrate’s immune system is composed by the innate immune system(defense responses mediated by germline encoded components that directly recognize components of potential pathogens)and by the adaptive immune system which consists of T-and B-lymphocytes[60].Many different innate immune mechanisms are deployed for host defense,a unifying theme of innate immunity is the use of germline-encoded pattern recognition receptors for pathogens or damaged self-components,such as the toll-like receptors,nucleotidebinding domain leucine-rich repeat(LRR)-containing receptors,retinoic acid-inducible gene I-like RNA helicases and C-type lectin receptors[61],like the CRCP(CGRP receptor component(CRCP)).

Fig.6　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for famacha(FAM)trait

Fig.7　Metabolic pathway involving genes present for egg count per gram of feces(EPG)and famacha(FAM)traits

Some of these genes(Additional file 1:Table S6)have already been documented in others species and been associated with immune response.Beside the functions described above,they also present some particularities,i.e.the EPBH1(EPH Receptor B1),which is involved in the GPCR pathway and developmental biology.The first process involves the G protein-coupled receptors(GPCRs)that constitute a large protein family of receptors that recognize molecules outside the cell and activate inside signal transduction pathways and,finally,cellular responses[62].

Fig.8　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for white blood cells(WBC)trait

The GATA3(GATA binding protein 3)gene also acts on the defense response,reacting to the presence of a foreign body or to the occurrence of an injury,restricting the injury/damage extension or preventing/recovering it from the infection caused by the foreign body.The GATA3 gene also positively regulates T cell differentiation and any process that activates or increases the frequency,rate or extent of T cell differentiation.An additional gene having a peculiar function is the SAMHD1(SAM domain and HD domain 1),which is also associated with the regulation of the innate immune responses.The SLA2(Src-likeadaptor 2)gene acts in the T cell regulation and negative regulation of B cell activation,and in any process that stops,prevents,or reduces the frequency,rate or extent of B cell activation.

The genes in the enrichment pathways(Additional file 2),such as the MYPN(myopalladin),PDGFRL(platelet derived growth factor receptor like),ROR1(receptor tyrosine kinase-like orphan receptor 1),CNTN1(Contactin 1)and FANK1(fibronectin type 3,and ankyrin repeat domains 1)are associated with the Immunoglobulin-like domains that are related in both sequence and structure,and can be found in several diverse protein families.Ig-like domains are involved in a variety of functions,including cellcell recognition,cell-surface receptors,muscle structure and the immune system[63].

Additional file 1:Table S7 and S8 show the genes with functions associated with immune response,innate,and adaptative immune response or those participating　in　the　regulation　of　innate　immune response for the genomic regions associated with RBC and PLT traits,respectively

The genomic regions and genes associated with RBC are presented in Additional file 1:Table S7 and illustrated in Fig.9.The KEGG pathays analysis revealed PI3K-Ark and toll-like receptor signaling pathways significantly enriched for WBC trait(Additional File 2).The CD109 gene(CD109 antigen)is a GPI-linked cell surface antigen expressed by CD34+acute myeloid leukemia cell lines,T-cell lines,activated T lymphoblasts,endothelial cells,and activated platelets[64].This gene was also reported by[26],indicating that CD109 gene potentially contributes to resistance to gastrointestinal nematodes in sheep.

The IL2RB gene(interleukin 2 receptor),which is involved in T cell-mediated immune responses,is present in three forms with respect to ability to bind interleukin 2.The low affinity form is a monomer of the alpha subunit and it is not involved in signal transduction.The intermediate affinity form consists of an alpha/beta subunit heterodimer,while the high affinity form consists of an alpha/beta/gamma subunit heterotrimer.Both the intermediate and high affinity forms of the receptor are involved in receptor-mediated endocytosis and transduction of mitogenic signals from interleukin 2[provided by RefSeq,Jul 2008].Kondo et al.[65]showed that a clonogenic common lymphoid progenitor,a bone marrowresident cell that gives rise exclusively to lymphocytes(T,B,and natural killer(NK)cells),can be redirected to the myeloid lineage by stimulation through exogenously expressed interleukin(IL)-2 and GM-CSF(granulocyte/macrophage colony-stimulating factor)receptors.As the IL2RB is one of the receptors that act in IL-15 expression,it has been proposed to be a critical cytokine for NK　celldevelopment.Thisgene　can　affectthis redirections in cytokine signaling and can regulate cellfate decisions.Additionally,a critical step in lymphoid commitment is downregulation of cytokine receptors that drive myeloid cell development.

