Structural analysis and haplotype diversity in swine LEP and MC4R genes

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1 J. Anim. Breed. Genet. ISSN - OIGINAL ATICLE Structural analysis and haplotype diversity in swine LEP and MC genes M. D Andrea, F. Pilla, E. Giuffra, D. Waddington & A.L. Archibald University of Molise, Dip. S.A.V.A., Campobasso, Italy Parco Tecnologico Padano CESA, Via A. Einstein, Lodi, Italy Division of Genetics and Genomics, oslin Institute (Edinburgh), oslin Midlothian, Scotland, UK Keywords Casertana pig breed; haplotypes; leptin; melanocortin- receptor. Correspondence Mariasilvia D Andrea, University of Molise, Dip. S.A.V.A., Via De Sanctis snc, Campobasso, Italy. Tel: + ; Fax: + ; dandrea@unimol.it eceived: February ; accepted: September Summary Knowledge about structural variation of candidate genes could be important to improve breeding selection scheme and preserve genetic variability in livestock species. Leptin (LEP) and melanocortin- receptor (MC) genes are involved in the energetic pathway and are obvious candidate genes for fatness. By sequencing LEP and MC genes in pigs belonging to lean (Large White and Duroc), fat (Meishan and Casertana) breeds and also Wild Boar, polymorphic sites, of which were novel, were found in the Leptin sequence while only the previously described mutation was found in the MC gene. A total of LEP haplotypes were observed and their distribution was unequal among the breeds. The phylogenetic analysis showed two haplotype branches distinguishing between lean and fat breeds. Introduction The analysis of genetic variability of livestock reflects their evolution and provides valuable information for breed conservation and management (Bruford et al. ). Moreover, knowledge about structural genetic variability in candidate genes for economically important traits, can contribute to understanding the genetic control of such complex traits. The leptin gene (LEP) is the homologue of the murine Lep gene also known as Obese or Ob. LEP encodes a protein of amino acids expressed in adipose tissue, which plays a key role in food intake. Genetic defects in the murine and human leptin genes are associated with obesity traits (Zhang et al. ). The function of leptin has been studied in other species, where a relationship between a positive energy balance and circulating levels of leptin has been observed (Cusin et al. ; Kolaczynski et al. ; Barb et al. a,b). In swine, seven polymorphisms were found in the LEP gene, and association analyses with production traits have produced differing results (Jiang & Gibson ; Kennes et al. ; Chen et al. ; Szydlowski et al. ; de Oliveira Peixoto et al. ). In concert with leptin, the melanocortin- receptor (MC) has a key role in regulating feed intake and energy balance (Kim et al. ; Huston et al. ). The missense mutation AspAsn originally described by Kim et al. () has been tested for associations with performance in different pig populations (Kim et al. ; Park et al. ; Stachowiak et al. ; Bruun et al. ; Jokubka et al. ; Kim et al. ; Meidtner et al. ) but with conflicting results that brought some authors to conclude that either MC could not be the causative mutation but it is closely related to the true quantitative trait nucleotide (QTN) or there may be an epistatic interaction between quantitative trait loci (QTL) (Bruun et al. ). Beside all, it is clear that MC is associated with growth and leanness in most populations. The Casertana (CT) pig is a local breed from the Southern Italy characterized by slow growth and massive accumulation of backfat (Pietrolà et al. ). It has been dismissed mainly for economic reasons, Journal compilation ª Blackwell Verlag, Berlin. No claim to original US government works J. Anim. Breed. Genet. ()

