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A 14.5 kb-long transgene containing the complete caprine β-lactoglobulin gene transcription unit as well as 6.1 kb and 3.7 kb of the 5′- and 3′-flanking regions, respectively, was microinjected into pronuclear stage mouse embryos. Four lines of transgenic mice were obtained, three of them expressing the transgene in their mammary glands during lactation. Western blot analysis of caprine β-lactoglobulin in the milk of hemizygous transgenic animals demonstrated the presence of the exogenous protein at concentrations up to 0.5 mg ml−1 of mouse milk

Suppressive subtractive hybridization libraries from oviduct at 62 h post-mating of two lines of rabbits divergently selected for uterine capacity were generated to identify differentially expressed genes. A total of 438 singletons and 126 contigs were obtained by cluster assembly and sequence alignment of 704 expressed sequence tags (ESTs), of which 54% showed homology to known proteins of the non-redundant NCBI databases. Differential screening by dot blot validated 71 ESTs, of which 47 showed similarity to known genes. Transcripts of genes were functionally annotated in the molecular function and the biological process gene ontology categories using the blast2go software and were assigned to reproductive developmental process, immune response, amino acid metabolism and degradation, response to stress and apoptosis terms. Finally, three interesting genes, PGR, HSD17B4 and ERO1L, were identified as overexpressed in the low line using RT-qPCR. Our study provides a list of candidate genes that can be useful to understanding the molecular mechanisms underlying the phenotypic differences observed in early embryo survival and development traits., This study was funded with project AGL2005-07624-C03.

[Background]: The ELOVL fatty acid elongase 6 (ELOVL6), the only elongase related to de novo lipogenesis, catalyzes the rate-limiting step in the elongation cycle by controlling the fatty acid balance in mammals. It is located on pig chromosome 8 (SSC8) in a region where a QTL affecting palmitic, and palmitoleic acid composition was previously detected, using an Iberian x Landrace intercross. The main goal of this work was to fine-map the QTL and to evaluate the ELOVL6 gene as a positional candidate gene affecting the percentages of palmitic and palmitoleic fatty acids in pigs., [Methodology and Principal Findings]: The combination of a haplotype-based approach and single-marker analysis allowed us to identify the main, associated interval for the QTL, in which the ELOVL6 gene was identified and selected as a positional candidate gene. A polymorphism in the promoter region of ELOVL6, ELOVL6:c.-533C>T, was highly associated with the percentage of palmitic and palmitoleic acids in muscle and backfat. Significant differences in ELOVL6 gene expression were observed in backfat when animals were classified by the ELOVL6:c.-533C>T genotype. Accordingly, animals carrying the allele associated with a decrease in ELOVL6 gene expression presented an increase in C16:0 and C16:1(n-7) fatty acid content and a decrease of elongation activity ratios in muscle and backfat. Furthermore, a SNP genome-wide association study with ELOVL6 relative expression levels in backfat showed the strongest effect on the SSC8 region in which the ELOVL6 gene is located. Finally, different potential genomic regions associated with ELOVL6 gene expression were also identified by GWAS in liver and muscle, suggesting a differential tissue regulation of the ELOVL6 gene., [Conclusions and Significance]: Our results suggest ELOVL6 as a potential causal gene for the QTL analyzed and, subsequently, for controlling the overall balance of fatty acid composition in pigs., This study has been funded by MICINN projects (AGL2008-04818-C03 and AGL2011-29821-CO2) and the Innovation Consolider-Ingenio 2012 Program (CSD2007-00036, Center for Research in Agrigenomics). J. Corominas was funded by a Formación de Personal Investigador (FPI) PhD grant from Spanish Ministerio de Educación (BES-2009-018223), Y. Ramayo by a Formación del Profesorado Universitario (FPU) PhD grant (AP2008-01450) and A. Puig was funded by a Personal Investigador en Formación (PIF) PhD grant from the Universitat Autónoma de Barcelona (458-01-1/2011).