Fig.9　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for red blood cells(RBC)trait

隧道斜井：拱顶（腰）处检测厚度（设计厚度40cm）在28～32cm，缺陷纵向长度约为1.0m，缺陷环向长度约为0.8～1.0m。

为进一步强化测绘地理信息统计质量，需要对其实际定位进行明确，并在此基础上适当开展试点探索工作。在实际工作中，为提升统计数据质量，除了要用发展的眼光来审视测绘地理信息监测，还要在实践中不断提出行之有效的改进思路，对统计数据方法进行完善，使资源得到最大程度上的整合与优化。为此，要从以下两个方面入手：其一，对统计数据工作思路进行协调统一，加强对测绘地理信息工作的重视程度，相关人员能够结合测绘地理信息客观规律，不断提高对统计数据的认知，做好定位后，给出相应指导意见。其二，在选取试点地区时，要具备一定代表性，结合当地实际情况，因地制宜，发挥示范作用，使地理信息监测更加具有针对性。

The CXCR4 gene may influence the immune system under physiologic and pathologic conditions through negative regulation of MHC class II expression,possibly through PKA and SRC kinase[67].In a study,Contento et al.[68]observed that while human T cell activation by antigen-presenting cells is taking place,the CCR5 and CXCR4 chemokine receptors are recruited into the immunological synapse,where they deliver costimulatory signals.This gene also participates in the intestinal immune network for IgA production pathway.

The ASCC3,CRLF2RB,FNDC3A,FNDC4,IFNAR1,IFNAR2,LEPR,MYLK,NCAM2,PVRL4SLAMF1,TXNIP,CD109,ACAN,NTRK3,CCDC141,LEPR,MYLK,PVLR4,IL12RB2,IGSF10,and JAM2 genes(Additional file 1:Table S7)are domains with an Ig-like fold that can be found in several proteins in addition to immunoglobulin molecules.For example,Ig-like domains occur in several different types of receptors(such as various T-cell antigen receptors),several cell adhesion molecules,MHC class I and II antigens,as well as the hemolymph protein hemolin,and the muscle proteins titin,telokin and twitchin(IPR013783).

The KEGG pathway analysis revealed platelet activation,toll-like receptor and PI3K-Ark signaling pathways for PLT(Additional file 2).The CXCL1,CXCL8 and CXCL10 genes identified for PLT(Additional file 1:Table S8;Fig.10)belong to a subfamily of chemokines that basically are structurally related to molecules that regulate cell trafficking of various types of leukocytes through interactions with a subset of 7-transmembrane and G protein-coupled receptors.They also play a fundamental role in the development,homeostasis,and functionality of the immune system[69].

Different genes were also identified for PLT,in these regard,the Fc-gamma receptors(FCGRs),such as FCGR1A,FCGR3A,and FCGR2B,that are integral membrane glycoproteins which exhibit complex activation or inhibitory effects on cell functions after aggregation by complexed immunoglobulin G(IgG)[70].These genes encode proteins that play an important role in the immune response,a receptor for the Fc portion of immunoglobulin G,and participate in the removal of antigen-antibody complexes from the circulation,as well as other antibody-dependent responses.It is also involved in the phagocytosis of immune complexes and in the regulation of antibody production by B-cells,respectively[provided by RefSeq,Jul 2008;RefSeq,Jun 2010].

where freq.A,freq.a,freq.B and freq.b are the frequencies of alleles A,a,B and b,respectively,and freq.AB,freq.ab,freq.aB and freq.Ab are the frequencies of haplotypes Ab,ab,aB and Ab in the population,respectively.The expected frequency of haplotype AB(freq.AB)is calculated as the product between freq.A and freq.B.The r2takes values close to zero when the alleles A and B segregate independently.The freq.AB higher or lower than the expected value indicates that these two loci in particular tend to segregate together and are in LD,with a maximum value for r2of one.

