A Missense Mutation in PPARD Causes a Major QTL Effect on Ear Size in Pigs

Chinese Erhualian is the most prolific pig breed in the world. The breed exhibits exceptionally large and floppy ears. To identify genes underlying this typical feature, we previously performed a genome scan in a large scale White Duroc × Erhualian cross and mapped a major QTL for ear size to a 2-cM region on chromosome 7. We herein performed an identical-by-descent analysis that defined the QTL within a 750-kb region. Historically, the large-ear feature has been selected for the ancient sacrificial culture in Erhualian pigs. By using a selective sweep analysis, we then refined the critical region to a 630-kb interval containing 9 annotated genes. Four of the 9 genes are expressed in ear tissues of piglets. Of the 4 genes, PPARD stood out as the strongest candidate gene for its established role in skin homeostasis, cartilage development, and fat metabolism. No differential expression of PPARD was found in ear tissues at different growth stages between large-eared Erhualian and small-eared Duroc pigs. We further screened coding sequence variants in the PPARD gene and identified only one missense mutation (G32E) in a conserved functionally important domain. The protein-altering mutation showed perfect concordance (100%) with the QTL genotypes of all 19 founder animals segregating in the White Duroc × Erhualian cross and occurred at high frequencies exclusively in Chinese large-eared breeds. Moreover, the mutation is of functional significance; it mediates down-regulation of β-catenin and its target gene expression that is crucial for fat deposition in skin. Furthermore, the mutation was significantly associated with ear size across the experimental cross and diverse outbred populations. A worldwide survey of haplotype diversity revealed that the mutation event is of Chinese origin, likely after domestication. Taken together, we provide evidence that PPARD G32E is the variation underlying this major QTL.


Introduction
The external ear is part of the auditory system and plays a vital role in collecting sound as the first step in hearing. Multiple congenital anomalies have been documented for human external ears. For instance, microtia, characterized by a small and abnormally shaped outer ear, occurs in approximately one in 8,000-10,000 births. However, only in a minority of cases has a genetic or environmental cause been found [1]. The domestic pig services as not only an agriculturally important animal for meat production but also an important large-animal model for human medicine [2]. Thousands of years of selective breeding has created diversity of phenotypes in pigs, such as ear size in Erhualian and White Duroc breeds. Erhualian is the most prolific pig breed and exhibits unusually large and floppy ears as breed character ( Figure 1). Historically, the large-ear feature of Erhualian pigs had been favored by owners for the traditional sacrificial culture [3]. White Duroc is one of worldwide-popular boar line and has small and erect ears ( Figure 1). We have created a four-generation White Duroc 6 Erhualian resource population, in which phenotypic traits related to ear size have been recorded in 1,027 adult F 2 animals and 560 adult F 3 individuals (Table S1). We mapped a major QTL for ear size around 58 cM on SSC7 ( Figure S1) using a genome scan on the White Duroc 6 Erhualian cross [4], which confirmed the previously reported QTL affecting ear size in a Large White 6 Meishan F 2 resource population [5]. The significant QTL had a small confidence interval of 2 cM and explained more than 40% of phenotypic variance. The aim of this study was to identify the genetic determinant underlying this major QTL.

