Sequencing , Genomic Structure , Chromosomal Mapping and Association Study of the Porcine ADAMTS 1 Gene with Litter Size

A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif (ADAMTS1) plays a critical role in follicular rupture and represents a major advance in the proteolytic events that control ovulation. In this study, a 9,026-bp DNA sequence containing the full coding region, all 8 introns and part of the 5’and 3’ untranslated region of the porcine ADAMTS1 gene was obtained. Analysis of the ADAMTS1 gene using the porcine radiation hybrid panel indicated that pig ADAMTS1 is closely linkage with microsatellite marker S0215, located on SSC13q49. The open reading frame of its cDNA covered 2,844 bp and encoded 947 amino acids. The coding region of porcine ADAMTS1 as determined by sequence alignments shared 85% and 81% identity with human and mouse cDNAs, respectively. The deduced protein contained 947 amino acids showing 85% sequence similarity both to the human and mouse proteins, respectively. Comparative sequencing of three pig breeds revealed one single nucleotide polymorphism (SNP) within exon 7 of which a G-C substitution at position 6006 changes a codon for arginine into a codon for proline. The substitution was situated within a PvuII recognition site and developed as a PCR-RFLP marker for further use in population variation investigations and association analysis with litter size. Allele frequencies of this SNP were investigated in seven pig breeds/lines. An association analysis in a new Qingping female line suggested that different ADAMTS1 genotypes have significant differences in litter size (p<0.01). (


INTRODUCTION
A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif (ADAMTS) is a novel family of extracellular proteases found in both mammals and invertebrates (Tang, 2001).ADAMTS1 was first cloned in the cachexigenic colon 26 adenocarcinoma subline in vivo (Kuno et al., 1996).Later research showed that ADAMTS1 plays an important role in follicles via progesterone receptor (PR)-dependent pathways (Robker et al., 2000).Female fertility is impaired in ADAMTS1 knockout mice, and this is accompanied by obvious abnormalities of the uterus and ovaries (Shindo et al., 2000).In addition, there is evidence that progesterone-and PR-dependent functions in cumulus cells are essential for expansion of cumulus-oocyte complexes (COC) in the pig, possibly through the action of ADAMTS1 (Shimada et al., 2005).All these data suggest that ADAMTS1 plays a critical role in follicular rupture and represents a major advance in understanding of the proteolytic events that control ovulation.
Variations in ADAMTS1 gene could be used as genetic markers to select animals for breeding.However, there is still no report on how a genetic variant of this gene affects litter size in pigs.The purpose of our research was to characterise the pig ADAMTS1 gene and determine whether single nucleotide polymorphisms (SNP) of exon 7 in porcine ADAMTS1 gene are associated with litter size.

Experimental animals
In this study, 6 pig breeds were used for allele

ABSTRACT :
A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif (ADAMTS1) plays a critical role in follicular rupture and represents a major advance in the proteolytic events that control ovulation.In this study, a 9,026-bp DNA sequence containing the full coding region, all 8 introns and part of the 5'and 3' untranslated region of the porcine ADAMTS1 gene was obtained.Analysis of the ADAMTS1 gene using the porcine radiation hybrid panel indicated that pig ADAMTS1 is closely linkage with microsatellite marker S0215, located on SSC13q49.The open reading frame of its cDNA covered 2,844 bp and encoded 947 amino acids.The coding region of porcine ADAMTS1 as determined by sequence alignments shared 85% and 81% identity with human and mouse cDNAs, respectively.The deduced protein contained 947 amino acids showing 85% sequence similarity both to the human and mouse proteins, respectively.Comparative sequencing of three pig breeds revealed one single nucleotide polymorphism (SNP) within exon 7 of which a G-C substitution at position 6006 changes a codon for arginine into a codon for proline.The new Qingping female line pigs that was established by mating five Duroc boars to twenty-five Qingping sows were selected from Hubei province in China.116 new Qingping female line pigs, which were 1 to 2 years old, were selected for association analysis.A total of 247 litter records included total number born (TNB) and the recorded number of piglets born alive (NBA) averaged from 1 to 4 parities.

DNA preparation
Genomic DNA was isolated from white blood cells as described by Xiong (1999).After isolation, the DNA pellet was dissolved in TE buffer and stored at -20°C.

