QTL mapping of egg albumen quality in egg layers

RESEARCH Open Access QTL mapping o egg albumen quali y in egg laye s Me i Honka ukia 1* , Ma ia Tuiskula-Haa is o 1 , Jesus A ango 3 , Jonna Tabell 1 , Ma hias Schmu z 2 , Rudol P eisinge 2 and Johanna Vilkki 1 Abs ac Backg ound: A esh, good quali y egg has a i m and gela inous albumen ha ancho s he yolk and es ic s g ow h o mic obiological pa hogens. As he egg ages, he gel-like s uc u e collapses, esul ing in hin and unny albumen. Occasionally hin albumen is ound in a esh egg, gi ing he imp ession o a low quali y p oduc . A mapping popula ion consis ing o 1599 F 2 hens om a c oss be ween Whi e Rock and Rhode Island Red lines was se up, o iden i y loci con olling albumen quali y. The pheno ype o albumen quali y was e alua ed by albumen heigh and in Haugh uni s (HU) measu ed on h ee consecu i e eggs om each F 2 hen a he age o 40 weeks. Fo he ine-mapping analysis, albumen heigh and HU we e used simul aneously o elimina e con ibu ion o he egg size o he pheno ype. Resul s: Linkage analysis in a small popula ion o se en hal -sib amilies (668 F 2 ) wi h 162 mic osa elli e ma ke s sp ead ac oss 27 ch omosomes e ealed wo genome-wide signi ican egions wi h addi i e e ec s o HU on ch omosomes 7 and Z. In addi ion, wo pu a i e genome-wide quan i a i e ai loci (QTL) egions we e iden i ied on ch omosomes 4 and 26. The QTL e ec s anged om 2 o 4% o he pheno ypic a iance. The genome-wide signi ican QTL egions on ch omosomes 7 and Z we e selec ed o ine-mapping in he ull se composed o 16 hal -sib amilies. In addi ion, hei exis ence was con i med by an associa ion analysis in an independen comme cial Hy-Line pu e line. Conclusions: We iden i ied ou chicken genomic egions ha a ec albumen quali y. Ou esul s also sugges ha genes ha a ec albumen quali y ac bo h di ec ly and indi ec ly h ough se e al di e en mechanisms. Fo ins ance, he QTL egions on bo h ine-mapped ch omosomes 7 and Z o e lapped wi h a p e iously epo ed QTL o eggshell quali y, indica ing ha eggshell memb anes may play a ole in albumen quali y. Backg ound Eggs o he able egg ma ke should be mic obiologically sa e and look good. The numbe o eggs o p ocessing egg p oduc s has inc eased du ing ecen yea s, emphasizing he impo ance o high quali y and good p ocessing p ope ies. Good quali y albumen has a i m jelly-like s uc u e ha keeps he yolk in he cen e o he egg. Albumen quali y s a s o degene a e immedia ely a e he egg is laid and hinning is a na u al p ocess du ing s o age. Fo b eeding pu poses, albumen quali y is measu ed in Haugh uni s (HU), exp essed as a unc ion o egg weigh and he albumen heigh (AH) o a b oken egg [1]. The egg indus y is pa icula ly in e es ed in unc ional p ope ies such as coagula ion and oaming, which makes quali y assessmen complex [2]. Gene ic backg ound can explain, in pa , di e ences in albumen quali y among indi iduals and b eeds [3]. The a e age he i abili y o albumen quali y is mode a e, eaching 0.30 [4]. I has also been demons a ed ha si es ha e a highe in luence on he he i abili y o AH and HU han dams, which indica es a sex-linked e ec [4]. Among en i onmen al ac o s, managemen and egg s o age condi ions ha e a subs an ial impac on main aining albumen heigh , while eed composi ion has only a mino e ec on albumen quali y [2,5]. Fu he mo e, i uses in he ep oduc i e ac may lead o he p oduc ion o wa e y whi e eggs and ex emely poo quali y albumen [5]. Va ious causes o albumen de e io a ion ha e been sugges ed. Impe ec ions can eme ge du ing he ea ly * Co espondence: [email p o ec ed] 1 MTT Bio echnology and Food Resea ch, Jokioinen 31600, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle Gene ics Selec ion E olu ion © 2013 Honka ukia e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 h p://www.gsejou nal.o g/con en /45/1/31 o ma ion o albumen in he ep oduc i e ac bu also a e o iposi ion. A po en ial explana ion o he decline in quali y wi h ime is linked wi h eggshell and memb ane ai s. An in ac eggshell wi h good inne and ou e memb ane s uc u es plays an impo an ole in albumen quali y, pa icula ly du ing s o age, p e en ing e apo a ion and escape o me abolic gases h ough he shell po es. CO 2 leak is known o change