Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Transcription

1 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Genetics Selection Evolution RESEARCH Open Access Genome-wide ssocition nd genomic prediction for host response to porcine reproductive nd respirtory syndrome virus infection Nichols J Boddicker 1, Angelic Bjorkquist 1, Rymond RR Rowlnd 2, Jon K Lunney 3, Jmes M Reecy 1 nd Jck CM Dekkers 1* Astrct Bckground: Host genetics hs een shown to ply role in porcine reproductive nd respirtory syndrome (PRRS), which is the most economiclly importnt disese in the swine industry. A region on Sus scrof chromosome (SSC) 4 hs een previously reported to hve strong ssocition with serum viremi nd weight gin in pigs experimentlly infected with the PRRS virus (PRRSV). The ojective here ws to identify hplotypes ssocited with the fvorle phenotype, investigte dditionl genomic regions ssocited with host response to PRRSV, nd to determine the predictive ility of genomic estimted reeding vlues (GEBV) sed on the SSC4 region nd sed on the rest of the genome. Phenotypic dt nd 60 K SNP genotypes from eight trils of ~200 pigs from different commercil crosses were used to ddress these ojectives. Results: Across the eight trils, heritility estimtes were 0.44 nd 0.29 for virl lod (VL, re under the curve of log-trnsformed serum viremi from 0 to 21 dys post infection) nd weight gin to 42 dys post infection (WG), respectively. Genomic regions ssocited with VL were identified on chromosomes 4, X, nd 1. Genomic regions ssocited with WG were identified on chromosomes 4, 5, nd 7. Aprt from the SSC4 region, the regions ssocited with these two trits ech explined less thn 3% of the genetic vrince. Due to the strong linkge disequilirium in the SSC4 region, only 19 unique hplotypes were identified cross ll popultions, of which four were ssocited with the fvorle phenotype. Through cross-vlidtion, ccurcies of EBV sed on the SSC4 region were high (0.55), while the rest of the genome hd little predictive ility cross popultions (0.09). Conclusions: Trits ssocited with response to PRRSV infection in growing pigs re lrgely controlled y genomic regions with reltively smll effects, with the exception of SSC4. Accurcies of EBV sed on the SSC4 region were high compred to the rest of the genome. These results show tht selection for the SSC4 region could potentilly reduce the effects of PRRS in growing pigs, ultimtely reducing the economic impct of this disese. Bckground For decdes the swine industry hs een ttling the economiclly importnt disese of porcine reproductive nd respirtory syndrome (PRRS), cused y the PRRS virus (PRRSV) ut success hs een limited. Since the infection mechnisms of the virus re not fully understood, vccine development is chllenge [1]. There hs een no genetic selection of pigs for PRRS resistnce or tolernce due to the lck of good DNA mrkers for mrker-ssisted selection nd lck of good indictor trit for indirect selection. * Correspondence: jdekkers@istte.edu 1 Deprtment of Animl Science, Iow Stte University, Ames, Iow 50011, USA Full list of uthor informtion is ville t the end of the rticle Recently, quntittive trit locus (QTL) ws identified on Sus scrof (SSC) 4 tht explins considerle mount of the totl genetic vrince for serum viremi nd weight gin of wened piglets following experimentl infection [2]. In susequent work, the effect of this ~1 M region ws identified in three unrelted popultions, encompssing five experimentl chllenge trils of 200 wened pigs [3]. Mny of the single nucleotide polymorphisms (SNPs) in this ~1 M region re in very high linkge disequilirium, which mkes identifiction of the custive muttion difficult; single tg SNP (SNP WUR ) ppers to trck the effect of the region with the B llele eing ssocited with PRRS tolernce [2,3]. The fore mentioned 2014 Boddicker et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly credited.

2 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 2 of 14 studies did not thoroughly investigte the rest of the genome, which my contin pertinent informtion for host response to PRRSV infection. The ddition of three trils to the dt nlyzed in Boddicker et l. [3] hs potentilly incresed the power in order to find these, likely smller, effects. The ojectives of the current study were to (1) reestimte genetic prmeters for host response to experimentl PRRSV infection in growing pigs y including three dditionl, unrelted popultions, (2) investigte dditionl genomic regions ssocited with weight gin nd viremi in response to PRRSV chllenge, (3) determine if there is smller informtive hplotype lock within the ~1 M region on SSC4 ssocited with host response to PRRSV infection, (4) determine which hplotypes within the SSC4 region re ssocited with the fvorle effects of the QTL for viremi nd weight gin, nd (5) investigte ccurcies of genomic estimted reeding vlues (GEBV) for host response to PRRS sed on the SSC4 region nd the rest of the genome, y using cross-vlidtion. Methods The Knss Stte University Institutionl Animl Cre nd Use Committee pproved ll experimentl protocols for this study. Study design A detiled description of the design, dt collection, nd moleculr techniques used in the PRRS host genetic consortium trils hs een previously pulished [4]. Briefly, ech chllenge tril of ~200 commercil pigs involved trnsporting nimls t wening (18 28 dys of ge) to Knss Stte University, where they were sujected to PRRSV chllenge. Within tril, pigs were from the sme high helth frm, except for trils 5 nd 8, which ech included pigs from two frms. All frms were free of PRRSV, Mycoplsm hyopneumonie, nd swine influenz virus. Upon rrivl, pigs were rndomly plced into pens of 10 to 15 