Primer design, polymerase chain reaction (PCR) and sequencing
The sequences of cDNAs of human and mouse ADAMTS1 genes (GenBank accession numbers: NM_006988 and NM_009621, respectively) were used to search against the porcine EST databases by BLAST (http://www.ncbi.nlm.nih.gov/blast/).Six porcine ESTs (GenBank accession numbers: BP440774, AJ683364, DN127702, BP458319, CJ018555 and BX919340) sharing >80% identity to the corresponding human and mouse cDNAs were obtained and assembled into a contig.Based on the contig sequence, a set of primers was designed to amplify the DNA sequence of porcine ADAMTS1 gene (Table 1).The PCR was performed in 25 μl reaction volume containing: 1×PCR buffer, 1.5 mM MgCl 2 , 250 μmol/L dNTP, 5 ppm of each PCR primer, 2 units Taq DNA polymerase (Biostar International, Canada) and 200 ng genomic DNA as template.PCR was run in the GeneAmp PCR system 9600 (Perkin-Elmer Co., USA).The conditions for PCR were as follows: initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 40 sec, annealing temperature for 40 sec, 72°C for 1 min and a final extension for 10 min at 72°C.The purified PCR products were cloned into the pGEM-T vector (Promega, USA) and sequenced.These primers yielded twelve overlapping PCR products that produced a consensus of 9,026-bp DNA sequence of pig ADAMTS1gene (Genbank accession number DQ177331).

Chromosomal localization of ADAMTS1
The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel consisting of 118 hybrid clones (Yerle et al., 1998) was employed for localization of ADAMTS1 to the pig chromosome.Primer pair 10 (Table 1) yielded a 703-bp product designed for mapping.The conditions for PCR were as follows: initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 40 sec, 59°C for 40 sec, 72°C for 1 min and a final extension for 10 min at 72°C.The PCR fragment was sequenced to verify the correctness of the sequence.PCR products were separated on a 2% agarose gel stained with 0.5 μg/ml ethidium bromide.The radiation hybrid PCR was analyzed with the IMpRH mapping tool (Milan et al., 2000) available from http://imprh.toulouse.inra.fr/.

Detection of PCR-PvuII-RFLP
Comparison of the sequences of ADAMTS1 in different pig breeds by using BLAST (http://www.ncbi.nlm.nih.gov)revealed one SNP within exon 7 that spanned a PvuII restriction site and comprised a G-C substitution at position 6006 which changed the codon for arginine into proline.For the PCR-RFLP assays, 8.5 μl of PCR products were digested with 5 units PvuII (TaKaRa, Tokyo, Japan) in 1×digestion buffer added in a total volume of 10 μl which, following digestion for 4 h at 37°C, was electrophoresed on a 1.5% agarose gel and visualized under UV light.

Statistical analysis
A total of 247 litter records from 116 sows were used for association analyses between different genotypes and litter sizes.TNB and NBA were analyzed with a model including fixed effects of month of farrowing, ADAMTS1 genotype, random effects of animal, parity effect and residual (Lukovic et al., 2007).The association between genotypes and recorded traits was analyzed using the general linear model (GLM) procedure (SAS Institute Inc., Cary, NC, USA).

Cloning, sequencing and genomic organization of ADAMTS1
A 9,026-bp DNA containing the full coding region, all 8 introns and part of the 5'and 3' untranslated region of the porcine ADAMTS-1 gene was obtained (Genbank accession number DQ177331).
By comparing with human ADAMTS1 sequence (NT_011512), the exon/intron boundaries of porcine ADAMTS1 gene have been determined.The sequences of exon/intron boundaries are consistent with the reported consensus sequences for splice donors (GT) and acceptors (AG) (Breathnach and Chambon, 1981) as shown in Table 2.The open reading frame of ADAMTS1 cDNA covered 2,844 bp and encoded 947 amino acids with a calculated molecular mass of 103 kDa.The coding region of porcine ADAMTS1 as determined by alignments shared 85% and 81% identity with human and mouse cDNAs, respectively, while the 947 deduced amino acids showed 85% sequence similarity both to the human and mouse proteins, respectively.
The hydropathy plot and the TMAP prediction derived from the World-Wide Web service showed that porcine ADAMTS1 protein included no transmembrane region and  there were many cysteine residues and four putative Nglycosylation sites in the protein which indicated that the ADAMTS1 gene product was a putative cysteine-rich secretory glycoprotein.Sequence analyses of porcine ADAMTS1 protein by ExPASy (http://au.expasy.org/)revealed that the porcine ADAMTS1 protein consisted of one disintegrinlike domain, one zinc-dependent metalloproteinase and three thrombospondin type I repeats (TSP1) (Figure 1).All these structural domains have very high similarity (>90%) among mammalian species by ClustalW website (http://www.ebi.ac.uk/clustalw/index.html).