albumen pH owa ds alkaline alues [6]. Albumen quali y, among o he egg quali y and p o- duc ion ai s, is a ypical quan i a i e ai ha has been s udied by QTL mapping. Ne e heless, among he a ail- able da a on chicken QTL, he e a e ela i ely ew QTL ha a ec albumen quali y [7]. In he ChickenQTLdb, 16 dis inc QTL loca ions a e associa ed wi h HU, AH o albumen weigh (AW). In he da abase, QTL egions ha in luence albumen quali y a e loca ed on ch omo- some 1 (HU be ween posi ions 48.17 and 53.13 Mb; AH and HU be ween 90.35 and 123.03 Mb) [8,9], ch omo- some 2 (HU be ween 5.31 and15.36 and be ween 31.23 and 38.97 Mb and AH be ween 80.69 and 104.34 Mb) [9,10], ch omosome 3 (AW a posi ion 106.44 Mb) [11] and ch omosome 4 (AH and AW a 9.45 Mb, AW be ween 62.18 and 75.89 and AW a ~80 Mb) [11,12]. In addi ion, genome-wide associa ion s udies e ealed signi ican SNP associa ions on ch omosomes 1, 3, 5, 18, 19, 23 and Z wi h ea ly o la e AH [13] and o he in e es ing associa ions ha e been epo ed on ch o- mosomes 7, 8, 9, 14, 20 and 24 [14]. These s udies ha e e- ealed ha se e al o e lapping genomic egions a e in ol ed in a ious egg quali y ai s, bu he ela ionships be ween hese egions ha e no ye been de ailed a he biological le el. Simple ex -based sea ches in he chicken genome [15] o genes associa ed wi h egg whi e yielded 20 hi s, eigh o which e e ed o known QTL egions, he emainde being anno a ed albumen ela ed genes, such as LYZ (lysozyme, ch 1: 37.29 Mb), LYG2 (lysozyme G-like 2, ch 1: 136.64 Mb), PRL (p olac in, ch 2: 59.7 Mb), SERPINB6 (se ine (o cys eine) pep idase inhibi o , clade B, membe 6e, ch 2: 68.85 Mb), CALB1 (calbindin 1, 28kDa, ch 2: 129.15 Mb), CST3 (cys a in C,ch 3: 16.49 Mb),SPP1(sec e ed phosphop o ein 1, ch 4: 47.10 Mb),MUC6(mucin 6, oligome ic mucus/gel- o min, ch 5: 16.121 Mb), MUC5B (o omucin, alpha subuni ,ch 5: 15.95 Mb) and TF ( ans e in, ch 9: 5.62 Mb). Al hough s udies on he egg albumen p o eome ha e signi ican ly expanded he lis o iden i ied albumen p o eins [16,17], and he mechanisms in ol ed in egg whi e hinning ha e been s udied a he p o ein le el [18], no causal a ia ions in albumen genes ha e ye been iden i ied. In his s udy, we used an F 2 in e c oss be ween he wo egg-laye lines, Whi e Rock and Rhode Island Red, in o de o iden i y QTL ha a ec albumen quali y. Mapping was pe o med in h ee s eps: (1) iden i ica ion o QTL in a small F 2 popula ion, (2) ine-mapping o hese QTL in a la ge F 2 popula ion, and (3) e i ica ion o he QTL in a comme cial line, Hy-Line. We also in es iga ed possible links be ween albumen quali y and eggshell p ope ies based on o e lapping QTL esul sin heChickenQTLdb(on hechickengenome build WASHUC2). Me hods Mapping popula ions Fo mapping, an F 2 popula ion was c ea ed be ween wo comme cial egg-laye lines om Lohmann Tie zuch i.e. Rhode Island Red and Whi e Rock. The c ossed lines di e ed in albumen quali y; he a e age HU was 56.31 o Rhode Island Red and 69.29 o Whi e Rock (Table 1). P ac ical managemen was simila o ha used in p e ious QTL s udies [19]. The ull mapping popula ion was made up by ecip ocal c osses be ween 14 Rhode Island Red indi iduals (six males and eigh emales) and 15 Whi e Rock indi iduals (six males and nine emales). The F 1 gene a ion consis ed o 16 males and 96 emales, leading o 16 hal -sib amilies. A genome scan was pe o med wi h 162 mic osa elli e ma ke s sp ead ac oss 27 o he 39 chicken ch omosome pai s using a subse o se en hal -sib amilies wi h 668 F 2 indi iduals. The ull F 2 mapping popula ion o 1599 indi iduals was used o ine-mapping. In o de o eanalyze he QTL egions, a comme cial egg laying chicken popula ion (Hy-Line) was used. The Hy-Line popula ion consis ed o 290 males belonging o pa e nal hal -sib amilies (3.5 males pe hal -sib amily). Pheno ypes Albumen quali y was e alua ed in he F 2 mapping popula- ion o h ee consecu i e eggs, each wi hin 24 h o laying, o each hen a he age o 40 weeks. To measu e albumen quali y, he egg was weighed, b oken on a glass pla e and he heigh o he hick albumen (AH) was measu ed wi h a mic ome e . AH was ans o med in o Haugh uni s (HU) wi h a s anda dized unc ion o he AH and egg weigh , and co ec ed wi h a cons an [1]. In he whole-genome scan, only he co ec ed HU es ima ions we e used, bu bo h AH and HU es ima ions we e used o exclude con ibu ion o he egg weigh o he pheno ype and o make he di e en da ase s compa able. In he comme cial Hy-Line popula ion, albumen quali y was assessed by AH wice du ing he p oduc ion pe iod i.e. a 26 weeks o age (= ea ly) and again a 42 o 44 weeks o age (= la e). The pheno ypes a e p esen ed as si e-daugh e a e ages and a e desc ibed in Table 1. Geno yping DNA p epa a ion and geno yping o mic osa elli e ma ke s we e ca ied ou as desc ibed by Tuiskula-Haa is o e al. Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 2 o 8 h p://www.gsejou nal.o g/con en /45/1/31 [20], who also epo ed ma ke maps and in o ma ion con en s along he ch omosomes. Fo ch omosome 7, geno yping o he en i e F 2 mapping popula ion was pe o med wi h i e mic osa elli e ma ke s co e ing 96 cM (MCW361, ADL326, MCW183, MCW236 and ADL315, see Table 2), h ee o which (ADL326, MCW183 and MCW236) we e also geno yped in he Hy-Line popula ion. Fo ine-mapping on ch omosome Z, a selec ed a se o SNP ma ke s co e ing he QTL egion (MCW258-MCW241) we e used ins ead o mic osa elli e ma ke s. An Illumina BeadXp ess [21] eade was used o geno ype mul iplex SNP in bo h mapping popula ions, as epo ed in [22]. In he F 2 popula ion, 20 in o ma i e SNP ma ke s we e selec ed o linkage analysis o he QTL egions (Table 3). In he Hy-Line popula ion, 12 SNP ma ke s we e geno yped on ch omosome Z, o which six we e included in he QTL egion (Table 3). Because di e en SNP seg ega ed in he di e en popula ions, he ma ke se s analyzed we e no comple ely iden ical in he di e en popula ions. S a is ical analysis Ma ke maps we e cons uc ed wi h CRI-MAP [23] using p ocedu es TWOPOINT, BUILD, FLIPS and CHROMPIC. QTL analyses we e pe o med using he leas squa es me hod ia he web-based G idQTL so wa e [24]. Signi icance h esholds o QTL analysis we e de e mined empi ically by pe mu a ion, and con idence in e als we e based on boo s apping. The leng h o he ch omosomes was aken in o accoun when de ining he signi icance h esholds. Mo e in o ma ion on he models used and how he signi icance le els and con idence in e als we e de i ed is a ailable in [19]. Fine-mapping in he F 2 popula ion used he same so wa e o he ch omosome 7 da a, whe eas a cus om-made eg ession p og am [22] was applied o he ch omosome Z da a. The signi icance le els o he linkage analysis we e ob ained using a pe mu a ion p ocedu e as explained in [19]. Fo he comme cial Hy-Line, mic osa elli e ma ke as- socia ions we e es ed wi h a non-pa ame ic K uskal– Wallis es , because he geno yping da a comp ised a single gene a ion and linkage analysis could no be ap- plied. Le μ ij be he ai mean o geno ype jin ma ke i, and i=1,…,3. The o al amoun o di e en geno ype g oups pe ma ke is N i and depends on he ma ke , so ha j=1,…,N i . The null hypo hesis o be es ed is hen H 0i :μ i1 =μ i2 =…=μ iNi e sus: H 1i :μ ik ≠μ il o a leas one pai o geno ypes k≠l and k, l ≤N i . Table 1 Desc ip i e s a is ics o he pheno ypes analyzed in he s udy o he F 2 and Hy-Line popula ions Popula ion N T ai and uni A e age S anda d de ia ion Min-Max Rhode island ed 84 AH, a e one week s o age, mm 5.8 0.39 4.5-7.3 Whi e ock 82 AH, a e one week s o age, mm 6.1 0.42 4.6-7.7 F 2 1599 HU, Haugh uni s 85.0 6.89 59.6-105.8 AH, mm 7.3 1.15 2.9-11.8 Hy-Line 299 AH ea ly, mm 8.4 0.44 7.32-9.57 Hy-Line 221 AH la e, mm 8.0 0.55 5.73-9.37 The pheno ypes o pa en al lines (Rhode Island Red and Whi e Rock) a e b eeding alues o AH a e one week s o age. Table 2 Bes esul s om he ini ial QTL mapping based on 668 F 2 indi iduals and HU Ch Ma ke map Ma ke s lanking he QTL and hei genomic posi ions F- a ios and co esponding bounda ies o p-le el (CI leng h in b acke s) Addi i e e ec (SE) Dominance e ec (SE) R 2 7MCW361-(1)- ADL326-(54)-MCW183-(31)- MCW236-(10)-ADL0315 MCW183-MCW236 (24.25-29.72 Mb) F = 8.32 (T01 8.76; T05 7.17) (CI = 57 cM) 14.0 (3.45) 1.83 (5.32) 0.04 26 MCW355-(13)-MCW285-(35)-ADL885 MCW285-ADL285 (2.50-4.91 Mb) F = 6.04 (T05 7.01; T10 4.61) (CI = 49 cM) 12.51 (5.24) 27.22 (11.70) 0.02 4MCW47-(43)-MCW5-(37)-ADL266-(15)-LEI94-(6)- MCW284-(12)-ADL331-(11)-MCW170-(5)-MCW180-(12)- MCW122-(6)-LEI119-(15)-MCW99-(9)-LEI73 