pigs. After 7-dy cclimtion period, pigs etween 25 nd 35 dys of ge (dy 0), were experimentlly infected intrmusculrly nd intrnslly with 10 5 (TCID50) of NVSL , highly virulent PRRSV isolte [5]. Blood smples were collected t 6, 0, 4, 7, 11, 14, 21, 28, 35, nd 42 dys post-infection (dpi). Body weight ws mesured t 0, 7, 14, 21, 28, 35, nd 42 dpi. Pigs were euthnized t 42 dpi. Trils 7 nd 8 were stopped t 35 dpi due to fcility vilility. Viremi ws mesured using semi-quntittive TqMn PCR ssy for PRRSV RNA, s descried in Boddicker et l. [2]. Assy results were reported s the log 10 of PRRSV RNA copies per ml of serum. Er tissue ws collected from ll pigs for DNA isoltion. Tissue or genomic DNA from the sires of pigs in trils 1 through 3 nd from ville sires nd dms for trils 4 through 8 ws supplied y the reeding compnies. Tissues or DNA smples were sent to GeneSeek Inc. (Lincoln, Nersk) for genotyping with Illumin s Porcine SNP60 BedChip (Sn Diego, Cliforni). Dt from eight trils of up to 200 pigs were nlyzed (Tle 1). Trils 1 through 3 were descried in Boddicker et l. [2] nd included pigs of the sme cross from single reeding compny. Trils 4 nd 5 were descried in Boddicker et l. [3] nd included two unrelted popultions from different reeding compnies. Trils 6 through 8 re unique to this pper nd were sourced from three dditionl reeding compnies, with pigs tht were unrelted to those in Boddicker et l. [2,3]. Pigs in tril 7 were sourced from the sme reeding compny s those in tril 4 ut were produced using different sire nd dm lines. Tle 1 provides n overview of the popultion structure y tril. See Boddicker et l. [2] for further detils on trils 1 through 3 nd Boddicker et l. [3] for further detil on trils 4 nd 5. Pigs in tril 6 were purered Lndrce (LR). Pigs in tril 7 were from Pietrin sires nd Lrge White (LW)/LR/Yorkshire dms. Tril 8 pigs were from Duroc sires nd Yorkshire/LR dms. Across ll eight trils, 175 pigs died efore 42 dpi (Tle 1). Ded pigs were necropsied nd susequent gross nd microscopic pthology y ord-certified pthologist identified PRRS ssocited disese s the mjor source of mortlity, except for tril 6. Deth loss ws high in tril 6, 46% y dy 42, due to secondry cteril infections, s identified y pthology, including Escherichi coli, Streptococcus suis, Stphylococcus ureus, nd Mycoplsm hyopneumonie. Phenotypic trits nd pedigree Detils on the phenotypic trits nlyzed re in Boddicker et l. [2,3]. Briefly, VL ws quntified s the re under the curve for log-trnsformed serum viremi t 0, 4, 7, 11, 14, nd 21 dpi. Weight gin to 42 dpi (WG) ws clculted s ody weight (BW) t 42 dpi minus BW t dy 0 dpi; for trils 7 nd 8 WG35 ws clculted. Mortlity ws defined s deth prior to the end of the experiment. Edits for trils 1 3 re in Boddicker et l. [2] nd in Boddicker et l. [3] for trils 4 nd 5. Briefly, edits for VL removed 34 individuls from the first 3 trils nd 15 for trils 4 nd 5. For WG, 47 nd 18 individuls were removed, respectively. Edits removed 88 individuls from trils 6 through 8, with 86 due to deth prior to 21 dpi nd two with missing viremi dt. For WG42, 111 individuls were removed from trils 6 through 8, ll due to deth prior to 42 dpi. The totl numer of individuls ville fter edits is in Tle 2. Recorded pedigrees (sire nd dm) were checked nd corrected using SNP genotypes, s descried in Boddicker et l. [3]. Briefly, prent-offsping mismtch frequencies

3 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 3 of 14 Tle 1 Popultion structure of trils 1 through 8 WUR Numer offspring per fmily Totl numer of offspring Tril Breed 1 N frequency 4 Min Men Mx Brrows Gilts Totl Ded ,3 Sires LR Dms LW Sires Duroc Dms LW/LR/Y Sires Duroc Dms LR/Y Sires LR Dms LR Sires Pietrin Dms LW/LR/Y Sires Duroc Dms Y/LR LR Lndrce, LW Lrge White, Y Yorkshire; 2 trils 1 through 3 consisted of the sme cross from one reeding compny; 3 trils 1 nd 2 were Lndrce y Lrge White crosses; for tril 3, 121 piglets were from Lndrce sires y Lrge White dms crosses nd 63 piglets were from the reciprocl cross of Lrge White sires y Lndrce dms; 4 frequency of the fvorle llele (B) t SNP WUR on SSC4; for trils 1 3, dm genotypes were not provided, so frequencies were determined using ordered genotypes of the offspring; 5 ded prior to 42 dys fter infection. were clculted s the numer of SNPs for which the prent nd offspring hd opposing homozygous genotypes divided y the totl numer of polymorphic SNPs for which the prent nd offspring were oth homozygous. If prent-offspring pir hd mismtch frequency of less thn 2%, then the nmed prent ws ccepted. Otherwise, offspring genotypes were compred to ll possile prents nd the most likely prent ws chosen. The originl pedigrees provided y the reeding compnies were mostly correct ut few full- nd hlf-si fmilies were ressigned to different sires nd dms. Sttisticl nlyses Heritilities nd vrinces due to litter were estimted sed on vlidted nd corrected pedigree reltionships, s determined from SNP genotypes (see Boddicker et l. [3] for detils), with single-trit niml model using the softwre ASREML [6]. Sex nd the interction of tril nd prity of the sow were included s fixed fctors nd pen within tril, niml, nd litter s rndom effects. Piglets were orn from prities 1 to 7. Prities 3 through 7 were comined into one prity clss. The effect of frm of origin for trils 5 nd 8 ws not significnt (p > 0.43) nd ws excluded from the nlyses. The genetic correltion etween trits ws estimted using ivrite niml model with the sme fixed nd rndom fctors s used in the single-trit models. Genome-wide ssocition nlyses Associtions of SNP genotypes with phenotypes were nlyzed y fitting ll SNPs simultneously using Byesin genomic selection methods [7], s