Fig.10　Manhattan plot of the additive genetic variance explained by windows of 10 adjacent SNPs for platelet(PLT)trait

For　HCT　trait(Table　S9),genes　like　IGF1R,IFNAR1,IFNAR2,IL12RB2,IL2RB,C2F2RB,PVRL4,LEPR,MYLK,SLAMF1,ADGRF3,and TXNIP were identified.These genes encode domains with an Iglike fold that can be found in several proteins in addition to immunoglobulin molecules.For example,Ig-like domains occur in several different types of receptors(such as T-cell antigen receptors),many cell adhesion molecules,MHC class I and II antigens,as well as the hemolymph protein hemolin,and the muscle proteins titin,telokin and twitchin.

从理论上出发,对于盆腔炎症,需进行药敏试验,根据试验结果,选择对应药物,然而实际上临床在未得到试验结果前需予以抗菌治疗,因此需临床医师根据实践经验,选择抗菌药。一般而言,症状较轻者口服第2代或第3代头孢菌素、喹诺酮类便可,无需住院治疗,症状严重伴有卵巢输卵管脓肿、盆腔腹膜炎等者,需住院治疗。

The IL1A and IL1B genes have the protein encoded as a member of the interleukin 1 cytokine family.This cytokine is a pleiotropic cytokine involved in several immune responses,inflammatory processes,and hematopoiesis[provided by RefSeq,Jul 2008].Also the IL1B gene,initially discovered as the major endogenous pyrogen,induces prostaglandin synthesis,neutrophil influx and activation,T-and B-cells activation,cytokine production,antibody production,fibroblast proliferation and collagen production.Also it was observed a function related to ligand-binding domain that displays similarity to C2-set immunoglobulin domains(antibody constant domain 2)in the LEPR gene.It has a specific effect on T lymphocyte responses,differentially regulating the proliferation of naive and memory T-cells.

For HCT and HGB traits,genes related to immune response were identified,i.e.the RAC2 gene,that seems to increase the resistance to parasites.In order to understand the function of RAC2 GTPase in regulating the cellular immune response,Williams et al.[71]assayed the effects of hemocytes in parasitized RAC2 mutant larvae,as well as characterized the effectofover-expressingRAC2　genein　hemocytes.These authors reported that this gene has an important role in the cellular immune response,being necessary for hemocyte spreading and cell-cell contact formation　duringimmunesurveillanceagainstthe parasitoid L.boulardi.When an invading parasitoid is recognized as a foreign body,circulating hemocytes should recognize them and attach to the egg chorion.After the attachment,thehemocytesshould　then spread out to form a multilayered capsule surrounding the invader.In RAC2 mutants this process was disrupted.Besides of that,this gene also acts on the B cell receptor signaling pathway,like the PPP3CA gene.

The HSPD1 gene encodes a mitochondrial protein that plays a role as a signaling molecule in the innate immune system.Zanin-Zhorov et al.[72]reported that this gene,as well as a synthetic peptide derived from it,acted as a costimulatory of human regulatoryCD4-positive/CD25　(IL2RA)positive T cells(Tregs),which inhibit lympho proliferation and IFNG and TNF secretion by CD4-positive and CD8-positive T cells.The authors concluded that the self-molecule HSPD1 can downregulate adaptive immune　responsesby　upregulating　Tregsthrough TLR2 signaling.

The linkage disequilibrium(LD)between two SNPs was evaluated using r2as follows:

Conclusions

The traits indicating gastrointestinal parasites resistance shown adequate genetic variability to respond to selection in Santa Inês breed,and it is expected a higher genetic gain for famacha when compared to the others traits.The level of LD estimated for markers separated by less than 1 Mb indicates that the Ovine SNP12k BeadChip will likely be a suitable tool for identifying genomic regions associated with those traits related to gastrointestinal parasite resistance.

Several candidate regions related to immune system development and activation,inflammatory response,regulation of lymphocytes,and leukocytes proliferation were found in this study.These candidate regions and CG may help in the selection of animals with higher resistance to parasites,and consequently,reduce the anthelmintic drugs costs,also the production losses linked to the use of it.Furthermore,when reducing the use of anthelmintic drugs,a potential reduction in waste problems regarding meat and milk discharge due to drugs residues should be minimized,reducing the impact upon the environment.