MCW122-LEI119 (76.43-80.94 Mb) F = 6.49 (T05 8.02; T10 5.02) (CI = 146 cM) 12.83 (3.66) 4.84 (5.74) 0.02 ZADL117-(22)-MCW331-(12)-MCW55-(6)-MCW258-(28)- LEI171-(4)-ADL201-(6)-MCW241-(5)-LEI229-(1)-MCW154-(1)- MCW246-(5)-LEI254-(1)-MCW294-(3)-ROS117-(3)-LEI111-(1)- LEI144-(1)-LEI121-(28)-LEI75-(5)-MCW269 MCW258-MCW241 (21.40-34.26 Mb) F = 39.20 (T0001 19.03; T00119.98; T01 9.55; T05 6.52) 15.28 (2.44) NA 0.02 Ch ch omosome numbe ; dis ances be ween ma ke s (in pa en heses) a e based on Haldane’s mapping unc ion; ma ke s geno yped in he comme cial line a e indica ed in bold; he ma ke s lanking he QTL and hei loca ions a e indica ed in Mb (WASHUC2), ollowed by F- a ios and co esponding bounda ies o p- alues, whe e T0001 o T10 ep esen p-le el h esholds om 0.0001 o 0.05 (bounda y o genome-wide mapping, T10 is a sugges i e le el QTL ); CI con idence in e al, SE s anda d e o ; R 2 = p opo ion o pheno ypic a iance explained by QTL o he addi i e e ec . Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 3 o 8 h p://www.gsejou nal.o g/con en /45/1/31 Table 3 Fine-mapping esul s wi hin di e en popula ions using di e en mapping me hods and ma ke composi ions Ch Pop N Me hod Ma ke composi ion and genomic posi ion Ma ke s lanking he QTL/associa ed ma ke T ai p- alue E ec R 2 ZF 2 1599 QTL linkage mapping by cus om made p og am s16765819(29.091.210)- s14687314(30.458.261)- s14691747(30.806.242)- s13799822(31.307.747)- s14762832(31.855.782)- s16766794(31.956.374)- s16766752(32.044.710)- s16766685(32.277.106)- s16766334(33.023.048)- s14761691(33.305.926)- s13795456(33.672.729)- s14761341(33.749.560)- s14761196(33.997.081)- s16767662(34.996.569)- s16110154(35.510.105)- s16767980(36.026.140)- s16110443(36.236.898)- s16111109(36.960.473)- s16132282(39.449.000)- s16684439(42.958.949) s14761341- s16767662 HU < 0.0001 12.57 (1.77) 0.03 (a 33.75-35.00 Mb) peak a posi ion o s14761196 AH < 0.0001 1.87 (0.30) 0.02 Hy-Line 290 PLINK s14700116(1.119.301)- s16741325(1.411.493)- s14067906(1.567.603) s16726302(1.876.555)- s14067572(1.991.047)- s14067220(2.364.494)- s16765819(29.091.210)- s13816749(30.040.859)- s13795687(32.123.345)- s14761341(33.749.560)- s14761196(33.997.081) s14763225(34.275.437) s16785819 a 29.091.210 AH-ea ly 0.03 3.86 (0.04) 0.06 7F 2 1599 QTL linkage mapping by G idQTL MCW361-(1)- ADL326-(54)-MCW183-(31)- MCW236-(10)-ADL0315 lanked by MCW183-MCW236 (24.52-29.72 Mb) AH < 0.01 2.26 0.02 LOD 6.90 (0.40) (CI = 40–75 cM) HU <0.01 14.16 0.02 LOD 6.83 (2.52) Hy-Line 90-290 Single ma ke s associa ions ADL326 alias anly in epea and SOCS box con aining 18 (5.152.776); ADL326 AH-ea ly 0.0046 NA NA MCW183 (24.245.453); MCW236 (29.724.317) AH-la e 0.0024 NA NA Ch ch omosome numbe , Pop popula ion, N numbe o indi iduals in he popula ion; s a is ically he mos p obable posi ions o he QTL a e indica ed acco ding o he posi ions o he lanking SNP ma ke s in Mb (on WASHUC2); R 2 = p opo ion o pheno ypic a iance o he e ec . Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 4 o 8 h p://www.gsejou nal.o g/con en /45/1/31 Ch omosome Z was analyzed wi h PLINK [25]. Da a we e checked o geno yping quali y and Ha dy-Weinbe g equilib ium (none o he ma ke s we e excluded) be o e analysis. Basic associa ion es ing o quan i a i e ai s and adjus men o mul iple compa isons we e used. Resul s The whole-genome scan using low-densi y ma ke maps and se en hal -sib amilies om he mapping popula ion (668 o 1599 F 2 indi iduals) de ec ed ou QTL egions ha a ec egg whi e quali y (HU) (Table 2). A genome-wide signi ican QTL (p < 0.05) was ound on ch omosome 7 be ween mic osa elli e ma ke s MCW183 and MCW236 (a he genomic posi ions 24.24 Mb and 29.72 Mb, espec i ely). The addi i e e ec o he locus was 14.0 HU and accoun ed o 2% o he pheno ypic a iance. A highly signi ican QTL (p < 0.0001) was de ec ed on ch omosome Z be ween mic osa elli e ma ke s MCW258 and MCW241 (a 21.40 Mb and 34.26 Mb, espec i ely) [see Addi ional ile 1].The e ec o his locus was 15.28 HU. A sugges i e (5% ch omosome-wide) QTL was ound on ch omosome 4, be ween ma ke s MCW122 and LEI119 (76.43 and 80.94 Mb, espec i ely), which explained 2% o he ai a ia ion, he e ec being 12.83 HU. Simila ly, a pu a i e QTL