implemented in the softwre GenSel [8], using the following mixed model: y ¼ X þ Xk j z j α j δ j þ ε where y = vector of phenotypic oservtions, X = incidence mtrix relting fixed fctors to phenotypes, = vector of fixed fctors of sex, pen within tril, nd the interction of tril nd prity clss, z j = vector of the genotype covrite for SNP j (j =1 to k) sed on the numer of B lleles using Illumin s (Sn Diego, Cliforni) genotype clling (coded 0, 1, 2, or equl to the tril verge for missing genotypes), α j = llele sustitution effect for SNP j, nd δ j = indictor for whether SNP j ws included (δ j =1) or excluded (δ j = 0) in the model for given itertion of the Mrkov chin Monte Crlo (MCMC). Pen within tril Tle 2 Trit mens nd estimtes (±SE) of heritility nd litter effects (proportions of phenotypic vrince) for virus lod nd weight gin fter infection otined from single trit pedigree-sed ASREML nlyses Trit 1 n Men SD 2 Heritility Litter Phenotypic correltion Genetic correltion VL, units ± ± ± ± 0.20 WG42, kg ± ± VL virl lod clculted s re under the curve of log-trnsformed viremi etween 0 nd 21 dpi nd WG42 weight gin from 0 dpi to 42 dpi; 2 stndrd devition clculted s the squre root of the sum of niml, litter, nd residul vrinces from the joint ASREML nlysis of ll 8 trils.

4 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 4 of 14 ws included s fixed fctor, s opposed to rndom effect in ASREML, ecuse the current version of GenSel does not llow dditionl rndom effects. A totl of itertions were run for ech nlysis, with the first 5000 itertions discrded s urn-in. The proility of δ i = 0 ws set equl to π = The Byesin model ws implemented using method Byes-B [7]. Genomic regions ssocited with trits were identified using 1 M, non-overlpping windows using uild 10.2 of the swine genome ( Sus%20scrof%2C%20whole%20genome%20shotgun%20 sequence, ccessed Novemer 1, 2011). Hplotype nlyses The QTL previously identified on SSC4 included 38 SNPs, two of which hd 0 clled genotypes [2]. The remining 36 SNPs in the ~1 M region on SSC4 were nlyzed to remove uninformtive groups of SNPs, with the gol of reducing the size of the ~1 M region. Linkge disequilirium (LD) etween SNPs in the ~1 M region ws determined using Hploview [9]. Using univrite niml model in ASREML tht included sex, the interction of tril nd prity of the sow, nd genotype for SNP WUR s fixed fctors nd pen within tril, niml, nd litter s rndom effects, ech of the remining 35 SNPs ws fitted s n dditionl covrite, one t time, to determine if the dditionl SNP ccounted for effects not cptured y SNP WUR The threshold for discrding SNP from the region ws p < Prentl nd offspring genotypes for the 36 informtive SNPs in the ~1 M region on SSC4 were ordered using the PHASE softwre [10], seprtely for ech tril. Hplotypes with high proilities, s identified y PHASE softwre, were nlyzed with MEGA5 softwre [11] to estlish phylogenetic tree. The neighor-joining method, with the p-distnce option, ws used to crete the tree. Hplotypes identified in the ~1 M region nd for the reduced region were nlyzed. Using the results from the phylogenetic tree for the reduced region, hplotypes tht crried the B llele t SNP WUR were llocted to two groups sed on phylogenetic distnce from one nother. The min effect of hplotype group ws fitted s fixed clss fctor in the ovementioned ASREML nlysis to determine which hplotypes were ssocited with the desirle phenotypes of VL nd WG. Due to the pprent dominnce effect of the B llele, n individul hd to hve t lest one copy of hplotype tht crried the B llele to e plced in tht group. If the two groups of hplotypes tht crried the B llele were not significntly different from ech other ut were significntly different from the group of hplotypes tht crried the A llele, oth B hplotypes groups were ssumed to crry the fvorle llele of the custive muttion. A similr procedure ws used for the A hplotypes to determine whether ll A hplotypes were ssocited with n unfvorle phenotype. Cross-vlidtion Accurcy of genomic predictions cross popultions ws evluted y cross-vlidtion, which involved trining on one popultion nd vlidting on nother popultion. Popultions were defined y tril, except for trils 1 through 3, which were considered s one popultion, since pigs included in those three trils were crossreds from the sme lines nd reeding compny. Ech popultion ws vlidted twice, using reduced nd full trining popultion (Tle 3). The trining popultions included ll popultions except for the popultion tht ws eing vlidted, with some exceptions. For the reduced trining popultions, only one of the first three trils ws used. Tril 3 ws used for this purpose ecuse it hd the highest estimtes of heritility of the three trils for oth VL nd WG (0.45 nd 0.50, respectively). For the full trining popultions, trils 1 through 3 were included when vlidting on trils 4 through 8. When vlidting on trils 1 nd 2, tril 3 ws included in the trining popultion, which resulted in some of the nimls in the vlidtion popultion to e relted to nimls in the trining popultion. To llow predictive ility of the rest of the genome, excluding the SSC4 region, to e ssessed, estimtes of llele sustitution effects for the trining popultion were cquired using the dditive Byesin model descried ove ut with genotype t SNP WUR included s fixed clss effect to ccount for the dditive nd non-dditive effects of the QTL in this region on VL nd WG [2]. Method Byes-CPi ws used rther thn Byes-B ecuse it ssumes homogenous vrince cross ll SNPs, which ppered pproprite given the results of the GWAS, nd estimtes prmeter π from the dt [7]. The strting vlue of π ws set to The GEBV of individul i in the vlidtion popultion for the rest of the genome (excluding the SSC4 region) ws predicted s: GEBV i ¼ Xk j¼1 z ij^α J where k = numer of SNPs included in the prediction ( SNPs), z ij = genotype covrite of SNP j for niml i (coded 0, 1, 2, or tril verge for missing genotypes), nd ^α j = llele sustitution effect estimte for SNP j sed on nlysis of the trining popultion. To estimte the ccurcy of predictions sed on the SSC4 region lone, the estimtes of the dditive nd dominnce effects of SNP WUR from the GenSel nlysis of the trining popultion were used to estimte the genotypic vlue sed on the oserved genotype for SNP WUR