Results/Discussion
Identical-by-descent analysis defines the major QTL within a 750-kb interval To fine map the QTL, we genotyped 1,027 adult F 2 animals and their 68 parents and 19 grandparents in the White Duroc 6 Erhualian cross using additional 17 SNP markers and 11 microsatellite markers in the QTL region. A final set of 33 markers covering the QTL region were then explored to deduce the QTL genotypes of F 1 sires by the marker-assisted segregation analysis as proposed previously [6]. We determined QTL genotypes of all 9 F 1 sires ( Figure S2). All 9 Q-bearing chromosomes for increased ear size shared a haplotype of ,1.2 Mb flanked by markers HMGA1 -TULP1. The shared haplotype was distinct from q-bearing chromosomes ( Figure 2).
These observations strongly suggest that the QTL is located in the 1.2-Mb interval.
Given the extremely divergent ear size phenotypes between Erhualian and White Duroc animals, we assumed that Q and q alleles were alternatively fixed in Erhualian and White Duroc founder animals; hence all Erhualian founder sows could share a chromosomal segment carrying the Q allele for increased ear size. To test this assumption, we reconstructed haplotypes of all 19 founder animals (2 sires and 17 dams) using 50 markers (15 microsatellites and 35 SNPs) in the QTL region. Almost all Erhualian founder sows shared a haplotype of ,750 kb within the refined 1.2-Mb interval ( Figure 2). As predicted, this shared haplotype was associated with increased ear size and presumably Q-bearing chromosomes. Two Erhualian founder sows carried a distinct haplotype (denoted as E q ), which was unexpected because it was contrast with our initial assumption. We then conducted a statistical analysis of F 2 animals in the White Duroc 6 Erhualian cross. The results revealed that the E q chromosome had an effect on decreased ear size similar to the White Duroc chromosome (D q ) and significantly different from the Erhualian Q-bearing chromosome (E Q ). The least-squares means (6 s.e.) of ear weight were 323.0764.55 for E Q E Q and 266.66618.9 for E Q E q (P = 0.04); 264.7163.52 for D q E Q and 236.98617.12 for D q E q (P = 0.06, Table 1). The shared E Q chromosome allowed us to refine the location of the major QTL to the 750-kb interval between markers UHRF1BP1 and TULP1 (Figure 2).
Selective sweep analysis refines the QTL to a 630-kb region Historically, Erhualian pigs had undergone selection for ear size because pigs with extraordinary large and floppy ears were favored for the ancient sacrificial culture in the Taihu region of East China [3]. Reduced genetic variation in the critical region containing the QTL was therefore predicted. To define the region of reduced genetic variation, we collected 211 animals representing all lineages in 3 Erhualian nucleus populations, 216 animals from 6 Chinese indigenous breeds and 119 independent animals from 3 Western worldwide-popular commercial breeds. Using these samples, we genotyped 6 microsatellite and 32 SNP markers in the 750-kb region. We found that 18 adjacent markers in a 630-kb region between markers UHRF1BP1 and FANCE showed dramatically reduced polymorphisms in all Erhualian pigs with nearly all major allele frequencies of more than 0.90. Notably, the 18 markers in the 630-kb region are monomorphic in the Erhualian nucleus population from Xishan county (n = 72). In comparison, the genetic polymorphisms of these markers were maintained in other Chinese, Western breeds, and wild boars ( Figure 3). The 630-kb region showing strong selective-sweep effects on Erhualian pigs was therefore predicted to contain the responsible locus. We further genotyped the 18 markers in the 630-kb region on 188 adult animals of Sutai pigs. This breed was developed after 18-genereation selection from a Duroc (50%) 6 Erhualian (50%) cross in 1986 [7], meaning that the breed has undergone 18 generations of meiosis reducing the extent of linkage disequilibrium between QTL and linked markers. The Erhualianoriginated haplotype of 630 kb showed significant (P = 0.009) association with increased ear size compared with other chromosomes in Sutai pigs ( Figure S3), thereby supporting the conclusion that this region harbors the causative gene.