was de ec ed on ch omosome 26 be ween ma ke s MCW285 and ADL285 (2.50 and 4.91 Mb, espec i ely). In con as o he o he iden i ied QTL, his QTL displayed a la ge dominance e ec . Va ious s a egies we e used o explo e he da a in g ea e de ail. Bo h AH and HU measu emen s we e used in o de o elimina e con ibu ion o he egg weigh o he pheno ype and o allow compa ison o di e en da ase s. All 16 hal -sib amilies we e geno yped wi h i e mic osa elli e ma ke s on ch omosome 7. Linkage analysis o his ull F 2 mapping popula ion inc eased he esolu ion and signi icance o he QTL, eaching he 1% genome-wide signi icance le el (Table 3). Howe e , he con idence in e als emained la ge because o he low-co e age map. The QTL on ch omosome Z was ine-mapped using he F 2 popula ion and linkage mapping wi h 20 SNP ma ke s co e ing he QTL egion. The mic osa elli e ma ke s o iginally used we e excluded om he analysis in o de o a oid bias om he e ogeneous in o ma ion con en a ising om he wo ypes o ma ke s. Inc easing hesamplesize( o he ullmappingpopula ion) made i possible o ocus on he egion con aining he QTL in he SNP analysis. The QTL a ec ing bo h AH and HU was loca ed in a egion be ween 33.75 and 34.99 Mb, lanked by he ma ke s s14761341 and s16767662 (Figu e 1). The QTL peak co-loca ed wi h he SNP ma ke s14761196. Resul s o AH and HU we e consis en , he only di e ence being ha he esul o HU was mo e signi ican han o AH(F=50.73 s.F=40.11).Wedid no ind any e idence o a QTL in ol ed in egg weigh bu he esul s sugges ed he p esence o QTL ha a ec eggshell s eng h (da a no shown). The comme cial Hy-Line popula ion was used o con i m he mos p omising QTL egions, on ch omosomes 7 and Z. On ch omosome 7, mic osa elli e ADL326 a posi ion 5.15 Mb was signi ican ly associa ed wi h bo h ea ly and la e AH (p = 0.0046 and 0.0024, espec i ely) be ween di e en geno ypes [see Addi ional ile 2]. Th ee seg ega ing alleles o med se en geno ype classes in he Hy-Line popula ion, wi h on a e age 11.5 indi iduals in each g oup. On ch omosome Z, ma ke s16765819 a posi ion 29.09 Mb was signi ican ly associa ed wi h ea ly and la e AH (p = 0.0003 and 0.004, espec i ely). This locus was loca ed a he dis al end o he QTL egion iden i ied wi h he F 2 popula ion. Al hough s16765819 was he ou e mos s udied ma ke , i was signi ican in bo h popula ions. Discussion We used an F 2 in e c oss be ween he wo egg-laye lines, Whi eRockandRhodeIslandRed, oiden i yQTL associa ed wi h albumen quali y and de ec ed ou genomic egions on ch omosomes 4, 7, 26 and Z. The QTL on ch omosome 26 has no been epo ed be o e. E idence o he QTL on ch omosome 4 (be ween posi ions 76.43 and 80.94 Mb) is indi ec ly suppo ed by he QTL epo ed o albumen weigh in wo o he s udies, espec i ely be ween posi ions 62.18 and 75.89 Mb in [12] and a 80 Mb in [11]. The QTL on ch omosome 7 be ween posi ions 24.24 Mb and 29.72 Mb co-loca ed wi h he HU40 associa ion epo ed by Liu e al. [14], using wo expe imen al egg- ype lines, Whi e Legho n and dwa b own laye s. This same egion o e lapped wi h a QTL ha a ec s eggshell s eng h be ween posi ions 24.25 and 31.83 Mb iden i ied by Sasaki e al. [26]. In addi ion o hese, Abash e al. [13] de ec ed a signi ican 3-SNP window o ea ly shell quali y a a posi ion be ween 25.1 and 26.2 Mb. Fu he mo e, Liu e al. [14] iden i ied pu a i e associa ions wi h eggshell hickness (EST40) be ween 27.47 and 29.06 Mb. I should be no ed ha all hese s udies used b own laye lines bu belonging o di e en b eeds [13,14,26]. P e iously, i was shown ha he QTL o eggshell hickness [26] and shell s eng h [19] a posi ion 35 Mb o e lapped on ch omosome Z. Ou esul s e eal ha he genomic egion, a ound 29–35 Mb, also in luences albumen quali y. The ac ha his egion is on chicken ch omosome Z ag ees wi h he adi ional knowledge ha si es ha e a la ge e ec han dams on albumen quali y [27]. A posi i e co ela ion be ween albumen and eggshell quali ies has been epo ed [4]. In addi ion, se e al s udies ha e sugges ed ha QTL associa ed wi h albumen quali y o e lap wi h a shell- ela ed QTL, which canno be Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 5 o 8 h p://www.gsejou nal.o g/con en /45/1/31 conside ed as pu e coincidence. Two hypo heses