Additional files

Genomic regions associated with egg counts per gram of feces(EPGlog)in Santa Inês sheep;Table S5.Genomic regions associated with famacha(FAM)in Santa Inês sheep;Table S6.Genomic regions associated with blood cell count(WBC)in Santa Inês sheep;Table S7.Genomic regions associated with red blood cells(RBC)in Santa Inês sheep;Table S8.Genomic regions associated with platelet(PLT)in Santa Inês sheep;Table S9.Genomic regions associated with hematocrit(HCT)in Santa Inês sheep;Table S10.Genomic regions associated with blood cell count(HGB)in Santa Inês sheep.(XLS 74 kb)Additional file 2:Table S11.KEGG pathways(P＜0.05)for counting eggs per gram of feces(EPGlog),famacha(FAM),white blood cells(WBC),red blood cells(RBC),platelets(PLT),hematocrit(HCT),and hemoglobin(HGB)traits obtained from the DAVID software;Table S12.Gene Ontology terms for biological processes significantly(P＜0.01)related with counting eggs per gram of feces(EPGlog),famacha(FAM),white blood cells(WBC),red blood cells(RBC),platelets(PLT),and hematocrit(HCT)traits.(XLS 68 kb)

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Abbreviations

BCS:Body condition score;EPGlog:Egg counts per gram of feces;FAM:Degree of anemia assessed by the famacha card;FC:Feces consistency;FS:Fur score;HCT:Hematocrit;HGB:Hemoglobin;LD:Linkage disequilibrium;PLT:Platelets;RBC:Red blood cell count;WBC:White blood cell count

Acknowledgements

To Fapesp,(Sao Paulo Research Foundation,grants#2010/05516-7,#2011/00396-6 and#2014/07566-2).MP Berton,E Peripolli received scholarship from Coordination for the Improvement of Higher Education Personnel(CAPES;Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)in conjunction with the Postgraduate Program on Genetics and Animal Breeding,Universidade Estadual Paulista,Faculdade de Ciências Agrárias e Veterinárias(FCAV,Unesp).F.B held productivity research fellowships from The Brazilian National Council for Scientific and Technological Development(CNPQ)

老三这时也看到了孔老一，连滚带爬冲了过来，也不说话，见面就跪：“哥，哥，咱爸过山去了。”说罢便伏地嚎啕大哭说罢便伏地号啕大哭。

Availability of data and materials

The phenotypic and genomic information used in this study belongs to a private sheep breeding program company,so we do not have authorization to share the data.

Funding

Sao Paulo Research Foundation-FAPESP grants#2010/05516-7,#2011/00396-6 and#2014/07566-2.

Authors’contributions

MPB,RMOS,EP,NBS did the manuscript writing and discussion of the results.JFM,MSA,BVG,in Genomic modeling and genomic analysis.GB in concepts,writing and modeling.PSO in phenotypic data collection and genotyping.JPE in data management.FB JBSF in conception,funding,modeling,genomic analysis and coordination.All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval

All experimental procedures involving animals were approved by the Ethical Committee of FZEA/USP CEUA n°7,718,021,216.

Author details

1Departamento de Zootecnia,Faculdade de Ciências Agrárias e Veterinárias,Universidade Estadual Paulista,Via de acesso Prof.Paulo Donato Castellane,s/no,Jaboticabal,SP CEP 14884-900,Brazil.2Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria INIA,Crta.de la Coruña,km 7,5-,28040 Madrid,Spain.3Departamento de Producción Agraria,School of Agricultural,Food and Byosystems Engineering,Universisdad Politécnica de Madrid,Campus Ciudad Universitaria Avda.Complutense 3-Avda.Puerta Hierro,28040 Madrid,Spain.4Instituto Nacional de Investigación Agropecuária(INIA),Ruta 50 Km.12,Colonia,Uruguay.5Faculdade de Zootecnia e Engenharia de Alimentos,Nucleo de Apoio à Pesquisa em Melhoramento Animal,Biotecnologia e Transgenia,Universidade de São Paulo,Rua Duque de Caxias Norte,225,Pirassununga,SP CEP 13635-900,Brazil.

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