5 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 5 of 14 Tle 3 Trining nd vlidtion popultions used for cross-vlidtion of genomic prediction for virus lod (VL) nd weight gin (WG) Vlidtion popultion Reduced trining popultion Full trining popultion Tril N for VL 1 N for WG 2 Trils N for VL 1 N for WG 2 Trils N for VL 1 N for WG , 5, 6, 7, , 4, 5, 6, 7, 8 1,059 1, , 5, 6, 7, , 4, 5, 6, 7, 8 1,059 1, , 5, 6, 7, , 5, 6, 7, , 2, 3, 5, 6, 7, 8 1,218 1, , 4, 6, 7, , 2, 3, 4, 6, 7, 8 1,229 1, , 4, 5, 7, , 2, 3, 4, 5, 7, 8 1,290 1, , 4, 5, 6, , 2, 3, 4, 5, 6, 8 1,219 1, , 4, 5, 6, , 2, 3, 4, 5, 6, 7 1,225 1,189 1 VL virus lod clculted s re under the curve of log-trnsformed viremi etween 0 nd 21 dys post infection (dpi); 2 WG weight gin from 0 dpi to 42 dpi. of ech individul in the vlidtion popultion. Accurcy of prediction, defined s the correltion etween the GEBV nd the true BV, were estimted s the correltion etween the GEBV nd djusted phenotypes in the vlidtion popultion divided y the squre root of heritility (Tle 2) otined when nlyzing ll eight trils together [3]. Here, djusted phenotypes were phenotypes djusted for estimtes of fixed effects (sex, pen within tril, nd the interction of tril nd prity clss) within the vlidtion popultion. Estimtes of ccurcies of GEBV cross ll eight trils were then otined y correlting djusted phenotypes with GEBV devited from their respective tril mens, nd dividing y the squre root of heritility. Results nd discussion Genetic prmeters Estimtes of heritility, litter effects, nd phenotypic nd genetic correltions for VL nd WG re in Tle 2. Heritilities for VL nd WG were moderte t 0.44 nd 0.29, respectively, nd were of similr mgnitude s those reported y Boddicker et l. [3] using suset (trils 1 5) of the dt nlyzed here. In comprison to the estimtes from the first five trils, the proportion of vrince explined y litter effects decresed for VL (0.11 to 0.09) nd incresed for WG42 (0.09 to 0.12). These estimtes of heritility provide solid evidence tht genetic improvement of host response to PRRSV infection is possile. Phenotypic nd genetic correltions etween VL nd WG were moderte nd negtive. Further nlysis of the SSC4 region SSC4 effects in trils 6 8 Results of single-mrker nlyses for the most significnt SNP in the SSC4 region (WUR ) tht ws identified y Boddicker et l. [2] for VL nd WG42 for ll trils comined, nd trils 6 through 8 individully, re in Figure 1. Results for SNP WUR for trils 1 through 3 re in Boddicker et l. [2] nd in Boddicker et l. [3] for trils 4 nd 5. This SNP ws significnt for WG42 (Figure 1A) for trils 6 (p < 0.01) nd 8 (p < 0.004), where susceptile AA nimls gined 4.8 kg nd 3.5 kg less, respectively, thn AB nimls. Although the effect ws in the sme direction for tril 7 for WG42, there ws no sttisticl significnce etween the genotypes for this tril (p > 0.57). For VL (Figure 1B), the SNP ws significnt for trils 6 (p < 0.005) nd 7 (p < 0.001) ut not for tril 8 (p > 0.56). For tril 8, the effects of the SNP on VL were, however, in the sme direction s for the other trils (i.e. incresed VL for AA nimls compred to AB nimls). The non-significnt effects of the WUR SNP on WG in tril 7 nd on VL in tril 8 re proly flse negtives ecuse the effect of the SNP ws significnt for the other trit in ech of these trils; it is less likely tht the significnt effects in trils 7 nd 8 re flse positives ecuse the effect is present in the other trils for oth trits [3]. These results show tht the effect of this region ws present in six unrelted popultions from five different reeding compnies. Reducing the region on SSC4 The region on SSC4 spns se pirs nd includes totl of 38 SNPs tht re on the 60 k SNP pnel, including two tht re fixed nd two for which no genotypes were clled. Figure 2 shows LD plot of the 34 polymorphic SNPs cross trils 1 through 8. Hploview identified five hplotype locks, including lock (Block 2) of 15 SNPs tht spnned 487 k tht hd very high LD mongst most SNPs. This lock hrors SNP WUR , which cptured over 99% of the effects of the SSC4 region on VL nd WG [2]. In generl, LD is not expected to e the sme etween reeds nd unrelted popultions over such long distnces. As n exmple, Amrl et l. [12] found very different LD ptterns etween LW popultion, Ningxing ( Chinese reed), nd the Europen wild or, cross three different genomic regions. Differences in LD cn result from muttion,