2 M. D Andrea et al. Structural analysis and haplotype diversity in swine LEP and MC genes i.e. because of its long time to reach its mature body weight and preference of consumers for lean meat. As an autochthonous breed it could be linked to specialized local products as a result of tradition and practice; moreover it represents useful genotypes and may be a reservoir of alleles that could be important for future breeding objectives and research. In this paper, we explored the genetic variation of LEP and MC segregating in CT versus Large White (LW), a major commercial breed. Additionally, two reference sequences from Duroc (DU), Meishan (Me) and Wild Boar (WB) were included in the database. Material and methods LEP and MC sequences were successfully obtained from animals, including LW; CT; two WB, two DU and two Me pigs. DNA was isolated from blood using a phenol chloroform method. To analyse the seven LEP and one MC previously identified polymorphic positions, the primers and the amplification conditions were carried as described previously (Jiang & Gibson ; Kim et al. ; Kennes et al. ). Moreover, to obtain the complete sequence of the two genes, LEP and MC overlapping PC primers were designed using the primer software ( on the EMBL-Bank (European Molecular Biology Laboratory Nucleotide Sequence Database) LEP U and AF sequences and MC AB sequence. The amplifications were conducted in ll using ng of genomic DNA, x buffer,. mm MgCl,. mm dntp,. lm of each primer and. U Taq polymerase (oche Taq DNA Polymerase, oche Diagnostic, Pleasanton, CA, USA). The thermal cycling conditions were different in accordance with the primers. In Table are reported the different primers used to amplify LEP (PLF to PbisL) and MC (PMF to PM) and some specific Leptin sequence primers necessary to overcome the sequence problems in samples from animals heterozygous for ins/ (PF to P). Moreover, the amplified fragment sizes and their localization within the genes are reported (Table ). The amplicon products were purified using the QIAquick PC purification kit (Qiagen, Inc., Valencia, CA, USA) and sequenced by standard protocols on a ABI PISM (Applied Biosystems, Foster City, CA, USA). The sequences were aligned to the reference swine sequences in the NCBI database (LEP: U and AF; MC: AB). The NCBI Map Viewer web resource ( mapview) was used to scan for mutations in functional sites. Haplotype analysis was performed using phase version. program (Stephens et al. ; Stephens & Scheet ). Phylogenetic and molecular evolutionary analyses were conducted using mega software version. with Maximum Parsimony and bootstrap options (Kumar et al. ). esults and discussion A total of bp were sequenced for the LEP gene, which included intron, exon, intron, exon and the UT known in swine (Soares & Guimarães ), and a total of polymorphisms were detected including single nucleotide polymorphisms (SNPs) of which only seven had been previously reported in pigs (Jiang & Gibson ; Kennes et al. ), five etions (nucleotide position: C, T, AAG, AG and C of the reference sequence U) and four insertions one of which was also associated with an inversion (nucleotide position: inscccinvtgc, AA, GGGTGGACG- TGG, AA of the reference sequence U) never reported before. All the new polymorphisms were found in either introns or the UT region. The CT LEP gene sequence containing all the polymorphisms has been deposited in the EMBL data base; accession number: AJ. None of the polymorphic LEP sites were revealed as potentially functional using the NCBI Map Viewer analysis. A total of haplotypes were inferred by phase (Tables and ). Despite the high number of mutations, only five haplotypes (A, B, C, D, E) were represented more than once in the CT and LW animals (Table ). The B haplotype was found only in CT. The unrooted tree showed two major branches (L is the third), where haplotypes B and closely related variants, including three Me variants, cluster together (Figure ). The B and E haplotypes could be considered as the CT haplotypes with the presence of the A, C and N haplotypes in CT animals most likely to have arisen from crossbreeding with other breeds to improve performance. Several of the nodes in the tree are not well supported by the bootstrap testing, but the tree formed from the five multiply represented haplotypes alone has two tight clusters (B, E) and (A, C, D), supported by / bootstrap samples (not presented). Casertana and LW are mostly represented by the A, B, C and D haplotypes, but with very different frequencies, with the B haplotype only found in the CT breed. This finding reflects population history and bottleneck effects typical of small traditional populations (S. Palermo, E. Capra, M. Torremorrell, M. Dolzan,. Davoli, C.S. Haley & E. Giuffra, in Journal compilation ª Blackwell Verlag, Berlin. No claim to original US government works J. Anim. Breed. Genet. ()