Author Summary
A central but challenging objective in current biology is to dissect the genetic basis of quantitative traits. Numerous quantitative trait loci (QTL) have been uncovered in model and farm animals, providing unexpected insights into the biology of complex traits. However, only a few causal variants underlying the QTL have been explicitly identified. By using a battery of genetic and functional assays, we herein show that a major QTL effect on pig ear size is most likely caused by a single base substitution in an evolutionary conserved region of the PPARD gene. The protein-altered mutation is of functional significance and explains a proportion of variation in ear size across diverse pig breeds. A worldwide survey showed that the mutant allele for increased ear size was derived from a common ancestor in Chinese pigs, likely after domestication. These findings establish, for the first time, an essential role of PPARD in ear development and highlight the great potential of naturally occurring mutations in farm animals to gain insights into mammalian biology. Moreover, the knowledge of the PPARD causal mutation adds to the limited list of quantitative trait genes and quantitative trait nucleotides characterized in domesticated animals.
Positional candidate gene analysis: discovery of a nonconservative missense mutation in PPARD concordant with the QTL genotypes of founder animals The 630-kb region encompasses 9 annotated genes (ANKS1A, DEF6, FANCE, PPARD, SCUBE3, TAF11, TCP11, UHR1BP1 and ZNF76) in the human homologous region. RT-PCR was performed to detect expression levels of these genes in ear tissues of piglets. Four genes including PPARD, FANCE, TAF11 and ZNF76 were highly expressed, whereas transcripts of other genes were almost absent in ear tissues (data not shown). Of the 4 genes, PPARD (peroxisome proliferator-activated receptor delta) is a ligand-modulated transcription factor belonging to the nuclear receptor superfamily and plays crucial roles in diverse biologically important processes [8]. For instance, PPARD play a pivotal role in modulating cell differentiation in both keratinocytes and sebocyte of skin [9]. PPARD also serves as a key regulator in fat metabolism; it triggers fat burning and enhances energy uncoupling in adipose tissues and skeletal muscle [10][11][12]. Moreover, PPARD is a key player in Wnt/b-catenin pathway [13], which has essential roles in diverse cellular activities including chondrocyte proliferation and differentiation [14]. The external ear is composed of skin, cartilage, connective tissues and fat. Given its crucial role in skin homeostasis, cartilage development and fat metabolism, PPARD stood out as a prime positional candidate for the major QTL. We monitored the relative mRNA expression of PPARD in ear tissues of Erhualian and Duroc pigs at four different ages by real-time RT-PCR. The expression levels were higher in samples at early ages (days 0, 45 and 90) compared with adult samples (day 300). However, no significant difference of expression levels was found in ear tissues between large-eared Erhualian and small-eared Duroc pigs ( Figure S4).
To search for causative mutations, we first sequenced the entire coding region of the PPARD gene using ear mRNA of two White Duroc and two Erhualian animals and identified only one nonsynonymous mutation. The G to A mutation caused a glycine to glutamic acid substitution at codon 32 (GU565977) in the conserved intrinsically disordered domain of the PPARD protein predicted by SMART (http://smart.embl-heidelberg.de/). The intrinsically disordered domain is a distinctive and common characteristic of eukaryotic hub proteins like multifunctional nuclear receptors and serves as a determinant of protein interactivity [15]. Comparison of amino acids of this protein domain across mammals revealed that glycine is well conserved in mammalian PPARDs (Figure 4), while the derived glutamic acid occurs only in alleles increasing ear size in pigs. We thus speculated that the nonconservative substitution probably changes the PPARD interactivity with other protein partners and consequently affects the gene's regulation function. Genotypes of F 1 sires (9 heterozygotes) and F 0 animals (17 homozygotes and 2 heterozygotes) at the mutation site were 100% concordance with their QTL genotypes. The potentially altered function and QTL concordance of PPARD G32E corresponded to the hypothesis that this SNP may be the causative mutation underlying the major QTL.
PPARD G32E is a functional variant mediating downregulation of b-catenin and its target gene expression PPARD is involved in the Wnt/b-catenin signaling pathway that regulates diverse cellular functions. In the nucleus, PPARD interacts with b-catenin binding to TCF/LEF transcription factors that stimulate transcription of target genes important for multiple cellular activities including cartilage development and organogenesis [14]. To demonstrate functional significance of PPARD G32E, we cotransfected the 293T cells with the lentiviral expression vectors of wild-type or mutant PPARD and a TCF/LEF-driven luciferase reporter construct. A Renilla luciferase expression vector was used for the normalization of transfection efficiency. Overexpression of mutant PPARD led to a 40% decrease (P,0.05 compared with the wild-type treatment) in TCF/LEF reporter activity ( Figure 5A), indicating the G32E mutation mediates down-regulation of b-catenin downstream genes. To examine a direct functional role of PPARD G32E in target genes of b-catenin, we treated pig ear-derived primary fibroblast cells with the lentiviral PPRAD expression vectors and monitored the mRNA levels of b-catenin and its known downstream (c-myc) [16] and upstream (Sox9) [17] genes along with GAPDH as a loading control by real time quantitative RT-PCR. The mRNA levels of b-catenin and c-myc were reduced respectively by 4.1-fold and 11.5fold (P,0.001) in mutant PPARD transfectants compared with the cells transfected with wild-type PPARD. Western blot analysis showed that both b-catenin and c-myc protein levels were decreased by the mutant PPARD treatment ( Figure 5B), thereby confirming the results of mRNA and luciferase reporter analyses. Sox9 mRNA expression in mutant PPARD transfectants was only slightly decreased to 1.1-fold of the wild-type PPARD treatment; the result was validated by Western blot ( Figure 5B). GAPDH was used as a protein loading control for total cell lysate, which was not affected by both wild-type and mutant PPARD treatments ( Figure 5B). Altogether, we conclude that PPARD G32E is a functional variant that mediate down-regulation of b-catenin and its target gene expression in the Wnt/b-catenin signaling pathway. Wnt/b-catenin signaling has been firmly demonstrated to suppress adipogensis [18][19]. The fact that PPARD is a key modulator of lipid production in the skin [9] and that PPARD G32E inhibits b-catenin expression led us to assume that the mutation stimulates lipid production and storage that are required for enlarged ear size.
PPARD G32E is significantly associated with ear size across the experimental intercross and outbred populations To confirm the effect of PPARD G32E on ear size, we performed a standard association test, a marker-assisted association test and an F-drop test [20] in the White Duroc 6 Erhualian cross. The SNP showed greatly significant (P,0.0001) association with ear weight and ear size in the standard association test. In the marker-assisted association test, the SNP was more significant (P,0.001) for these traits compared with the QTL effect. After fitting this polymorphism in the QTL model, the great QTL effect disappeared with F-value drop rations of less than 0.03 (Table S2). These results were in agreement with the hypothesis that the SNP is the causative mutation for the major QTL affecting ear size. Nevertheless, we cautioned the results because variants closely linked with a causative mutation also lead to strong association in F 2 resource populations due to the high level of linkage disequilibrium between founder breeds [20].
To obtain additional supporting evidence, we further genotyped the G32E mutation on 667 mature pigs from 4 Chinese local breeds (Erhualian, Hang, Yushan Black and Bama Xiang) and 3 synthetic commercial lines (Sutai, Suzhong, Sujiang) with phenotypic data of ear size. These populations show a wide range of ear size and segregate for the mutation. The association analyses confirmed the effect of PPARD G32E on ear size. The 32E allele was significantly associated with increased ear size across the tested breeds (P,0.05; Table 2). Chinese local pig breeds have low levels  of linkage disequilibrium extending up to only 0.05 cM [21]. The concordantly significant association across Chinese breeds thereby strengthened the hypothesis that PPARD G32E is the responsible locus for ear size. The effects of PPARD G32E differ in their magnitude in the tested breeds; one reason is that the effects are context-dependent and are influenced by different genetic backgrounds and environments. Another possibility is that PPARD G32E is only responsible for part of the effect on ear size in Erhualian pigs.