can be p oposed i.e. ei he genes common o bo h albumen quali y and eggshell quali y exis in hese ch omosomal egions o he shell- ela ed QTL has pleio opic e ec s on albumen quali y (o ice e sa). One possible common ac o connec ing albumen quali y and shell cha ac e is he eggshell memb anes, which a e wa e p oo ba ie s agains me abolic gases and wa e . The ou e memb ane i.e. cu icle, ep esen s he ou e mos pe meabili y con ol [28]. Ba ie p ope ies depend on he shell po es and dis ibu ion o he cu icle o e he su ace o he egg. The cu icle memb ane can be pa ially o o ally absen [28]. Because egg whi e quali y de e io a es du ing s o age, he ba ie p ope ies o shell memb anes a e likely o a ec egg whi e quali y signi ican ly, a leas a e o iposi ion. Acco ding o one heo y, egg whi e hinning is caused by a change in pH in he albumen e.g. [18]. This al e a ion a ises by he e apo a ion o gases h ough he shell. Mo eo e , he cu icle con ibu es o eggshell hickness [29] and hus he ecu en co-loca ions o QTL associ- a ed wi h eggshell quali y and albumen quali y migh be explained by he key oles played by he eggshell and memb anes o p e en albumen de e io a ion. Acco ding o ano he heo y, mul i unc ional genes con ol egg ai s such as albumen and shell p ope ies. This mul ipu pose unc ioning hypo hesis is suppo ed, o ins ance, by he esul s o Hincke e al. [30] and Jonche e e al. [31], who bo h showed ha some egg whi e p o eins a e ound also in he shell (o shell memb anes). Ne e heless, i seems ha egg albumen quali y and shell ai s a e connec ed a he gene le el. Many p omising candida e genes o albumen quali y a e p esen in he QTL egions de ec ed. Some o he mos ele an candida es om each QTL egion a e discussed below. The egion de ec ed on ch omosome 7 be ween posi ions 23 and 30 Mb seems o be impo an o egg quali y ai s and includes a clus e o cell shape and adhesion genes, such as ITGB3 (in eg in, be a 3 (pla ele glycop o ein IIIa, an igen CD61), 23.38 Mb), DES (desmin, 23.73 Mb), VIL1 ( illin 1, 24.09 Mb), TNS4 ( ensin 4, 24.09 Mb), ITGB5 (in eg in, be a 5, 29.46 Mb) and MUC13 (mucin 13, cell su ace associa ed, 29.51 Mb). In pa icula , MUC13 is a e y a ac i e candida e o con olling albumen quali y because he o omucin p o ein, consis ing o mucin subuni s, is belie ed o be esponsible o he gel-like s uc u e o he esh albumen [16,17,28,32,33]. The QTL peak on ch omosome Z co-loca ed wi h SNP ma ke s14761196 a posi ion 33.99 Mb ha lies wi hin an uncha ac e ized gene, KIAA1797.Thisgeneis exp essed widely in ep oduc i e and sec e o y issues. Bo h he independen comme cial Hy-Line popula ion and he F 2 mapping popula ion showed a e y signi ican associ- a ion wi h ma ke s16765819 a posi ion 29.09 Mb on s13799822 s14691747 s14762832 s16766794 s16766752 s16766334 s14761691 s13795456 s14761341 s14761196 s16767662 s16110154 s16767980 s16110443 s16111109 s16684439 s16766685 s16132282 Figu e 1 QTL g aph o ch omosome Z. The analysis ocused on he QTL egion de ec ed p e iously (ch omosome loca ion in cM); he ull F 2 mapping popula ion (1599 indi iduals), SNP ma ke s (named acco ding o he s-code) and HU pheno ype da a we e used; he highes F- alue o he QTL (50.73) co-loca ed wi h SNP ma ke s14761196. Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 6 o 8 h p://www.gsejou nal.o g/con en /45/1/31 ch omosome Z ha is loca ed in he bo de egion o he QTL. Ma ke s16765819 is loca ed nea o he PTPRD (p o ein y osine phospha ase, ecep o ype, D). O he pu a i e candida e genes nea he QTL peak a e, o ins ance, GCNT1 (glucosaminyl (N-ace yl) ans e ase 1, co e 2 36.9 Mb), which plays a ole in mucin biosyn hesis (KEGG en y 427260) and UBQLN1 (ubiquilin 1, 39.4 Mb), which is equi ed du ing p o ein deg ada ion. The numbe o SNP on ch omosome Z di e ed be ween he popula ions s udied. Wi hin he QTL egion ine- mapped on ch omosome Z, six SNP we e in o ma i e in he Hy-Line popula ion, bu only h ee o hese we e common in he wo mapping popula ions. Al hough his complica es compa isons and in e p e a ion o he esul s be ween he popula ions, ou esul s we e in line and we e unambiguous o he comme cial line. Clus e s o signi ican ly associa ed ma ke s, as on ch o- mosomes 7 and Z, could be due ei he o a high