3 Structural analysis and haplotype diversity in swine LEP and MC genes M. D Andrea et al. Table LEP and MC amplification and sequencing primers, fragment sizes and localization Amplification primers Sequence Length in bp and gene position of the amplicon eference sequence Annealing condition PLF ATA CCC AGC CCA GGG GAC int U Touch down PL TCT CCA GGC TTT TAT GAG GA U C for s PLF TGA TCC TCA TAA AAG CCT GGA int & ex U PL TCC TGG TGA CAA TCG TCT TG U PLF GTT TCC AGG CCC CAG AAG ex U PL GAA ATG TCA CTG ATC CTG GTG A U PLF AGG GTC ACC GGT TTG GAC T ex part. U PL ACC ACC TCC GTG GAG TAG AG U PLF AAG CCT CCC TCT ACT CCA CG part. ex & UT U PL AAA GGC TGG TGT TTT GCT TC U PLF AAG CAA AAC ACC AGC CTT TC UT U PL GGG GCT GAG CAC AAT AGA TG U PbisLF TGA CAC CAA AAC CCT CAT CA part. ex & int U Touch down PbisL TCA GCT GTC ACC AGG AAG AA U C for s PbisLF TCT TCC TGG TGA CAG CTG AA int U PbisL GTC TGT GCT GGG AGC TGT CT U PbisLF GCC CAT GTT CCC ACA CTA AC int U PbisL CAA AGC CAC AAC CGA AAA CT U PbisLF GTG GCT TTG ATA GCA CCC AG int U PbisL CAG CCA CGA CTG TCT GTT TC U PbisLF ACA GAC AGT CGT GGC TGG TT int & part. ex U PbisL TTT CTG GAA GGC AGA CTG GT U PbisLF AAA CAA GGA GGC ATG GGT TT UT U PbisL GCT ATC CTG CTT CAA AGG GA U PbisLF TCC CTT TGA AGC AGG ATA GC UT U PbisL CAA ACA AAA CAG CCT CCT CC U PLF AAG TGT TTG CTG GAA GAG CG UT AF PL TTT CAG GGG GCA AAG GTA AT AF PLF TGC AGA CAG CTC CGA TTA GA UT U PL GGA CAC GCT GGA TCT GTC AT U PbisLF TTA CAG GAA GGC AGA CAG CTC int U Touch down PbisL GTT AGT GTG GGA ACA TGG GC U C for s PbisLF ATC AAG CAG GGT TCC ATC TG UT U PbisL CCC ATG GAT GGT ACT GGA AA U PMF AAA GAA GCA GAG GAG GAG CC UT AB Touch down PM CCT CAG CGA TTT TCT CCA AG AB C for s PMF CTT GGA GAA AAT CGC TGA GG part. UT - ex AB PM GTG CAG ACT GCC CAG ATA CA AB PMF CCC CTT GGA AAA GGC TAC TC part. ex AB PM GAC AAA TCA CAG AGG CCA CC AB PMF ATG TTC CTC ATG GCC AGA CT part. ex UT AB PM CAG GGA GAA TGA GCA TGGT TTT AB Further LEP primers specific for sequencing Sequence Sequence eference sequence PF TTA TCC TCC TTC TTC CC U CTA CAG ATT AGA ACA TTC C U P GTA GAG GGT TGT ATA GG U PF AGA AGA GGC ATC TGG AG U P GTT TGG AGG AGA CAG A U PF AGG AAGTGT GTT GGT GG U P GAC CAT CTG CTA AAG CC U Journal compilation ª Blackwell Verlag, Berlin. No claim to original US government works J. Anim. Breed. Genet. ()

4 M. D Andrea et al. Structural analysis and haplotype diversity in swine LEP and MC genes Table LEP haplotypes in the population analysed: CT, LW, Me, D, WB intron intron ex UT HAP INS INV ins ins A T C C A A T T C G G n n G T G C C T G G G G C G A G A A A T C C A G A G n A C C C G T C G C C T n G G n C T T T C G C G G B G T * G G C C T * * y y A C A T T C A * * * * * * * * * * C * * G A G A y T * * T * G * * * T G y A * y A C * * T * T * * C T C * A A T T C * * n n G T G C C T G * * * * * * * * * * T * * A G A G n A * A C * T * * * C T n G * n C T * * C * C * * D * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * C * * * * * * * * * * * * * * C * * * * * * E G * * G G C C T * * y y A C A T T C A * * * * * * * * * * C * * G A G A y T * * T * G * * * T G y A * y A C T * T * T * * F * * * A A T T C T * n n G T G C C T G T A * G A G * G C G * * A A * * * * * T * * * * T T T * * n G A n C T C * C A * * C G * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * H * * * G G C C T G * y y A C A T T C A G G * C G A * A A A T * C * G A G n A C * C * T C G C C T * * G * * * T * * G C * G I * * * * * * * * * * * * * * * * * * * * * * * A G * G C G C * A * * * * * T * * * A G * * * T G * * A * * * C * * A T * C J * * * * * * * * * * n n G T G * * * * * * A * G A * A A A * * C G * * * * A * A * G T * * * C T * * G * * * T * * G C * G K * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * y * * C * * * * * * * * y * * * * * * * T * T * * L * * T A A T T C * * * * * * * C C T G * * G * A G A G * * * T * A * * * n T * * * A G * T T T G n * A * * * C * C * C A * M T * C * * * * * * * y y A C A T T C A * * * * G A G A * * T C * * * * * * A * * * G T * G C C T * * G * * * T * * * * G * N * * * * * * * * * * n n G T G C C T G * * * * * * * * * * C * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * O * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * T * * * A G * T T T G * * * * * * * * * * * * * P * * * * * * * * * * * * * * * * * * * * * * * * * * * * * T * * * * * * * A * * * G T * G C C T * * * * * * * * T * T * * Q * * * * * * * * * * * * * * * * * * * * * * * * * * * C G * * * * * * * * * * A * * * * * * * * * * * * * * * * C * C * * * * * * * * * * * * * * * * * * * * * * * * G * * * * A A * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * LEP, leptin gene; CT, Casertena; LW, Large White; Me, Meishan; WB, Wild Boar. Position referred to the sequence U. *Matches at the same position with the previous nucleotide or, etions and insertion. y ¼ Presence of mutation. n ¼ Absence of mutation. Journal compilation ª Blackwell Verlag, Berlin. No claim to original US government works J. Anim. Breed. Genet. ()

