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Genome Res. May 2007; 17(5): 586–593.
PMCID: PMC1855175

Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1)

Abstract

The number of vertebrae in pigs varies and is associated with meat productivity. Wild boars, which are ancestors of domestic pigs, have 19 vertebrae. In comparison, European commercial breeds have 21–23 vertebrae, probably owing to selective breeding for enlargement of body size. We previously identified two quantitative trait loci (QTL) for the number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7. These QTL explained an increase of more than two vertebrae. Here, we performed a map-based study to define the QTL region on SSC1. By using three F2 experimental families, we performed interval mapping and recombination analyses and defined the QTL within a 1.9-cM interval. Then we analyzed the linkage disequilibrium of microsatellite markers in this interval and found that 10 adjacent markers in a 300-kb region were almost fixed in European commercial breeds. Genetic variation of the markers was observed in Asian local breeds or wild boars. This region encoded an orphan nuclear receptor, germ cell nuclear factor (NR6A1, formerly known as GCNF), which contained an amino acid substitution (Pro192Leu) coincident with the QTL. This substitution altered the binding activity of NR6A1 to its corepressors, nuclear receptor-associated protein 80 (RAP80) and nuclear receptor corepressor 1 (NCOR1). In addition, somites of mouse embryos demonstrated expression of NR6A1 protein. Together, these results suggest that NR6A1 is a strong candidate for one of the QTL that influence number of vertebrae in pigs.

Wild boars, which are the ancestors of modern domestic pigs, uniformly have 19 vertebrae. In comparison, European commercial breeds have 21–23 vertebrae (King and Roberts 1960). These breeds have long been selectively bred for enlargement of body size to increase meat production and improve reproductive performance. This process presumably increased the number of vertebrae. In mammals, the vertebral formula shows developmental constraint (Narita and Kuratani 2005). The number of cervical vertebrae is fixed at seven, and the total number of thoracic and lumbar vertebrae tends to be 19, although the respective counts vary among species. For example, in the Monotremata, Marsupialia, Lagomorpha, Rodentia, and Artiodactyla, the total number of thoracic and lumbar vertebrae is conserved at 19, which is thought to be the primitive form. In comparison, this number is increased in the Perissodactyla (e.g., horse, 24 vertebrae) and Carnivora (e.g., dog, 20 vertebrae) and that of the Primata is decreased to 17. However, these changes are lineage specific, and variation is restricted in each species, as is seen in the Primata (Pilbeam 2004). In light of these findings, it is interesting that the number of vertebrae in pigs varies from 19 to 23 within a single species.

In previous papers, we reported two quantitative trait loci (QTL) affecting the number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7; these QTL were identified using nine F2 families produced by crossing between breeds of European, Asian, and miniature pigs (Wada et al. 2000; Mikawa et al. 2005). These two QTL acted independently without an epistatic effect, and each had mainly an additive effect. For the QTL on SSC1, all the alleles of European commercial pigs used in the experimental families increased the number of vertebrae by 0.44–0.68 per allele. For the QTL on SSC7, some European alleles similarly increased the number of vertebrae (0.38–0.68). The combined effect of the two QTL accounted for an increase of more than two vertebrae. In F2 populations in which alternative alleles for both QTL were fixed in founder breed pigs, the proportions of phenotypic variance in the number of vertebrae explained by the QTL on SSC1 and SSC7 were similar, at ~30% (Mikawa et al. 2005). In the current report, we describe our map-based study of the QTL on SSC1 and present a 300-kb region that is almost fixed in a variety of European commercial breeds. We also suggest that an orphan nuclear receptor, germ cell nuclear factor (NR6A1, formerly known as GCNF) is a strong candidate for the gene underlying this QTL, in light of analyses of polymorphism, function, and expression.