numbe o causa i e polymo phisms o o a single causa i e poly- mo phism in he egion wi h a high le el o LD [33]. The e ec o each associa ed SNP in he clus e migh ha e a small e ec , bu wi hin he egion, he haplo ypes could ha e a la ge o e all e ec on he ai s udied. Many esul s sugges ha accumula ion o a ia ions a he same locus is a ela i ely common mechanism [33]. Conclusions We iden i ied ou genomic egions ha a ec albumen quali y in chicken o which hose on ch omosomes 4 and 26 a e no el. The QTL on ch omosomes 7 and Z o e lapped wi h p e iously iden i ied QTL o shell qual- i y, which sugges s he exis ence o possible common ac o s o bo h albumen and shell quali y. The esul s o his s udy a e cong uen wi h he gene al assump ion ha mul i- ac o ial causes a e in ol ed in egg albumen hinning. The genes ha con ol albumen quali y a e di e se and ac ei he di ec ly o indi ec ly ia di e en mechanisms. Egg whi e hinning is an in ica e p ocess ha can ake place anywhe e du ing he p ocess, s a ing om albumen o ma ion in he magnum o egg s o age a e o iposi ion. Addi ional iles Addi ional ile 1: HU QTL esul s wi hin 668 F2 on ch omosome 7 and Z du ing he ini ial scan wi h he low-co e age ma ke map. Sho desc ip ion: QTL on ch omosomes 7 and Z de i ed by a mul i-ma ke eg ession me hod (Y-axis = F- a io, X-axis = loca ion in cM). Addi ional ile 2: Signi ican geno ype- ai associa ion plo s ob ained om he comme cial line (Hy-Line) wi h di e en ma ke s and pheno ypes. Sho desc ip ion: Ma ke associa ion es in comme cial line wi h a non-pa ame ic K uskal-Wallis es . Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Au ho s’con ibu ions MH designed he geno yping wo k, pa icipa ed in he s a is ical analysis (QTL mapping and associa ion analyses) and w o e he manusc ip . JT pe o med he geno yping. MT-H con ibu ed o he design o he s udy and o da a collec ion and analyses. JA, MS and RP con ibu ed o he design o he s udy, p o ided pheno ypic da a and animal samples. JV supe ised he s udy and edi ed he manusc ip . All au ho s ead and app o ed he inal manusc ip . Acknowledgemen s This p ojec was unded by Lohmann Tie zuch GmbH. We a e g a e ul o Lau a Lau amäki o unning he expe imen s in he poul y house. The memo y o Lau a is deeply e e ed. We a e hank ul o Anneli Vi a o managing he labo a o y, Tiina Jaakkola o p ecise echnical assis ance and Daniel Fische o guidance in s a is ical analysis. Au ho de ails 1 MTT Bio echnology and Food Resea ch, Jokioinen 31600, Finland. 2 Lohmann Tie zuch GmbH, Cuxha en, Ge many. 3 Hy-Line In e na ional, P.O. Box 310, Dallas Cen e , IA 50063, USA. Recei ed: 16 No embe 2012 Accep ed: 13 July 2013 Published: 16 Augus 2013 Re e ences 1. Haugh RR: The Haugh uni o measu ing egg quali y. U.S. Egg and Poul y Magazine 1937, 43:552–573. 2. Sil e sides FG, Budgell K: The ela ionships among measu es o egg albumen heigh , pH, and whipping olume. Poul Sci 2004, 83:1619–1623. 3. Sco TA, Sil e sides FG: The e ec o s o age and s ain o hen on egg quali y. Poul Sci 2000, 79:1725–1729. 4. Poggenpoel DG: Co ela ed esponse in shell and albumen quali y wi h selec ion o inc eased egg p oduc ion. Poul Sci 1986, 65:1633–1641. 5. Hun on P: Poul y p oduc ion. Wo ld Animal Science C9. London: Else ie ; 1995. 6. Jin YH, Lee KT, Lee WI, Han YK: E ec s o s o age empe a u e and ime on he quali y o eggs om laying hens a peak p oduc ion. Asian-Aus J Anim Sci 2011, 24:279–284. 7. Hu ZL, Pa k CA, F i z ER, Reecy JM: QTLdb: A comp ehensi e da abase ool building b idges be ween geno ypes and pheno ypes. In P oceedings o he 9 h Wo ld Cong ess on Gene ics Applied o Li es ock P oduc ion: 1–6 Augus 2010. Leipzig; 2010 [h p://www.kong essband.de/wcgalp2010/ asse s/h ml/0017.h m] 8. Tuiskula-Haa is o M, de Koning DJ, Honka ukia M, Schulman NF, Maki-Tanila A, Vilkki J: Quan i a i e ai loci wi h pa en -o -o igin e ec s in chicken. Gene Res 2004, 84:57–66. 9. Hansen C, Yi N, Zhang YM, Xu S, Ga o a J, Cheng HH: Iden i ica ion o QTL o p oduc ion ai s in chickens. Anim Bio echnol 2005, 16:67–79. 10. Honka ukia M, Tuiskula-Haa is o M, de Koning DJ, Vi a A, Maki-Tanila A, Vilkki J: A egion on chicken ch omosome 2 a ec s bo h egg whi e hinning and egg weigh . Gene Sel E ol 2005, 37:563–577. 