PPARD G32E has a unique origin of Chinese pigs likely after domestication
To reveal the ancestral state and allele frequency of PPARD G32E in diverse pig breeds, we genotyped the mutation in a panel of 1,166 animals representing 31 domestic breeds and Chinese and European wild boars. Overall, the derived 32E allele for increased ear size occurred at high frequencies (.0.80) in Chinese breeds with large and floppy ears. In contrast, the 32G allele for normal ear size was fixed in all wild boars, European local and commercial breeds, and occurred at low frequencies (,0.30) in Chinese indigenous breeds having small and erect ears. These results indicated that PPARD G32E may occur in Chinese pigs after domestication. We detected only one heterozygote in European local breeds ( Table 3). The animal was from Large Black pigs that exhibit large and floppy ears and have been influenced by Chinese breeds brought into England in the late 18009s [22].
We further analyzed the genetic variability and haplotype structure around the G32E mutation in a worldwide pig panel. A total of 868 animals representing 34 breeds were genotyped for 32 SNPs in a 77-kb region of PPARD. Again, the Erhualian breed showed a selective sweep signal as it had negative classical selection statistics Tajimas D and much smaller nucleotide variability (p N ) compared with other Chinese local breeds and Western commercial breeds (Table S3). Especially, the genetic variability at the 32 loci was wiped out in the Erhualian population from Xishan. Moreover, we plotted a distribution of the frequency of the derived 32E allele (P A ) against Tajimas D index to elucidate the existence of directional selection for the G32E mutation. When  P A = 0, Tajimas D was highly variable across breeds, likely due to demographic and/or sampling effects. In stark contrast, Tajima's D took highly negative values when P A .0.8 in Erhualian and other Chinese large-eared breeds as expected in a classical directional selection ( Figure S5). We reconstructed 16 major haplotypes with frequencies larger than 0.01 from the 32 SNPs genotyped. Of the 16 haplotypes, only one carried the derived 32E allele; it was at high frequencies in Erhualian pigs and intermediate frequencies in some floppy-eared Chinese breeds whereas absent in Western pigs and wild boars ( Table 4). The NJ phylogenetic tree illustrated that the typical haplotype of Erhualian pigs was generally divergent from other haplotypes ( Figure S6). These observations supported the assumption that the G32E mutation has a unique origin in Chinese breeds likely after domestication and has undergone selection in Erhualian pigs. We calculated linkage disequilibrium measures (r 2 ) between all pairs of loci and inferred haplotype blocks. Three and two haplotype blocks were identified in the PPARD region for Chinese indigenous pigs and Western commercial breeds, respectively. Only a single nevertheless larger block that spanned 53 kb and contained the G32E SNP was found in Erhualian pigs, reflecting a selection hitching effect ( Figure  S7). The G32E SNP was in high disequilibrium with very few of the SNPs analyzed (two with r 2 .0.8), and there was no observable trend between physical distance and disequilibrium measures for the G32E SNP and the rest of loci ( Figure S8).
Diverse pieces of evidence support the casualty of PPARD G32E for the QTL The elucidation of the genetic basis of multifactorial traits in domestic animals is still a big challenge, and few successful examples have been reported [23][24][25][26][27]. In this study, a battery of genetic and functional assays obtained diverse pieces of supporting evidence that the PPARD G32E substitution underlies the major QTL effect on ear size on SSC7. (1) The shared haloptypes of 9 F 1 sires segregating for the QTL spanned a region of ,1.2 Mb containing PPARD. (2) All Erhualian founder chromosomes shared a ,750 kb segment spanning PPARD that were associated with the Q allele for increased ear size. (3) Erhualian pigs showed an obvious selective sweep signal in a 630-kb region encompassing PPARD; the signal was concordant with the breeding history of the breed. (4) The 630-kb haplotype showed similar QTL effect on increased ear size in Sutai pigs that were developed after 18-generation selection in the Erhualian 6 Duroc cross. (5) Of the 4 genes expressed in ear tissues within the critical region, PPARD stood out a prime candidate for its established essential roles in skin homeostasis, cartilage development and fat metabolism. (6) Only one missense mutation (G32E) was identified in PPARD using White Duroc and Erhualian founder animals. The mutation caused a nonconservative amino acid change at the conserved intrinsically disordered domain and was of functional significance. (7) The G32E SNP was concordant with QTL genotypes of F 0 and F 1 animals in the White Duroc 6 Erhualian cross. (8) The G32E SNP showed strikingly significant association with ear size across the experimental cross and diverse outbred populations. (9) The derived allele for increased ear size occurred at high frequencies only in Chinese floppy-eared breeds. Altogether, these data led us to conclude that G32E in the PPARD gene has an important contribution to ear size in pigs. The results establish, for the first time, a direct and novel role of PPARD in ear development and may be of relevance for the pathogenesis of external ear abnormalities in humans.