Results

Defining the QTL region by using F2 families

In our previous studies, we mapped a QTL for the number of vertebrae on SSC1qter, between microsatellite markers SW1957 (151.6 cM) and SW1301 (175.8 cM) (Wada et al. 2000; Mikawa et al. 2005). To restrict the candidate region, three families in the previous study were reanalyzed by using microsatellite markers distributed densely throughout the QTL region (Mikawa et al. 2004). First, we performed an interval mapping using a subfamily of a Large White × Japanese wild boar population, for which construction three Large White female pigs (W1, W2, and W3) were used as parents. In the previous study, we reported that in two of them (W2 and W3) the QTL on SSC7 had no effects on number of vertebrae. In the subfamily derived from W2 and W3, which consisted of 207 F2 animals, the effect of increasing the number of vertebrae was attributable only to SSC1, and the proportion of phenotypic variance explained by the QTL on SSC1 was ~60%. As a result of interval mapping, the peak F-ratio (187.2) was detected between SJ344 (160.5 cM) and SJ861 (162.2 cM) (Fig. 1A). To evaluate the effect of sampling error on the estimated QTL position, we constructed a 95% confidence interval for the position showing the highest F-ratio, with 1000 repetitions of bootstrap sampling. We obtained the region from 159.5 to 163.6 cM as the 95% confidence interval for the QTL position (Fig. 1A).

Figure 1.
Dissection of a QTL region on SSC1 for vertebral number in F2 families. (A) Plots of F-ratio for interval mapping analyses in the Large White × Japanese wild boar population (red) and Jinhua × Duroc cross population (black). For the latter, ...

Another interval mapping was performed using a Chinese Jinhua × Duroc population, which was the largest population in our studies and consisted of 549 F2 animals; in this population, alternative alleles were fixed in founder breed pigs in the QTLs on both SSC1 and SSC7. When the genotype of a marker near the QTL detected on SSC7 was included as a covariate in the model for the analysis of SSC1, the peak F-ratio (171.2) was obtained at SJ344 (160.5 cM) on SSC1 (Fig. 1A). A bootstrap analysis (with 1000 repetitions) showed that the 95% confidence interval ranged from 159.8 to 161.4 cM (Fig. 1A). We think that the results of these two independent analyses provide sufficient evidence for us to deduce that the QTL was located in the region from 159.5 to 163.6 cM.

The third F2 family was produced by crossing Chinese Meishan females with a Göttingen miniature male. In this population, a QTL for vertebral number was detected on SSC1 but not on SSC7. QTL analysis showed that the Göttingen miniature sire was heterozygous on SSC1: one allele (Ver) had the effect of increasing vertebral number, whereas the other had no effect and was thought to be wild type (wt). The Meishan dams were homozygous wild type (wt/wt) (Mikawa et al. 2005). Among the eight F1 dams, five were homozygous wild type (wt/wt) and two were heterozygous (Ver/wt), but the genotype of the remaining animal was uncertain because of recombination within the QTL region that originated from the Göttingen miniature chromosome. By using microsatellite markers, we mapped the recombination site between SJ344 (160.5 cM) and SJ270 (161.1 cM). The genomic region proximal to SJ344 was from the Ver-containing chromosomal segment from the sire (Fig. 1B). The heterozygosity of the QTL of the F1 dam with the recombination was suggested by the segregation of the number of vertebrae in 18 F2 animals produced from the F1 dam. This segregation was confirmed through correction for the QTL effects from the F1 sire (Ver/wt) when we assumed that the Ver allele increased the number of vertebrae by 0.57, the average of the QTL effects in the experimental families in our previous study. We therefore judged that the QTL was located proximal to SJ270, and the results suggested that the QTL was located between SJ819 (159.2 cM) and SJ270 (161.1 cM). To further define the QTL, we then constructed a BAC contig for this region and developed microsatellite markers (Supplemental Table 1; Fig. 2A–C).

Figure 2.
Analyses of genomic structure and genetic variation of the QTL region. (A) Gene map of a part of the human chromosome 9qter. (B) BAC contig for the QTL region. Vertical lines indicate positions of STS (broken) and microsatellite markers (solid). BAC clones ...