6 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 6 of 14 Figure 1 Lest squres mens y genotype for SNP WUR cross ll trils 1 through 8 nd within trils 6, 7, nd 8 for weight gin from 0 to 42 dys post infection (A) nd virl lod (B). Virl lod ws clculted s re under the curve of log-trnsformed viremi from 0 to 21 dys post infection. Within tril, columns with different letter re significntly different t p < Numers of individuls within genotype re listed in the rs. recomintion, selection, nd drift. Block 2 is unique in the sense tht LD ws very high cross six unrelted popultions tht consisted of different reeds (Figure 2). Tle 4 shows the p-vlue for the effects of ech SNP in the SSC4 region on VL nd WG42, when the effect of SNP WUR is ccounted for in the model. The SNPs locted efore nd fter Block 2 ll hd p-vlues greter thn 0.05 for oth VL nd WG42, indicting tht these SNPs do not cpture significnt mount of dditionl vrition in VL or WG42 tht is not lredy ccounted for y SNP WUR Therefore, these SNPs re proly not in high LD with the custive muttion nd were excluded from the cndidte region. Mny of the SNPs within Block 2 lso hd p-vlues greter thn 0.05 ut, with the high LD present nd confounding mong SNPs, Block 2 ws not further reduced in size. Given these results, the ~1 M cndidte region ws reduced to 487 k region tht proly hrors the custive muttion. Hplotype nlysis A totl of 77 unique hplotypes were identified in the originl ~1 M region cross ll eight trils, 11 of which crried the B llele t SNP WUR (B hplotypes). After reducing the region to 487 k, only 19 unique hplotypes remined, of which four crried the B llele. Tle 5 shows the frequency of ech B hplotype, long with the trils ech hplotype ws present in. Eight of the originl eleven B hplotypes were comined into one hplotype (hplotype 17) fter the region ws reduced. This ws the most common hplotype nd it ws present in ll trils nd in every prentl reed. Therefore, it is proly the

7 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 7 of 14 Figure 2 Linkge disequilirium (r 2 ) plot of the 1 M region on Sus scrof chromosome 4 cross trils 1 through 8. Blck squres signify r 2 = 100% nd white squres signify r 2 = 0%. ncestrl hplotype, with the remining B hplotypes originting from recomintion or muttion since the reeds were estlished. Figure 3 shows the phylogenetic tree for hplotypes in the 487 k region, generted using Meg 5 softwre. Using only the unique hplotypes, the tree clerly grouped the hplotypes sed on the llele present t SNP WUR , with the B hplotypes grouped t the ottom of the tree in Figure 3. Strting with hplotype 17 in Figure 3, hplotypes were grouped y phylogenetic distnce sed on two cuts in the phylogenetic tree to determine whether ll B hplotypes crried the fvorle custive vrint. If n individul hd t lest one of the hplotypes to the right of the cut, the individul ws plced in the group to the right of the cut, ecuse of the identified dominnce mode of ction of the QTL [3]. To crete grouping 1, the tree ws cut to the left of hplotype 17B nd to the left of 18B, to crete the following hplotype groups: hplotype 17B vs. 15B plus 18B vs. ll A hplotypes. Only one copy of hplotype 19B ws present cross trils nd tht niml s other hplotype ws 17B, so this niml ws ssigned to hplotype 17. Other groupings were creted y moving the first cut up the tree, resulting in the groupings specified in Figure 4. Lest squres mens of the effect of ech hplotype group for ech grouping re in Figure 4. Results showed tht ll B hplotypes were ssocited with the fvorle phenotype, nd were significntly different from hplotypes tht crried the A llele (p < 0.001) for oth VL nd WG42, except for grouping 2. For grouping 2, hplotype 18 hd significntly lower VL thn the other B hplotypes ut ws not significntly different from B or A hplotypes for WG. Hplotype 18 hd totl of 21 copies (20 individuls) nd ws specific to tril 5; therefore, the results for group 2 my represent effects specific to this tril nd this my confound the estimtes of the effects of this hplotype. Hplotype 14, which ws t the se of the A hplotypes (Figure 3), ws not significntly different from the other A hplotypes (p > 0.83) nd tended towrds significnce from the B hplotypes (grouping 4, p < 0.09) for VL. For WG42, hplotype 14 ws not significntly different from the other B nd A hplotypes. Hplotype 14 only hd 11 copies nd the SE ssocited with the effect of hplotype 14 were lrge. Moving further up the tree, the group with A hplotypes 14, 13, nd 11 ws not significntly different from the other A hplotypes ut ws significntly different from the B hplotypes for oth VL nd WG. In summry, the phylogenetic nlysis segregted the hplotypes primrily y the llele t SNP WUR ,

8 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 8 of 14 Tle 4 Sttisticl significnce (p-vlues) of ssocitions of single nucleotide polymorphisms within the 1 M region on chromosome 4 fter ccounting for the effects of SNP WUR LD (r 2 ) with P-vlues Numer SNP nme Position WUR VL 2 WG 3 1 MARC MARC ALGA ASGA MARC Fixed 6 MARC Fixed 7 ALGA INRA < ASGA MARC LD = 1 11 WUR Used in model 12 ALGA H3GA ASGA ASGA LD = 1 16 MARC LD = 1 17 ALGA M1GA ASGA ALGA ASGA ASGA DRGA MARC ASGA M1GA M1GA M1GA ALGA MARC MARC H3GA ALGA ASGA ALGA MARC The olded re represents Block 2 of Figure 2; 2 VL virl lod clculted s re under the curve of log-trnsformed viremi etween 0 nd 21 dys post-infection; 3 WG weight gin from 0 dpi to 42 dpi. nd there ws significnt difference etween the A versus B hplotype groups. Due to the high LD in the 487 k region, reltively few hplotypes existed cross the different popultions for this region. This my indicte tht there is little recomintion in this region. In fct, cross ll ~1600 nimls evluted, only one recomintion could e identified within the ~1 M region on SSC4. This recomintion occurred etween SNPs 31 nd