Potential pleiotropic effects of PPARD G32E on diverse traits
The genomic region harboring PPARD G32E is of great interest in pig genetics, because significant QTL for diverse traits related to growth, carcass length, skeletal morphology and fat deposition have been consistently evidenced in the region using the current resource population and different crosses between Chinese Meishan and commercial breeds [28][29][30][31][32][33]. The overlapping QTL for multiple traits in the region led us to assume that there might be a single critical gene having pleiotrophic effects on these traits. We herein showed the causality of PPARD G32E for the QTL affecting ear size in the critical region. Given that PPARD serve as a crucial and multifaceted determinant of diverse biological functions including fat metabolism, cartilage development, chondrocyte proliferation and differentiation a European domestic pigs include Iberian (n = 10), Berkshire (n = 4), Large Black (n = 2), Mid White (n = 2), Chester White (n = 2), Old Spot (n = 2), Yorkshire (n = 2), Tamworth (n = 1), British Lop (n = 1), Hampshire (n = 1), Saddle Back (n = 1). All these animals are homozygous GG except a heterozygote detected in one of two Large Black pigs, which exhibits large or medium-size and floppy ears and are influenced by Chinese breeds brought into England in the late 1800's (Kijas et al. 1998) [22]. doi:10.1371/journal.pgen.1002043.t003 [8,[10][11][12][13][14], we thus speculate that PPARD is a strong candidate of the multiple significant QTL on SSC7 and that PPARD G32E might have pleiotropic effects on growth, carcass and fatness traits in pigs. Further investigations will be performed to validate the hypothesis in the future.