At present, a wide range of molecular sex-typing protocols in wild and domestic animals are available. In pigs, most of these methods are based on PCR amplification of X–Y homologous genes followed by gel electrophoresis which is time-consuming and in some cases expensive. In this paper, we describe, for the first time, a SYBR green-based quantitative real-time PCR (qPCR) assay using an X-linked gene, the glycoprotein M6B, for genetic sexing of pigs. Taking into account the differences in the glycoprotein M6B gene copy number between genders, we determine the correct sex of 54 pig samples from either diaphragm or hair follicle from different breeds using the 2−ΔΔCT method for relative quantification. Our qPCR assay represents a quick, inexpensive, and reliable tool for sex determination in pigs. This new protocol could be easily adapted to other species in which the sex determination was required., This study has been funded by the Innovation Consolider-Ingenio 2010 Program (CSD2007-00036, Centre for Research in Agrigenomics). Y. Ramayo-Caldas was funded by a FPU PhD grant (AP2008-01450).

A previous study allowed the identification of two QTL regions at positions 11–34 cM (QTL1) and 68–76 cM (QTL2) on porcine chromosome SSC12 affecting several backfat fatty acids in an Iberian x Landrace F2 intercross. In the current study, different approaches were performed in order to better delimit the quoted QTL regions and analyze candidate genes. A new chromosome scan, using 81 SNPs selected from the Porcine 60KBeadChip and six previously genotyped microsatellites have refined the QTL positions. Three new functional candidate genes (ACOX1, ACLY, and SREBF1) have been characterized. Moreover, two putative promoters of porcine ACACA gene have also been investigated. New isoforms and 24 SNPs were detected in the four candidate genes, 19 of which were genotyped in the population. ACOX1 and ACLY SNPs failed to explain the effects of QTL1 on palmitic and gadoleic fatty acids. QTL2, affecting palmitoleic, stearic, and vaccenic fatty acids, maps close to the ACACA gene location. The most significant associations have been detected between one intronic (g.53840T > C) and one synonymous (c.5634T > C) ACACA SNPs and these fatty acids. Complementary analyses including ACACA gene expression quantification and association studies in other porcine genetic types do not support the expected causal effect of ACACA SNPs., This work was funded by MICINN projects AGL2008-04818-C03/GAN and CSD2007-00036. María Muñoz was funded by a FPI Ph.D. grant from the INIA Institution.

[Background]: Real-time quantitative PCR (qPCR) is still the gold-standard technique for gene-expression quantification. Recent technological advances of this method allow for the high-throughput gene-expression analysis, without the limitations of sample space and reagent used. However, non-commercial and user-friendly software for the management and analysis of these data is not available., [Results]: The recently developed commercial microarrays allow for the drawing of standard curves of multiple assays using the same n-fold diluted samples. Data Analysis Gene (DAG) Expression software has been developed to perform high-throughput gene-expression data analysis using standard curves for relative quantification and one or multiple reference genes for sample normalization. We discuss the application of DAG Expression in the analysis of data from an experiment performed with Fluidigm technology, in which 48 genes and 115 samples were measured. Furthermore, the quality of our analysis was tested and compared with other available methods., [Conclusions]: DAG Expression is a freely available software that permits the automated analysis and visualization of high-throughput qPCR. A detailed manual and a demo-experiment are provided within the DAG Expression software at http://www.dagexpression.com/dage.zip., This work was supported by the Innovation Consolider-Ingenio 2010 Program (CSD2007-00036, Centre for Research in Agricultural Genomics), and by MICINN project AGL2011-29821-C02 (Ministerio de Economía y Competitividad).

This study aimed at identifying differential gene expression conditional on the fatty acid profile of the longissimus thoracis (Lt) muscle, a prime cut of economic relevance for fresh and cured pork production. A population of 110 Iberian (25%) × Landrace (75%) back-crossed pigs was used, because these two breeds exhibit extreme profiles of intramuscular saturated fatty acid, monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) contents. Total RNA from Lt muscle was individually hybridized to GeneChip Porcine Genome arrays (Affymetrix). A principal component analysis was performed with data from the 110 animals to select 40 extreme animals based on the total fatty acid profile and the MUFA composition (MAP). Comparison of global transcription levels between extreme fatty acid profile pigs (n = 40) resulted in 219 differentially expressed probes (false discovery rate, This work was funded by MICINN project AGL2008-04818-C03/GAN.