9 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 9 of 14 Tle 5 Hplotypes for the 487 k region (with ritrry numers corresponding to the phylogenetic tree in Figure 3) tht contin the B llele for SNP WUR on chromosome 4, long with the SNP sequence nd the numer of copies present in trils 1 through 8 Hplotype numer Hplotype SNP sequence 1 Numer of copies 17B GAGGCGAGGGCCCAG 423 All 18B GAGGCGCGGACAACG B GAGGCGCGGACCCCG B AGGGCGAGGGCCCAG Letter in old is SNP WUR Trils in which the hplotype ws present 32 in Figure 2, outside the 487 k region. Therefore, this is region with very little recomintion, explining the reltively few hplotypes tht were present cross ll trils. Additionl regions ssocited with VL The results from the GWAS for VL re presented in Figure 5A. The top ten 1 M windows tht explined the gretest percentge of genetic vrince re in Tle 6. The region on SSC4 explined the lrgest percentge of genetic vrince t 13.2%. The remining top 10 regions B B B B Figure 3 Phylogenetic tree of the hplotypes present cross trils 1 through 8 for the reduced 487 k region on Sus scrof chromosome 4. The trees were creted using the neighor-joining, p-distnce method in the Meg 5 softwre. Hplotypes with the B llele for SNP WUR re leled with B following the hplotype numer. quickly dropped to less thn 1% of the genetic vrince nd rnged from 1.24% to 0.39%. The second nd third rnked regions will e further discussed. A region on SSCX, which included two consecutive 1 M regions, ccounted for 1.73% of the genetic vrince cross ll trils for VL ut only 0.02% of the genetic vrince for WG (Figure 5A, Tle 6). There hve een no reports of QTL ssocited with trits relted to growth within 3 M up or down strem of this region ( The region does include one reported QTL for lood ph in Meishn nd Pietrin pigs infected with Srcocystis miescherin [13]. A region on SSC1 ccounted for 0.70% of the genetic vrince for VL cross ll trils nd ws the third highest region for VL (Figure 5A, Tle 6). This region lso explined only smll percentge of genetic vrince for WG (0.03%). Three QTL ssocited with helth trits hve een reported for this region, including C3c concentrtion, lkline phosphtse ctivity, nd white lood cell counts ( index). However, none of these QTL were identified under PRRS chllenge. Interesting cndidte genes within 2 M on either side of this 1 M window include DBC1 (deleted in ldder cncer 1) ndtumor necrosis fctor (TNF) receptor-ssocited fctor 1-like ( The DBC1 geneistumorsuppressorgenethtisssocited with progrmmed cell deth [14]. The TNF receptor-ssocited fctor 1-like gene is ssocited with ntivirl ctivity [15]. Additionl regions ssocited with WG The results from the GWAS for WG re presented in Figure 5B nd the top 10 windows tht explined the gretest percentge of genetic vrince re in Tle 6. Agin, the region on SSC4 explined the lrgest percentge of genetic vrince, t 9.1%. The percentge of genetic vrince explined y the remining regions rnged from 0.43% to 2.61%. A region on SSC5 ws the second highest nd ccounted for 2.6% of the genetic vrince cross ll trils (Figure 5B, Tle 6). This window does not pper to hve n ssocition with VL, s the percentge of genetic vrince for VL ws only 0.06%. Reports of QTL in this region ssocited with helth in the pig include QTL for cholesterol level, hptogloin concentrtion, lkline phosphtse ctivity, interleukin-2 level, nd interferon-gmm level ( Interleukin-2 nd interferon-gmm re oth cytokines tht respond to pthogen invsion of host cells. Interferongmm hs een shown to inhiit PRRSV repliction in mcrophges [16,17]. There were two reports of QTL ssocited with verge dily gin of helthy pigs tht spn the

10 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 10 of 14 A Virus Lod (re under the curve) n=363 n=28 n = 1023 n= , 18 A 17, A 17, 15, 18 n=20 c n=1023 n=391 n = 1023 n=391 A 17, 15, 18 n=11 n = A 17, 15, 14, 13, n=391 n = 452 n=571 A Grouping 1 Grouping 2 Grouping 3 Grouping 4 Grouping 5 Hplotypes Groups B 18 Weight Gin (kg) n=360 n=28 n=983 n=368 n=20 n=983 n=388 n =983 n = 388 n=11 n =972 n = 388 n =428 n= , 18 A 17, A 17, 15, 18 A 17, 15, A 17, 15, 14, 13, A Grouping 1 Grouping 2 Grouping 3 Grouping 4 Grouping 5 Hplotypes Groups Figure 4 Lest squres mens y hplotype group within grouping sed on cutting the phylogenetic tree for virul lod (A) nd weight gin (B). Hplotype numers refer to those identified in Figure 3; A refers to ll A hplotypes. Within grouping, columns with different letter re significntly different t p < Numers of individuls within ech hplotype group re listed in the rs. Blck rs represent hplotypes with the B llele for SNP WUR nd white rs represent hplotypes with the A llele for SNP WUR SSC5 region identified here ( org/cgi-in/qtld/ss/index). A 1 M window on SSC7 (M 27) ccounted for 1.45% of the genetic vrince in WG cross ll trils (Figure 5B, Tle 6) ut only 0.06% of the genetic vrince for VL. This region is within ~5 M region (M 24 29) tht contins severl Swine Leukocyte Antigen (SLA) genes within the swine Mjor Histocomptiility Complex ( ccessed Mrch, 2013). To dte, there hve een 19 reports of QTL ssocited with production trits in this region, 18 of which were ssocited with BW or verge dily gin from irth to mrket weight (pig genome ssemly 10.2, nimlgenome.org, ccessed Mrch, 2013). However, these QTL were identified using helthy, non-chllenged pigs. The QTL identified here is for WG under PRRSV chllenge ndcouldenewqtl;lterntelyoneofthepreviously reported QTL could lso e ssocited with WG under PRRSV chllenge. Additionl work is required to understnd the underlying mechnism. Cross-vlidtion Accurcies of predictions sed on the SSC4 region nd sed on the whole genome including the SSC4 region, were previously reported y [3], using trils 1 through 3 for trining nd trils 4 nd 5 for vlidtion. On verge, ccurcies of GEBV sed only on the SSC4 region were higher (0.24 for VL nd 0.33 for WG) thn ccurcies of GEBV sed on the whole genome, including SSC4 (0.20 nd 0.31, respectively). Given those results, the hypothesis here ws tht the rest of the genome hs much lower predictive ility thn the region on SSC4. Accurcies of predictions sed on SNP WUR nd GEBV for the rest of the genome excluding the SSC4 region re shown in Figure 6A nd B for VL nd WG.