Chromosomal mapping of pig ADAMTS1
Using the ImpRH panel, we determined that pig ADAMTS-1 was closely linked with microsatellite marker S0215 on SSC13q49 (distance of 76cR, LOD = 4.18, retention frequency = 18%) (Figure 2).The ADAMTS-1 gene has been mapped to chromosome 21q21-q22 in the human (Hurskainen et al., 1999).Our results are in agreement with comparative mapping data, as human chromosome 21q21-q22 is a syntenic region of porcine chromosome 13q49.

Frequencies of allele and genotype of different pig breeds
The digested products (pair 8) showed allelic fragments of 413 bp (allele A) or 314+99 bp (allele B) (Figure 3).The allele frequency analysis included 349 unrelated animals from seven breeds/lines (Table 3).

Association analysis
Results of tests for ADAMTS1 genotypes and litter size traits on the new Qingping female line are given in Table 4. Pigs with GG genotype had less TBA and NBA than pigs with CC genotype (p<0.05) and GC genotype (p<0.01),respectively.The results indicated that GC sows showed the highest and GG sows showed the lowest litter size values in multiple parities.

DISCUSSION
In this study, the candidate gene approach was used to  demonstrate associations between specific genes and litter size.Using this approach, polymorphisms in some defined genes such as the estrogen receptor (Li et al., 2004), follicle-stimulating hormone-β (Li et al., 2002) and Oviduct-specific Glycoprotein 1 (Niu et al., 2006) have all been reported to be associated with litter size in swine.So the candidate gene approach is a very efficacious method to find molecular markers.
ADAMTS1 plays an important role in follicles via progesterone receptor (PR)-dependent pathways which control ovulation (Robker et al., 2000).However, there have been no reports of the sequence, mapping and association study of ADAMTS1 gene in pigs.In our study ADAMTS1 gene was selected as a candidate gene to be associated with litter size in swine.
The genetic effect on reproductive traits was investigated in the new Qingping female line sows.Based on the data representing litter records from new Qingping female line sows, the NBA and TNB in multiple parities were higher for GC sows than for the sows of the other two genotypes.In view of these observations, it is tempting to suggest that GC sows have better performance than GG sows for litter size traits in Qingping pigs.
Nucleotide mutation can alter gene function, especially changing the coding region of the protein.In the experiments reported here, a polymorphism analyzed in ADAMTS1 gene was found in exon 7 which changes a codon for arginine into a codon for proline.
In this study, we determined that pig ADAMTS1 is closely linkage with microsatellite marker S0215 on SSC13q49.So far, two quantitative trait loci (QTLs) have been reported which contributed to the reproductive traits on Pig Chromosome 13.One of these contributed to ovulation rate (Rathje et al., 1997); the other contributed to number of stillborn (Cassady et al., 2001).Although ADAMTS1 gene was not located on either of these two QTLs, it is possible that ADAMTS1 is a genetic marker linked with other QTLs which contribute to the reproductive traits.This is the initial step to consider ADAMTS1 gene as a candidate gene for litter size.This polymorphism could be a potential genetic marker for litter size.The estimations presented are based on data from the new Qingping female line; however, in this study the populations are too small and the number of animals examined is limited.So, analyzing more animals is necessary to confirm the association between the ADAMTS1 genotype and reproductive traits in other purebreds and crossbreds.

Table 1 .
Primer pairs used in the analysis of the porcine ADAMTS1 gene

Table 3 .
Allele frequencies of the ADAMTS1 C6006G SNP in ten pig breeds/lines

Table 4 .
Effect of the ADAMTS1 genotypes on reproductive traits in new Qingping female line pigs Total number of pigs observed.Letter denoting significant difference between groups: a, b , p<0.05; A, B p<0.01.