Studies of the variation in recombination rate across the genome provide a better understanding of evolutionary genomics and are also an important step towards mapping and dissecting complex traits in domestic animals. With the recent completion of the porcine genome sequence and the availability of a high-density porcine single nucleotide polymorphism (SNP) array, it is now possible to construct a high-density porcine linkage map and estimate recombination rate across the genome. A total of 416 animals were genotyped with the Porcine SNP60BeadChip, and high-density chromosome linkage maps were constructed using CRI-MAP, assuming the physical order of the Sscrofa10 assembly. The total linkage map length was 2018.79 cM, using 658 meioses and 14 503 SNPs. The estimated average recombination rate across the porcine autosomes was 0.86 cM/Mb. However, a large variation in recombination rate was observed among chromosomes. The estimated average recombination rates (cM/Mb) per chromosome ranged from 0.48 in SSC1 to 1.48 in SSC10, displaying a significant negative correlation with the chromosome sizes. In addition, the analysis of the variation in the recombination rates taking 1-Mb sliding windows has allowed us to demonstrate the variation in recombination rates within chromosomes. In general, a larger recombination rate was observed in the extremes than in the centre of the chromosome. Finally, the ratio between female and male recombination rates was also inferred, obtaining a value of 1.38, with the heterogametic sex having the least recombination., This work was funded by MICINN projects AGL2008-04818-C03/GAN and CSD2007-00036. María Muñoz was funded by an FPI PhD grant from the INIA institution. Yuliaxis Ramayo-Caldas was funded by an FPU PhD grant from Spanish Ministerio de Educación.

BackgroundThe traditional strategy to map QTL is to use linkage analysis employing a limited number of markers. These analyses report wide QTL confidence intervals, making very difficult to identify the gene and polymorphisms underlying the QTL effects. The arrival of genome-wide panels of SNPs makes available thousands of markers increasing the information content and therefore the likelihood of detecting and fine mapping QTL regions. The aims of the current study are to confirm previous QTL regions for growth and body composition traits in different generations of an Iberian x Landrace intercross (IBMAP) and especially identify new ones with narrow confidence intervals by employing the PorcineSNP60 BeadChip in linkage analyses.ResultsThree generations (F3, Backcross 1 and Backcross 2) of the IBMAP and their related animals were genotyped with PorcineSNP60 BeadChip. A total of 8,417 SNPs equidistantly distributed across autosomes were selected after filtering by quality, position and frequency to perform the QTL scan. The joint and separate analyses of the different IBMAP generations allowed confirming QTL regions previously identified in chromosomes 4 and 6 as well as new ones mainly for backfat thickness in chromosomes 4, 5, 11, 14 and 17 and shoulder weight in chromosomes 1, 2, 9 and 13; and many other to the chromosome-wide signification level. In addition, most of the detected QTLs displayed narrow confidence intervals, making easier the selection of positional candidate genes.ConclusionsThe use of higher density of markers has allowed to confirm results obtained in previous QTL scans carried out with microsatellites. Moreover several new QTL regions have been now identified in regions probably not covered by markers in previous scans, most of these QTLs displayed narrow confidence intervals. Finally, prominent putative biological and positional candidate genes underlying those QTL effects are listed based on recent porcine genome annotation., This work was funded by MICINN projects AGL2008-04818-C03/GAN and CSD2007-00036. DPM was funded by a FPI Ph.D grant from the Spanish Ministerio de Educación (BES-2009-025417). YR was funded by a FPU Ph.D grant from the Spanish Ministerio de Educación (AP2008-01450).