Genetic variation of microsatellite markers in the QTL region

We attempted to fine-map the QTL, in accordance with the assumption that the genetic variation around this QTL would be reduced in commercial breed pigs because vertebral number is associated with body size, which has been a focus of selective pig breeding. To analyze the genetic variation, we collected 194 independent samples from five commercial breeds (Landrace, Large White, Yorkshire, Duroc, and Berkshire) and 40 samples from Asian local breeds (Meishan, Jinhua, and Japanese wild boar) as references. Using these samples, we genotyped 24 microsatellite markers in the 1.9-cM region between SJ819 and SJ270. The results revealed that SJ878 and SJ885 were nonpolymorphic in commercial breeds and that SJ641, SJ884, and SJ820 showed dramatically reduced polymorphism: The frequencies of all major alleles were >0.99. At SW705, located between SJ878 and SJ885, the frequency of the major allele was 0.93 and that of the second allele, which was two nucleotides smaller than the major allele, was 0.05 in commercial breeds (Fig. 2C; Table 1). In comparison, the genetic variation of these markers was maintained in the Asian breeds.

Table 1.
Genetic variations of microsatellite markers in the QTL candidate region on SSC1 for number of vertebrae

To define the region of reduced genetic variation, we isolated 11 novel microsatellite elements (underlined in Fig. 2D) by sequencing the 600-kb region between SJ854 and SJ872. Reanalysis with these 11 markers, in addition to the 24 markers described earlier, revealed that lack of genetic variation in commercial breeds occurred at 10 adjacent markers located in a 300-kb region between SJ641 and SJ820 (Fig. 2D,E; Table 1).

Candidate gene detection and polymorphism analysis

The region from SJ641 to SJ820 contained two nuclear receptor genes: NR5A1 (formerly AD4BP; adrenal 4-binding protein) and NR6A1 (formerly GCNF; germ cell nuclear factor) (Fig. 2D). NR5A1 participates in gonadal differentiation and steroidogenesis (Luo et al. 1994; Shen et al. 1994). NR6A1 is an orphan receptor that is expressed in the testis and ovary (Katz et al. 1997; Zhang et al. 1998; Yang et al. 2003). NR6A1 also is expressed in early embryos (Lan et al. 2002, 2003), and Nr6a1-deficient mouse embryos display serious defects in somitogenesis, generating a maximum of 13 (instead of 25) somites (Chung et al. 2001). We sequenced the coding regions of NR5A1 and NR6A1 from 11 European and 14 Asian breed pigs used as parents in the F2 experimental families in our previous study (Mikawa et al. 2005) and identified only one amino acid substitution, coincident with the QTL in NR6A1 (Fig. 3A) but not in NR5A1. This substitution (Pro192Leu; C → T at nucleotide 748 of AB248749) was fixed as leucine in the 194 pigs of commercial breeds in the DNA panel. Asian pigs had proline, which is conserved in human and mouse NR6A1. In the region between the NR5A1 and NR6A1 genes or their introns, several single-nucleotide substitutions were fixed alternatively in European and Asian breeds (data not shown), but we could not evaluate their functional association with the QTL effect.

Figure 3.
(A) Comparison of amino acid sequences of Nr6a1 hinge domains of pig, human, and mouse. We identified an amino acid substitution (Pro192Leu; C → T at nucleotide 748 of ...

Interaction of swine NR6A1 and corepressors

The amino acid substitution in NR6A1 was located in the hinge region between the DNA binding domain and the putative ligand-binding domain. The hinge domain of NR6A1 is reported to be essential for its interaction with the corepressors, nuclear receptor corepressor 1 (NCOR1) and nuclear receptor-associated protein 80 (RAP80) (Yan et al. 2002). To analyze the influence of Pro192Leu on the binding of NR6A1 to its corepressors we used two-hybrid systems. The pig leucine-type NR6A1 showed three times higher binding activity to pig RAP80 than did the proline-type protein (Fig. 3B). For NCOR1, the binding activity of the leucine form of NR6A1 was twice as high as that of the proline form when an NCOR1 C-terminal peptide (454 amino acid residues) containing the interacting domains (ID-I and ID-II [Yan and Jetten 2000]) was used (Fig. 3B).