11. Wa decka B, Olszewski R, Jaszczak K, Zieba G, Pie zchala M, Wicinska K: Rela ionship be ween mic osa elli e ma ke alleles on ch omosomes 1–5 o igina ing om he Rhode Island Red and G een-legged Pa igenous b eeds and egg p oduc ion and quali y ai s in F(2) mapping popula ion. J Appl Gene 2002, 43:319–329. 12. Sch eiweis MA, Hes e PY, Se a P, Moody DE: Iden i ica ion o quan i a i e ai loci associa ed wi h egg quali y, egg p oduc ion, and body weigh in an F2 esou ce popula ion o chickens. Anim Gene 2006, 37:106–112. 13. Abash B, Sand o d E, A ango J, Se a P, Ful on JE, O'Sulli an NP, Hassen A, Habie D, Fe nando RL, Dekke s JCM, Lamon SJ: Ex en and consis ency o linkage disequilib ium and iden i ica ion o DNA ma ke s o p oduc ion and egg quali y ai s in comme cial laye chicken popula ions. BMC Genomics 2009, 10:S2. 14. Liu W, Li D, Liu J, Chen S, Qu L, Zheng J, Xu G, Yang N: A genome-wide SNP scan e eals no el loci o egg p oduc ion and quali y ai s in whi e legho n and b own-egg dwa laye s. PLoS One 2011, 6:e28600. 15. The Na ional Cen e o Bio echnology In o ma ion, GENE: [h p://www.ncbi. nlm.nih.go /gene] 16. Mann K: The chicken egg whi e p o eome. P o eomics 2007, 7:3558–3568. Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 7 o 8 h p://www.gsejou nal.o g/con en /45/1/31 17. Mann K, Mann M: In-dep h analysis o he chicken egg whi e p o eome using an LTQ O bi ap Velos. P o eome Sci 2011, 9:7. 18. Omana DA, Liang Y, Ka NNV, Wu J: P o eomic analysis o egg whi e p o eins du ing s o age. P o eomics 2011, 11:144–153. 19. Tuiskula‐Haa is o M, Honka ukia M, P eisinge R, Schmu z M, De Koning DJ, Wei WH, Vilkki J: Quan i a i e ai loci a ec ing eggshell ai s in an F2 popula ion. Anim Gene 2011, 42:293–299. 20. Tuiskula-Haa is o M, Honka ukia M, Vilkki J, de Koning DJ, Schulman NF, Mäki-Tanila A: Mapping o quan i a i e ai loci a ec ing quali y and p oduc ion ai s in egg laye s. Poul Sci 2002, 81:919–927. 21. Illumina: [h p://www.illumina.com] 22. Honka ukia M, Tuiskula-Haa is o M, Ahola V, Uima i P, Schmu z M, P eisinge R, Ca e o D, Venne s öm P, A ango J, O'Sulli an N, Ful on J, Vilkki J: Mapping o QTL a ec ing incidence o blood and mea inclusions in egg laye s. BMC Gene 2011, 12:55. 23. G een P, Falls K, C ooks S: CRI-MAP Documen a ion. e sion 2.4. ; 1990 [h p:// sa .bio.cal ech.edu/sa _manuals/c imap-doc.h ml] 24. QTL Analysis using G id Technology: [h p://www.g idq l.o g.uk/] 25. Sasaki O, Odawa a S, Takahashi H, Ni asawa K, Oyamada Y, Yamamo o R, Ishii K, Nagamine Y, Takeda H, Kobayashi E, Fu ukawa T: Gene ic mapping o quan i a i e ai loci a ec ing body weigh , egg cha ac e and egg p oduc ion in F2 in e c oss chickens. Anim Gene 2004, 35:188–194. 26. Lo enz FW, Taylo LW: The inhe i ance o an albumen quali y cha ac e is ic o chicken eggs. J Ag ic Res 1940, 61:293–301. 27. Rose-Ma el M, Du J, Hincke MT: P o eomic analysis p o ides new insigh in o he chicken eggshell cu icle. J P o eomics 2012, 75:2697–2706. 28. Kusuda S, Iwasawa A, Doi O, Ohya Y, Yoshizaki N: Di e si y o he cu icle laye o a ian eggshells. J Poul Sci 2011, 48:119–124. 29. Hincke MT, Nys Y, Gau on J: The ole o ma ix p o eins in eggshell o ma ion. J Poul Sci 2010, 47:208–219. 30. Jonchè e V, Réhaul -Godbe S, Henneque -An ie C, Cabau C, Sibu V, Cogbu n LA, Nys Y, Gau on J: Gene exp ession p o iling o iden i y eggshell p o eins in ol ed in physical de ense o he chicken egg. BMC Genomics 2010, 11:57. 31. Hiidenho i J: O omucin.InBioac i e Egg Compounds. Edi ed by Huopalah i R, Lopez-Fandino R, An on M, Schade R. Heidelbe g: Sp inge -Ve lag Be lin; 2007:61–68. 32. O engenden M, Fen abil MA, Wu J: N-glycosyla ion o o omucin om hen egg whi e. Glycoconj J 2011, 28:113–123. 33. Bickel RD, Kopp A, Nuzhdin SV: Composi e e ec s o polymo phisms nea mul iple egula o y elemen s c ea e a majo -e ec QTL. PLoS Gene 2011, 7:e1001275. doi:10.1186/1297-9686-45-31 Ci e his a icle as: Honka ukia e al.:QTL mapping o egg albumen quali y in egg laye s. Gene ics Selec ion E olu ion 2013 45:31. Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo figu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi Honka ukia e al. Gene ics Selec ion E olu ion 2013, 45:31 Page 8 o 8 h p://www.gsejou nal.o g/con en /45/1/31