Ethics statement
All animal work was conducted according to the guidelines for the care and use of experimental animals established by the Ministry of Agriculture of China.

Fine mapping by identical-by-descent analysis
Microsatellite markers in the mapped interval were mined from the pig genome assembly (Build 9.2) at http://www.ensembl.org/ Sus_scrofa/Info/Index and were genotyped using standard procedures. Primers for amplification of microsatellite markers are given in Table S4. QTL genotypes of F 1 boars in the White Duroc 6Erhualian intercross were determined by marker-assisted segregation analysis as described previously [6]. Briefly, a Z-score was calculated for each F 1 sire; the score is the log10 of the H 1 /H 0 likelihood ratio where H 1 assumes that the boar is heterozygous at the QTL (Qq), while H 0 postulates that the boar is homozygous QQ or qq. Boars were considered to be Qq when Z .2, QQ or qq when Z ,22, and of undetermined genotype if 22,Z,2. The pedigree and management of the intercross population with phenotypic data of ear size have been described elsewhere [4]. Haplotypes of founder animals were reconstructed with the SimWalk2 program.

RT-PCR of candidate genes in ear tissues
Total RNA was extracted from pig tissues using the Rneasy Fibrous Tissue Mini Kit (Qiagen). To analyze expression of candidate genes in ears, products from the first strand-complementary DNA synthesis (TaKaRa) were amplified with primers given in Table S5. The quantification of the PPARD transcripts was performed by the comparative C t method (2 2DDCt ) using the primers and TaqMan probes shown in Table S5. Real-time PCR was done with the Universal PCR Master Mix using an ABI7900 instrument (Applied Biosystem). All samples were analyzed in triplicate. The b-actin gene was used as the internal reference gene.

Resequencing of PPARD cDNA and genotyping of PPARD G32E
The entire coding region of porcine PPRAD was re-sequenced using ear mRNA of two White Duroc and two Erhualian animals. Primer pairs listed in Table S6 were used to generate overlapping PCR amplicons. All PCR products were purified using the NucleoSpin Extract II kit (Macherey-Nagel) and sequenced using the same primers. The sequence traces were assembled and analyzed for polymorphisms using the SeqMan program (DNAS-TAR). The PPARD G32E mutation was genotyped using the ABI SNapshot protocol.  Luciferase report assay The coding region of pig PPARD was amplified with primers PPARD-Age-I-F (59-GAG GAT CCC CGG GTA CCG GTC GCC ACC ATG GAG CAG CCG CCG GAG-39) and PPARD-Age-I-R (59-TCA TCC TTG TAG TCG CTA GCG TAC ATG TCC TTG  TAG-39). The amplified cDNA was gel-purified and digested with AgeI and NheI (NEB). The restricted fragments were cloned to pGC-FU-EGFP-3FLAG lentiviral expression vector (Genechem). The sequence and orientation of the insert were verified by DNA sequencing. The expression of His-tagged PPARD in cultured cells was confirmed by Western blot analysis with anti-His antibody. The human 293T cells were infected with the lentiviral expression constructs of pig wild-type and mutant PPARD. The infected cells were seeded at a concentration achieving 80% confluence in 96-well plates 18 h before transfection. The cells were transiently transfected with TCF/LEF-Luc reporter vector (Cignal, SAB) along with a control Relina luciferase vector using Lipofectamine plus reagent. The cell lysates were obtained with 16 reporter lysis buffer (Promega) 48 h after transfection. The luciferase activity was assayed in a Berthold Auto Lumat LB953 luminometer (Nashua, NH) by using the luciferase assay system from Promega. The relative luciferase activity was normalized to the Relina luciferase activity in each sample.