Expression of Nr6a1 in mouse embryos

We analyzed the expression of Nr6a1 in mouse embryos (embryonic day [ED] 10.5). By using in situ hybridization, we detected only very faint signals for Nr6a1 mRNA in three tissues—the mandibular component of the first branchial arch, the lung bud, and the somites (Fig. 4), while its corepressors’ (Rxrip110, mouse homolog of RAP80, and Ncor1) mRNA were detected in a variety of tissues (Supplemental Fig. 1A,B). While the expression of Nr6a1 mRNA was so faint and hardly detectable, somites on both sides of the notochord were intensely immunostained for Nr6a1 protein (Fig. 5). Because of the many molecular mechanisms conserved in the embryonic development of mammals, we expect that the expression patterns of pig NR6A1 and its corepressors would be similar to those we noted in mice.

Figure 4.
Expression of Nr6a1 mRNA in embryonic day (ED)10.5 mouse embryos. Sense (A,B,D,F) and antisense (C,E,G) probe for Nr6a1 mRNA were hybridized to sections of ED10.5 mouse embryos. Faint signals for Nr6a1 mRNA were detected with antisense probe in the mandibular ...
Figure 5.
Immunohistochemical study of Nr6a1 protein in embryonic day (ED)10.5 mouse embryos. Frozen sections of ED10.5 mouse embryos were fixed by ethanol and incubated either with rabbit anti-Nr6a1 antibodies (A,B,D) or with rabbit IgG isolated from preimmune ...

Discussion

Domestic pigs in Europe have been under consistent selective breeding since the 19th century. European commercial breed pigs now grow faster and larger than Asian breeds or wild boars. The increase in vertebral number is a factor in these improvements. We suspected that selective breeding had reduced the genetic variation around the responsible locus, but we had not expected that fixation around the QTL for the number of vertebrae would affect such a wide (300-kb) region throughout a variety of commercial breeds. This result suggests that this QTL was the result of a single allele mutation rather than polymorphism and that the effect of the allele was very important for pig breeding. It is difficult to evaluate when and how the fixation occurred but it would occur at least before the establishment of present European commercial breeds (Large White, Yorkshire, Berkshire, Duroc, Hampshire, Landrace, etc.).

NR5A1 and NR6A1 are located in this 300-kb region. NR5A1 participates in gonadal differentiation and steroidogenesis and is expressed in primary steroidogenic tissues (Luo et al. 1994; Shen et al. 1994). Despite normal survival in utero, all Nr5a1-null mice die by postnatal day 8, and these animals lack adrenal glands and gonads and are severely deficient in corticosterone (Luo et al. 1994). Most of the in vivo function of NR6A1 is still unknown. Nr6a1 is expressed in the testis, ovary, and early embryos, and Nr6a1-deficient mouse embryos cannot survive beyond ED10.5, owing to cardiovascular defects and failure to establish an appropriate chorioallantoic connection. In addition, mutant embryos display serious defects in somitogenesis, generating a maximum of 13 (instead of 25) somites (Chung et al. 2001), although it is possible that malformation of somites was caused by the termination of other biological processes independent of somitogenesis. In Xenopus, NR6A1 may play a role in the formation of the anterior–posterior axis (David et al. 1998). In the human genome, a predicted gene, GPR144 (G protein-coupled receptor 144), is located proximal to NR5A1. We found that the pig genome contains GPR144-homologous sequences but not those corresponding to exons 1 and 20, where the start and stop codons, respectively, are located in the human homolog. Furthermore, in the pig sequences we found stop codons in the frame corresponding to the open reading frame of human GPR144. We therefore think that GPR144 is nonfunctional in pigs.