Long-chain acyl-CoA synthetase (ACSL) family members catalyse the formation of long-chain acyl-CoA from fatty acid, ATP and CoA, thus playing an important role in both de novo lipid synthesis and fatty acid catabolism. Previous studies in our group evaluated ACSL4 as a positional candidate gene for quantitative trait loci located on chromosome X in an Iberian × Landrace cross. A DQ144454:c.2645G>A SNP located in the 3′ untranslated region of the ACSL4 gene was associated with the percentages of oleic and monounsaturated fatty acids. The aim of the present work was to evaluate the functional implication of this genetic variant. An expression analysis was performed for 120 individuals with different genotypes for the DQ144454:c.2645G>A polymorphism using real-time quantitative PCR. Differences between genotypes were identified in liver, with the ACSL4 mRNA expression levels higher in animals with the G allele than in animals with the A allele. A SNP genome-wide association study with ACSL4 relative expression levels showed significant positions on chromosomes 6 and 12. Description of positional candidate genes for ACSL4 regulation on chromosomes 6 and 12 is provided., This study has been funded by MICINN project (AGL2008-04818-C03). J. Corominas was funded by a FPI PhD grant from the Spanish Ministerio de Educación (BES-2009-018223) and Y. Ramayo by a FPU PhD grant (AP2008-01450).

Leptin signalling plays a fundamental role in growth, fatness and body composition. The aim of this study was to investigate the porcine LEP gene sequence in an Iberian × Landrace experimental cross to identify polymorphisms associated with productivity and quality traits. Because of the documented effects on these traits of the LEPR c.1987C>T polymorphism, the LEP and LEPR c.1987C>T polymorphisms and their interactions have been jointly investigated. The LEP gene sequencing has allowed the identification of 39 polymorphisms, eight of which are novel. Three intronic SNPs, LEP g.1382C>T, LEP g.1387C>T and LEP g.1723A>G, have been genotyped, and association analyses have been carried out. Analyses of LEP g.1387C>T, fully linked to LEP g.1382C>T, have revealed additive effects on live and carcass weights and dominant effects on several backfat thickness measurements. Novel effects of both LEP and LEPR polymorphisms on fatty acid composition in subcutaneous fat have been detected, probably mediated through the effects on fatness. The results reported here suggest that the T alleles of both LEP g.1387C>T and LEPR c.1987C>T, which are fixed in the Iberian pigs, would lead to an increase in growth, fatness and saturated fatty acid content in fat, which could be explained by an increased feed intake., This work was funded by MICINN project AGL2008-04818-C03/GAN. Dafne Pérez-Montarelo was funded by an FPI PhD grant from the Spanish Ministerio de Educación (BES-2009-025417).

Domestication, modern breeding and artificial selection have shaped dramatically the genomic variability of domestic animals. In livestock, the so-called FAT1 quantitative trait locus (QTL) in porcine chromosome 4 was the first QTL uncovered although, to date, its precise molecular nature has remained elusive. Here, we characterize the nucleotide variability of 13 fragments of ∼500 bp equally spaced in a 2 Mb region in the vicinity of the FAT1 region in a wide-diversity panel of 32 pigs. Asian and European animals, including local Mediterranean and international pig breeds, were sequenced. Patterns of genetic variability were very complex and varied largely across loci and populations; they did not reveal overall a clear signal of a selective sweep in any breed, although FABP4 fragment showed a significantly higher diversity. We used an approximate Bayesian computation approach to infer the evolutionary history of this SSC4 region. Notably, we found that European pig populations have a much lower effective size than their Asian counterparts: in the order of hundreds vs hundreds of thousands. We show also an important part of extant European variability is actually due to introgression of Asian germplasm into Europe. This study shows how a potential loss in diversity caused by bottlenecks and possible selective sweeps associated with domestication and artificial selection can be counterbalanced by migration, making it much more difficult the identification of selection footprints based on naive demographic assumptions. Given the small fragment analyzed here, it remains to be studied how these conclusions apply to the rest of the genome., AO is recipient of FPI PhD fellowships from Ministerio de Ciencia e Innovación (MICINN), Spain; SERO is recipient of a Ramón y Cajal position (MICINN). This work was funded by Grants AGL2007-65563-C02-01/GAN and Consolider project (MICINN, Spain) to MPE, the China National 973 Project 2006CB102100 to LSH and CGL2009-0934 (MICINN, Spain) to SERO.