5 Structural analysis and haplotype diversity in swine LEP and MC genes M. D Andrea et al. Table LEP haplotypes in the population analysed: CT, LW, Me, D, WB UT H A P ins CT LW Me D WB Total A G T C T T T C G G G G n C A T n T T C C G n T C G n A G C G C G C G A C C B * C G C * * T A A A A y T G G y C C * * A y C T A y * * * A T A A A G T T C * T C T * * C G G G G n C A T n T T * * G n T C G n * * * G C G C G A C C D * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * E * C G C * * T * A A A y T G G y C C * * A y C T A y * * * A T A A A G T T F * * * * * * * A * * * * * * * * * * * * G * * C G n G A T G * * * * * * * G * * * * * * * * * * * * * * * * * * T * * * * * * * * * * * * * * * * * * H * T C T * * C G G G G n C A T n T T C * * n T * * * A G C * C G C G A C C I * C G C * * T A A A A y T G G y C C * * * y C * * * G A T * T A A A G T T J * T C T * * C G G G G n C A T n T T * * * n T * * * A G C * C G C G A C C K * C G C * * T A A A A y T G G y C C * * A y C T A y * * * A T A A A G T T L A * C * C C C G G G G n * * * * * * * T G * * C G n G A T G * * * * * * * M G T * T T T * * * * * * C A T n T T * C * n T * * * A G C * C G C G A C C N * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * O * * * * * * * * * * * * * * * * * * * * * y C * * * G A T * T A A A G T T P * C G C * * T A A A A y T G G y C C * * * n T * * * A G C * C G C G A C C Q * T C T * * C G G G G n C A T n T T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * LEP, leptin gene; CT, Casertena; LW, Large White; Me, Meishan; WB, Wild Boar. Position referred to the sequence U. *Matches at the same position with the previous nucleotide or, etions and insertion. y ¼ Presence of mutation. n ¼ Absence of mutation. ¼ Position referred to the sequence AF. Journal compilation ª Blackwell Verlag, Berlin. No claim to original US government works J. Anim. Breed. Genet. ()

6 M. D Andrea et al. Structural analysis and haplotype diversity in swine LEP and MC genes Casertana (CT) Large White (LW) Wild Boar (WB) Duroc (DU) Meishan (Me) C Q D A P M H J N O L F G I Figure Phylogenetic tree of haplotypes in the leptin (LEP) gene. Numbers are the number of bootstrap samples from supporting the corresponding node. K B E preparation). However, it is interesting that the B haplotype also identifies a divergent branch of the tree which is closely related to the three Asian variants carried by the Me and WB animals analysed (Figure ), and that both CT and Me animals are fat breeds distinct from LW and other European breeds highly selected for leanness, suggesting that artificial selection effects could account for the observed distribution of haplotypes. As for many traditional breeds, the CT is considered to be threatened by extinction; moreover, the breed has a niche for ham production in Southern Italy. Markers for the SNPs that identify the B haplotype of the Leptin gene should thus be included in marker panels for the genetic conservation of the existing populations. A total of bp were sequenced for the MC gene, including the only exon and bp upstream and downstream of it. Only one, previously described (Kim et al. ), mutation was found in MC, with frequencies of the G allele of and % in CT and LW respectively. All the Me, DU and WB pigs studied were homozygous for the G allele. The low polymorphism levels in the MC gene accord with the literature. MC is a subtype of seven-transmembrane G protein-coupled receptors that have conserved amino acid sequences, and thus the lack of polymorphisms could be explained by functional constraints and selection pressure (Kim et al. ; Huston et al. ; Kim et al. ). The MC data with its lack of polymorphism provides a valuable contrast to the LEP gene in which we have revealed many novel polymorphisms. In part, the MC data act as a control for the rigour of our experimental work and confirmation that the CT samples are not artefactual. The LEP gene could be considered as a hot spot gene. In the recent work from Stachowiak et al., a further four novel polymorphisms were found in a -bp fragment of the promoter region. The extensive polymorphisms found in LEP and not previously described may account for the lack of consistency in the results of association analysis between LEP polymorphisms and traits. The association analyse conducted to date were performed considering the single SNP instead of haplotype. In conclusion, whilst the results for association studies in swine between polymorphisms at the LEP locus and performance are inconsistent, the markedly greater level of polymorphism observed in the fat CT breed which shares this variation with other fat pigs (WB, Me) in contrast to the lower levels of variation found in lean breeds (LW and DU) would be consistent with unwitting selection on the LEP locus or linked loci in the pursuit of lean genotypes. Indeed, these data have some of the features of a signature of selection. The new LEP polymorphisms described here provide the tools for future more rigorous studies of associations between LEP genotypes and performance. Journal compilation ª Blackwell Verlag, Berlin. No claim to original US government works J. Anim. Breed. Genet. ()

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