11 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 11 of 14 A B Percentge of Genetic Vrince Percentge of Genetic Vrince M windows ordered y chromosome M windows ordered y chromosome Figure 5 Mnhttn plot of the genome-wide ssocition nlysis of virl lod (A) nd weight gin to 42 dys post infection (B). Results re sed on method Byes-B with π =0.99,ndshowthe percentge of genetic vrince tht is explined e ech non-overlpping 1 M window, leled y index numer of the first 1 M window nd ordered y chromosome (1 18, X, nd Y). Virus lod ws clculted s re under the curve of log-trnsformed viremi from 0 to 21 dys post infection. Accurcies were, on verge, much higher for SNP WUR thn for the rest of the genome. Accurcies of predictions of genotypic vlues sed on SNP WUR were very similr for the full nd reduced trining dt ut vried etween vlidtion trils from 0.08 to 0.51 for VL, with n ccurcy of 0.48 nd 0.54 when ll trils were nlyzed jointly, using the full nd reduced trining dt, respectively, nd from 0.06 to 0.75 for WG, with n ccurcy of ~0.34 for the joint nlyses. For GEBV sed on the rest of the genome nd the reduced trining dt, ccurcies centered round zero for VL, rnging from 0.23 to for individul vlidtion trils, with n ccurcy of 0.10 nd 0.04 for the joint nlysis with the full nd reduced trining dt, respectively. For WG, ccurcies sed on the rest of the genome tended to e positive, rnging from 0.14 to +0.31, with n ccurcy of 0.21 nd 0.07 for the joint nlysis with the full nd reduced trining dt. For the full trining popultions, ccurcies incresed sustntilly for trils 1 nd 2, except for WG for tril 2, Tle 6 Top ten 1 M windows tht explined the gretest percentge of genetic vrince for virus lod nd weight gin Chromosome Position 3 Rnk Genetic vrince (%) Virus lod X X Totl genetic vrince Weight gin Totl genetic vrince VL virus lod clculted s re under the curve of log-trnsformed viremi etween 0 nd 21 dys post infection (dpi); 2 WG weight gin from 0 dpi to 42 dpi; 3 Megse position within Sus scrof chromosome; 4 posterior men of the totl genetic vrince from the genome-wide ssocition nlyses. ut hd little impct on the other trils. These increses in ccurcy for trils 1 nd 2 were s expected ecuse the full trining dt included dt from the sme crossred genetics s used for vlidtion. Resons for the generlly oserved poor predictive performnce of GEBV cross popultions cn e ttriuted to dominnce, episttis, nd differences in linkge disequilirium [18]. Accurcies of GEBV re generlly lower when trining nd vlidtion popultions re more distntly relted. For exmple, Stchi et l. [19] prtitioned dt from Angus ulls into five groups using two methods; rndom lloction nd k-mens clustering, with the ltter iming t incresing reltionships within groups nd decresing reltionships etween groups. On verge, ccurcies of GEBV when cross-vlidtion in groups creted y rndom lloction were higher thn when groups were creted y k-mens clustering (0.65 vs 0.44). They concluded tht this ws due to closer

12 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 12 of 14 A 0.8 WUR Reduced WUR Full 0.7 Rest Reduced Rest Full Accurcy Joint Tril B WUR Reduced Rest Reduced WUR Full Rest Full Accurcy Joint Figure 6 Estimtes of ccurcy of genomic predictions in the vlidtion popultion. Accurcy sed on the correltion etween predictions nd phenotypes djusted for fixed effects, divided y the squre root of heritility, for predictions otined using reduced or full trining popultions (see Tle 3). Predictions were sed on the effect of SNP WUR (WUR) of Sus scrof chromosome (SSC) 4 only, nd sed on the rest of the genome excluding the effect of the SSC4 region (Rest), for virus lod (A, clculted s re under the curve of log-trnsformed viremi etween 0 nd 21 dys post infection) nd weight gin to 42 dys post infection (B). Tril reltionships etween trining nd vlidtion popultions when using rndom lloction. In the current study, the reduced trining popultions were not relted to the vlidtion popultions. However, when vlidting on trils 1 nd 2, tril 3 ws included in the full trining popultion, which resulted in some reltedness etween trining nd vlidtion popultions. Inclusion of tril 3 in the trining popultion resulted in incresed ccurcies for trils 1 nd 2 compred to the sence of tril 3 in the reduced trining popultion, which grees with the results of Stchi et l. [19]. However, SE were lrge for ll estimtes of ccurcy. Furthermore, the increse in reltionships etween trining nd vlidtion popultions ws confounded with the incresed size of the trining popultion. Conclusions Trits ssocited with PRRS following experimentl infection in growing pigs pper to hve strong genetic component, with moderte estimtes of heritility for VL nd WG, long with lrge QTL on SSC4. Strong LD is present in the SSC4 region. However, comining ll eight trils roke some of the LD in the ~1 M region, resulting in smller hplotype. This reduced hplotype lock could e useful to identify the custive muttion. A phylogenetic nlysis seprted the hplotypes y the llele t the SNP tht explins nerly ll the genetic vrince for VL nd WG tht is contriuted y the region, providing dditionl evidence tht SNP WUR is the most informtive SNP in the region (long with three other SNPs tht were in complete LD