We identified an amino acid substitution (Pro192Leu) in the hinge region of NR6A1, and this substitution is coincident with the QTL. Nuclear receptors are reported to bind coregulators and function as repressors or activators. For example, retinoic-acid and thyroid-hormone receptors bind NCOR1 (Horlein et al. 1995) or NCOR2 (SMRT [silencing mediator of retinoic acid and thyroid hormone receptors]) (Chen and Evans 1995) as corepressors. Yan and Jetten (2000) reported that NR6A1 binds NCOR1 but not NCOR2; they also isolated a novel nuclear protein, RAP80, which interacts with NR6A1 as a corepressor. The hinge domain of NR6A1 is essential for its interaction with NCOR1 and RAP80 (Yan et al. 2002). The amino acid substitution (Pro182Leu) in the hinge regions altered the binding activity of NR6A1 to NCOR1 and RAP80, although the biological significance of this effect is unknown.

These findings, as well as our evaluation of Nr6a1 expression in the somites of ED10.5 mouse embryos, suggested that NR6A1 was a strong candidate for the QTL. Because our map-based study excluded the promoter region of NR6A1 and because Pro192Leu was a functional polymorphism, this mutation may be the cause of the QTL. However, this hypothesis cannot exclude the possibility of single-nucleotide polymorphisms (such as those in introns or unknown genes including those for noncoding RNAs) that alter the regulation of expression of the QTL.

In pigs as livestock, meat productivity is associated with vertebral number; therefore isolation of the QTL for vertebral number is valuable. The information we present likely will be useful in breeding and construction of new pig lines, especially when Asian pigs are used as genetic resources. Furthermore, we propose a putative functional role for NR6A1 in somitogenesis. Vertebral morphology is associated with the expression pattern of Hox genes (Burke et al. 1995). Disruption of Gdf11, Mll, and Bmi1, which are located upstream of the Hox genes, shifts the expression boundaries of the Hox genes and alters vertebral morphology (Yu et al. 1995; Hanson et al. 1999; McPherron et al. 1999; Dubrulle et al. 2001). The “segmentation clock” is also essential in directing vertebral morphology and is closely linked to the Notch and Wnt signaling pathways (Cordes et al. 2004). The mouse mutation vestigial tail (vt) shows reduced transcription of Wnt3a and altered vertebral morphology (Greco et al. 1996). We think it will be important to investigate the involvement of NR6A1 in Hox expression patterning and regulation of the segmentation clock.

Recently, some progress has been made in understanding the targets of NR6A1 in the developmental process of early embryos. Sato et al. (2006) reported that NR6A1 binds the DR0 element (Chen et al. 1994) in the promoter region of Oct3/4 and recruits DNA methyltransferase for Oct3/4 silencing. OCT3/4 is required for early embryonic cells to maintain pluripotency (Nichols et al. 1998) and also plays a crucial role in the specification of the first embryonic lineage (Niwa et al. 2000). Hentschke et al. (2006) reported that NR6A1 is a repressor of CRIPTO, a coreceptor for the morphogen nodal. NR6A1-mediated repression of the CRIPTO promoter is also dependent upon the DR0 site. CRIPTO is required for correct orientation of the anterior–posterior axis in the mouse embryo (Ding et al. 1998). NR6A1 was originally identified as the GCNF expressed predominantly in germ line, but now its importance in embryonic development has been realized. We think that these findings support the idea that NR6A1 is a strong candidate of the QTL for the number of vertebrae.

Methods

QTL scanning for number of vertebrae on SSC1

A QTL scan on SSC1 was performed for the number of vertebrae in both the Large White × Japanese wild boar population and the Jinhua × Duroc cross population. An interval mapping based on the least-squares method developed for outbred population (Haley et al. 1994) was used, whereby a QTL was scanned every 0.1 cM on SSC1. At a QTL, we assumed that the grandparental breeds were fixed for alternative alleles, Q and q. Denoting the effect of QQ as a, the effect of Qq as d, and the effect of qq as –a, the phenotypic value of the ith F2 individual, yi, can be expressed as the following linear model:

equation image

where μ is the intercept of the model; ui is the coefficient for the additive effect of a putative QTL for the ith individual, which [denoting the probability of an individual being genotype AB as Pr(AB)] is given as Pr(QQ) – Pr(qq); vi is the coefficient for the dominance effect of a putative QTL, which is equal to Pr(Qq); and ei is the residual error. This model was fitted in the analysis of a QTL on SSC1 for a Large White × Japanese wild boar population in which only the QTL on SSC1 was found to segregate in our previous study. For another F2 population, i.e., a Jinhua × Duroc population in which two QTL had been detected, on SSC1 and SSC7, in our previous study, analysis of the QTL on SSC1 was performed using the modified model, which incorporated the genotype of a microsatellite marker (SW252) nearest to the QTL position on SSC7 as a covariate. The modified model can be written as

equation image

where wi is the coefficient for the additive effect of a QTL on SSC7, which is given as Pr(QQ) – Pr(qq) assuming that the QTL is located close to SW252; and b is the additive effect of the QTL. The results of our previous analysis suggested that the QTL on SSC7 was additive; therefore, we considered only the additive effect of the QTL.

For both populations, 95% confidence intervals for the QTL on SSC1 were obtained by bootstrap analysis of 1000 repetitions (Visscher et al. 1996). In these analyses, the map positions of our linkage map (Mikawa et al. 2004) were used.

Isolation of bacterial artificial chromosomes (BAC) clones and development of microsatellite markers

The QTL region of SSC1qter corresponds to a region of the human chromosome 9ter (Mikawa et al. 2004). Swine sequence-tagged sites (STSs) were developed from swine expressed sequence tags (ESTs) or swine whole-genome shotgun sequences, which were obtained by BLAST searches with the human gene sequences on the chromosome 9 and then confirmed to be the targeted sequences by using BLAST searches inversely against the human genome (Supplemental Table 1). BAC clones (Suzuki et al. 2000) were isolated with swine STS and BAC end sequences were used for chromosome walking. Microsatellite sequences were isolated from BAC clones by using a direct sequencing method reported previously (Fujishima-Kanaya et al. 2003). Genotyping of microsatellite markers was performed with the ABI Prism 3700 DNA Analyzer and GeneScan analysis software (Applied Biosystems).

Swine genomic DNA panel

For analysis of genetic variation in pigs, a DNA panel was constructed from 194 samples of European commercial-breed pigs and 40 samples of Asian local-breed pigs. For Berkshire samples (n = 76), DNA was prepared from samples of pork meat produced in Japan, the United States, and the United Kingdom. For samples from Duroc (n = 52), Landrace (n = 17), Large White (n = 23), and Yorkshire (n = 7) breeds, DNA was prepared from semen provided for artificial insemination in Japan. The donor male pigs were confirmed to be without consanguinity for three generations. For Hampshire breed samples (n = 19), DNA was prepared from animals bred at the experimental stations of Kumamoto and Okinawa prefectures in Japan. For Meishan samples (n = 18), DNA was prepared from animals bred at the National Institute of Livestock Breeding, Japan. For Jinhua samples (n = 12), DNA was prepared from animals bred at the Shizuoka Swine and Poultry Experiment Station in Japan. For Japanese wild boars, DNA was prepared from 10 animals from six different prefectures.

Plasmid construction for two-hybrid analysis

Swine RAP80 cDNA (AB248750) was cloned from testis cDNA by using the 5′RACE System (Invitrogen) and RT-PCR with primers designed from swine EST (BX923822, CF180155) homologous to human RAP80 cDNA. Swine NCOR1 partial cDNA (AB248751) containing ID-I and ID-II (Yan and Jetten 2000) was cloned using the 3′RACE System (Invitrogen) from the sequence of swine EST (BX915421, AU296541) homologous to human NCOR1. These DNA fragments were inserted into pBIND vector, in which the Renilla luciferase gene was located as an internal control (CheckMate Mammalian Two-Hybrid System, Promega). Proline-type and leucine-type NR6A1 cDNA (AB248749) were cloned from testis cDNA by using RT-PCR. From the NR6A1 cDNA, DNA fragments spanning the region from the hinge domain to the C terminus (nucleotides 687–1613 of AB248749, equivalent to the ΔN1 construct (Yan et al. 2002), were inserted into pACT vector (CheckMate Mammalian Two-Hybrid System, Promega).