Real-time RT-PCR and western blot analysis in cultured cells
The pig ear-derived fibroblast cells were transfected with pGC-FU-EGFP-3FLAG lentiviral expression vector (Genechem). Five days post-transfection, 1610 6 cells were harvested for qPCR and Western blot analysis. Total RNA was extracted from harvested cells using Trizol (Invitrogen). Two mg of total RNA was synthesized into cDNA with M-MLV reverse transcriptase (Promega) and oligo d(T). Real time PCR was performed on the cDNA using the SYBR Premix Ex Taq (TaKaRa) and primers listed in Table S7 in a TP800 Real Time System (TaKaRa). The quantification of transcripts was performed by the comparative C t (2 2DDCt ) method. All values were reported as mean 6 S.D. of triplicate assays of each cDNA sample. Rabbit anti-PPARD (Sigma), mouse anti-b-catenin (Abcam), rabbit anti-c-myc (Cellsignaling), mouse anti-Sox9 (Abcam) and mouse anti-GAPDH (Santa Cruz) antibodies were used in Western blots in a routine way. The specific immunoreactive bands were visualized using an ECL plus kit (GE Healthcare) and quantified with the Molecular Imaging Software (Kodak).

Association analysis
The entire White Duroc 6 Erhualian resource population was genotyped for the PPARD G32E mutation. Association of the mutation with ear size and weight was evaluated using standard association, marker-assisted association and F-drop test as described previously [20]. Association analyses were also performed on 667 animals representing 7 different breeds. Photographs were taken for one ear of each animal after the ear was fixed and covered with a ruler as an internal reference of the size. Ear size was calculated using the Qwin software (Laica). Significance was evaluated by the ttest in the GLM procedure of SAS 9.0.

Analysis of haplotype phylogenies and linkage disequilibrium
Genomic DNA pools of White Duroc (n = 2) and Erhualian (n = 2) animals were amplified with primers given in Table S6. All PCR products were purified with the Qiagen protocol and sequenced using the same PCR primers, revealing a subset of SNP markers in the genomic region of porcine PPARD. SNP markers were genotyped by iPLEX SEQUENOM MassARRAY platform. SNP genotype calls were filtered and checked manually, and aggressive calls were omitted from the dataset. Population genetics parameters including the mean number of pairwise differences across loci (p N ), Tajimas D, Fu and Li's D were estimated with DnaSP v5 [34]. Haplotypes were reconstructed with PHASE v2 [35]. Haplotype phylogenetic tree based on p-distance were drawn using MEGA4 [36]. The Haploview v4.1 program [37] was used to calculate linkage disequilibrium measures (r 2 and D') and to identify haplotype blocks. Figure S1 Plots of F-ratios indicating the major QTL for ear size at 58 cM on pig chromosome 7. Markers and distance in cM are given on the x-axis, and F-ratios are indicated on the left y-axis. The threshold for 1% genome-wide significant level is indicated by the dashed horizontal line. The confidence interval of 2 cM is marked by the dashed vertical line. LEW: left ear weight; REW: right ear weight; LEA: left ear area; REA: right ear area. (TIF) Figure S2 QTL genotypes of F 1 boars determined by markerassisted segregation analysis. The number of offspring in each sire family is given above the error bars. The right ear size measured in the pedigree is marked by cm 2 in left axis. A Z-score is given for each sire pedigree. Q alleles associated with increased ear size are marked by a diamond, q alleles by a circle. (TIF) Figure S3 Association of the Erhualian-originated haplotype in the critical 630-kb region with increased ear size in Sutai pigs. Rare haplotypes with frequencies of less than 0.01 were discarded for analysis. E denotes the Erhualian haplotype. (TIF) Figure S4 Real-time RT-PCR analysis of PPARD temporal expression in ear tissues of Erhualian and Duroc pigs. Tissue samples were collected from Erhualian and Duroc pigs at days 0, 4563, 9063, and 30063 for RNA extraction. Six animals were sampled from each breed at each period. Real-time PCR was performed in triplicate. PPARD expression levels normalized with b-actin are given (mean 6 s.e.). No significant difference was observed in PPARD expression levels between Erhualian and Duroc pigs at each stage. (TIF) Figure S5 Relationship between Tajima' D and frequency of the derived 32E allele. (TIF) Figure S6 NJ phylogenetic tree constructed with the 16 frequent PPARD haplotypes. The detail information about each haplotype is given in Table 4. Haplotype 1 is the only one containing the derived 32E allele for increased ear size and is the major haplotype of Erhualian pigs. (TIF)