13 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 13 of 14 with the WUR SNP) nd is likely in very high LD with the custive muttion. The most common B hplotype ws present in ll reeds nd lines represented in this study nd ws likely present efore the segregtion of the reeds. Cross-vlidtion for the SSC4 QTL resulted in high estimtes of ccurcy when vlidting cross popultions for VL nd WG, which cn enefit selection strtegies without infecting piglets with PRRSV. After ccounting for the SSC4 region, the rest of the genome hd little predictive ility for VL nd WG cross unrelted popultions. Additionl work is required to determine predictive ility within popultion. With the effect of the SSC4 region present in ll reeds nd lines nlyzed here, there is good possiility tht the effect is present in more popultions nd tht genetic progress for PRRS tolernce or resistnce cn e mde. However, dditionl work is needed to verify the effects of this region on chllenges with different PRRSV strins nd in the field. Other genomic regions ssocited with VL nd WG were identified lso ut with smll effects. In generl, these regions will proly not ply mjor role in reducing the economic impct of PRRS. The genomic region identified in this study cn e used for mrker-ssisted selection for response to PRRS in growing pigs. The implementtion of selection for these mrkers will not mke pigs completely resistnt to PRRS ut will increse the well-eing of the nimls nd reduce the finncil impct of PRRSV infections y limiting reductions in growth rte nd other negtive effects of PRRS following infection. Further work is required to investigte the effects of these QTL ginst different PRRSV strins nd in the field where pigs re sujected to mny dditionl environmentl stressors. Competing interests The uthors declre tht they hve no competing interests. Authors contriutions NJB conducted the sttisticl nlyses, interprettion of results, nd wrote the mnuscript, AB conducted the initil hplotype nlysis studies, RRRR conceived the study nd led the niml infection trils nd smple collection, JKL conceived the study nd coordinted the hndling, storge, nd smple preprtion for DNA, JMR helped conceive the study, coordinted dt se development nd helped with interprettion of nlyses, JCMD coordinted nd oversw sttisticl nlysis of the dt nd contriuted to interprettion of results nd writing the mnuscript. All co-uthors reviewed nd contriuted to development of the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements This project ws supported y the USDA NIFA PRRS CAP Awrd , the Ntionl Pork Bord, nd the NRSP-8 Swine Genome nd Bioinformtics Coordintion projects, nd the PRRS Host Genetics Consortium consisting of USDA ARS, Knss Stte Univ., Iow Stte Univ., Michign Stte Univ., Wshington Stte Univ., Purdue Univ., University of Nersk-Lincoln, PIC/Genus, Newshm Choice Genetics, Fst Genetics, Genetiporc, Inc., Genesus, Inc., IDEXX Lortories, nd Tetrcore, Inc. The uthors cknowledge technicl ssistnce to this project from Jun Pedro Steiel for the lgorithm to compute virl lod sed on re under the curve, Mx Rothschild for ssistnce with genotyping, Eric Fritz for dtse mngement, Ani Wolc nd Dinesh Thekkoot for sttisticl ssistnce, nd the l of Bo Rowlnd, specificlly Becky Eves, Mureen Kerrign, Ben Trile, Jessic Otrdovec, Brooke Bloomerg, Auree Gottlo, Lur O Brien, nd Rnjini Chnd for niml cre nd smple collection, nd the l of Jon Lunney, specificlly Smuel Arms nd Amer Tietgens, for preprtion of ll genomic DNA smples for SNP genotyping. Author detils 1 Deprtment of Animl Science, Iow Stte University, Ames, Iow 50011, USA. 2 College of Veterinry Medicine, Knss Stte University, Mnhttn, Knss 66506, USA. 3 United Stte Deprtment of Agriculture, Agriculturl Reserch Services, Beltsville Agriculturl Reserch Center, Beltsville, Mrylnd 20705, USA. Received: 29 August 2013 Accepted: 13 Decemer 2013 Pulished: 4 Mrch 2014 References 1. 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14 Boddicker et l. Genetics Selection Evolution 2014, 46:18 Pge 14 of Grrick DJ: Consequences of genomic prediction in cttle. Interull Bull 2010, 41: Stchi M, McClure MC, McKy SD, Rolf MM, Kim J, Decker JE, Txis TM, Chpple RH, Rmey HR, Northcutt SL, Buck S, Woodwrd B, Dekkers JCM, Fernndo RL, Schnel RD, Grrick DJ, Tylor JF: Accurcies of genomic reeding vlues in Americn Angus eef cttle using K-mens clustering for cross-vlidtion. Genet Sel Evol 2011, 43:40. doi: / Cite this rticle s: Boddicker et l.: Genome-wide ssocition nd genomic prediction for host response to porcine reproductive nd respirtory syndrome virus infection. Genetics Selection Evolution :18. Sumit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immedite puliction on cceptnce Inclusion in PuMed, CAS, Scopus nd Google Scholr Reserch which is freely ville for redistriution Sumit your mnuscript t