Mammalian two-hybrid analysis

CHO cells (2 × 105/well) were plated in six-well dishes and 20 h later transfected in F12 medium with a reporter plasmid, pG5luc (Promega), in which the firefly luciferase gene was located, and with pACT and pBIND expression plasmids (each 0.5 μg) by using 3 μl of FuGENE 6 transfection reagent (Roche Molecular Biochemicals). Cells were collected 48 h after transfection and assayed for both firefly and Renilla luciferases by using a Dual-Glo Luciferase Reporter Assay System (Promega). Renilla luciferase activity was used as an internal control to monitor transfection efficiency. Transfections were performed in triplicate, and each experiment was repeated three times.

In situ hybridization of Nr6a1 and corepressors in mouse embryo

In situ hybridization on mouse embryo (ED10.5) sections was performed as described previously (Hoshino et al. 1999). Mouse embryos were fixed in 4% paraformaldehyde and dehydrated in a step-wise manner with ethanol. Sections (6 μm) were made from wax-embedded preparations. For Nr6a1 probes, 1586–2084 bp of NM_010264 was amplified from testis RNA by PCR primers with T7 or SP6 promoter sequences. For probes to Ncor1 and Rxrip110 (mouse homolog of RAP80), 2319–2739 bp of NM_011308 and 467–893 bp of NM_011307 were amplified by PCR, respectively, as well. Digoxygenin-labeled RNA probes were prepared by in vitro transcription with a DIG RNA Labeling Mix (Roche Molecular Biochemicals). Hybridization was performed with probes at concentrations of 200–500 ng/mL in hybridization solution (50% formamide, 5× SSC, 1% SDS, 50 μg/mL tRNA, and 50 μg/mL heparin) at 55°C for 16 h. After hybridization, the specimens were washed, and this was followed by RNase treatment. After treatment with 0.5% blocking reagent (Roche Molecular Biochemicals) in TBST for 1 h, the samples were incubated with anti-DIG AP conjugate (Roche Molecular Biochemicals), and staining reactions were performed with NBT/BCIP (Roche Molecular Biochemicals), followed by counterstaining with nuclear fast red (Sigma-Aldrich).

Anti-NR6A1 polyclonal antibodies and immunohistochemistry

A NR6A1-specific polypeptide (amino acids 36–50, CQDELAELDPSTISV) was synthesized, purified by HPLC, conjugated to keyhole limpet hemocyanin, and then injected into rabbits to generate rabbit anti-NR6A1 antibodies. Preimmune and hyperimmune sera were collected and passed through HiTrap protein A HP affinity columns (Amersham) to generate rabbit preimmune immunoglobulin G (IgG) and rabbit anti-NR6A1 IgG, respectively. Frozen sections of mouse embryos (ED10.5) were fixed in ethanol at 4°C for 10 min, dried, and washed with distilled water. Samples were incubated for 2 h either with the preimmune IgG or with purified anti-NR6A1 antibodies. After extensive washing, these samples were incubated with biotinylated goat anti-rabbit secondary antibodies followed by horseradish peroxidase–streptavidin complex. Positive signals were visualized by incubation in peroxidase substrate using diaminobenzidine as the chromogen. Samples were then counterstained in 0.05% (wt/vol) methyl green (Sigma-Aldrich).

Acknowledgments

We thank A. Horiuchi, T. Yamaguchi, and Y. Nakazawa for construction of the Jinhua × Duroc population; M. Nii for construction of the Large White × Japanese wild boar population; and members of the DNA Marker Project and the Animal Genome Research Program (NIAS/STAFF) for their many years of support. This work was supported by grants to the Animal Genome and DNA Marker projects from the Ministry of Agriculture, Forestry and Fisheries, Japan, and by a grant-in-aid from the Japan Racing Association.

Footnotes

[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to EMBL/GenBank/DDBJ under accession nos. AB248749-AB248751, AP009124; accession nos. for STS are given in Supplemental Table 1].

Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.6085507

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