[Background]: Recent studies in pigs have detected copy number variants (CNVs) using the Comparative Genomic Hybridization technique in arrays designed to cover specific porcine chromosomes. The goal of this study was to identify CNV regions (CNVRs) in swine species based on whole genome SNP genotyping chips., [Results]: We used predictions from three different programs (cnvPartition, PennCNV and GADA) to analyze data from the Porcine SNP60 BeadChip. A total of 49 CNVRs were identified in 55 animals from an Iberian x Landrace cross (IBMAP) according to three criteria: detected in at least two animals, contained three or more consecutive SNPs and recalled by at least two programs. Mendelian inheritance of CNVRs was confirmed in animals belonging to several generations of the IBMAP cross. Subsequently, a segregation analysis of these CNVRs was performed in 372 additional animals from the IBMAP cross and its distribution was studied in 133 unrelated pig samples from different geographical origins. Five out of seven analyzed CNVRs were validated by real time quantitative PCR, some of which coincide with well known examples of CNVs conserved across mammalian species., [Conclusions]: Our results illustrate the usefulness of Porcine SNP60 BeadChip to detect CNVRs and show that structural variants can not be neglected when studying the genetic variability in this species., This work was funded by MICINN project AGL2008-04818-C03/GAN (Spanish Ministry of Science) and by Innovation Consolider-Ingenio 2010 Programme (CSD2007-00036 "Centre for Research in Agrigenomics"). Y. Ramayo-Caldas was funded by a FPU PhD grant from Spanish Ministerio de Educación (AP2008-01450). C.A. Souza was funded by a PhD grant from CAPES, Brazil.

We have investigated the origin of swine breeds through the joint analysis of mitochondrial, microsatellite, and Y-chromosome polymorphisms in a sample of pigs and wild boars with a worldwide distribution. Genetic differentiation between pigs and wild boars was remarkably weak, likely as a consequence of a sustained gene flow between both populations. The analysis of nuclear markers evidenced the existence of a close genetic relationship between Near Eastern and European wild boars making it difficult to infer their relative contributions to the gene pool of modern European breeds. Moreover, we have shown that European and Far Eastern pig populations have contributed maternal and paternal lineages to the foundation of African and South American breeds. Although West African pigs from Nigeria and Benin exclusively harbored European alleles, Far Eastern and European genetic signatures of similar intensity were detected in swine breeds from Eastern Africa. This region seems to have been a major point of entry of livestock species in the African continent as a result of the Indian Ocean trade. Finally, South American creole breeds had essentially a European ancestry although Asian Y-chromosome and mitochondrial haplotypes were found in a few Nicaraguan pigs. The existence of Spanish and Portuguese commercial routes linking Asia with America might have favored the introduction of Far Eastern breeds into this continent. © The Author 2009. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved., We are grateful to Carles Vila`, Carles Lalueza, and Ju- lio Rozas for critically reading the manuscript. We are also indebted to Maria San ̃ a for valuable comments about the process of pig domestication and to Francesc Calafell for helping in the statistical analyses. Many thanks to all of those contributing to sample collection: G. Aktan (Ay-FSafari), A.R. Jafari-Tabrizi (Iran Safari), S. Shushunov (Russian Hunting Agency), F. Pilla (University of Molise), J. Tibau (Institut de Recerca i Tecnologia Agroalimenta `- ries), J. Capote (Instituto Canario de Investigaciones Agrarias), A. Linden (University of Liege), J.L. Noguera (Institut de Recerca i Tecnologia Agroalimenta`ries), L. Silio ́ and M.C. Valdovinos (Instituto Nacional de Investi- gaciones Agrarias, Spain), E. Martı ́nez (Zoo of Madrid), N. Okumura (STAFF Institute Japan), G. Rohrer (United States Department of Agriculture), Z. Bosze (Agricultural Biotechnology Center, Hungary), M. Cumbreras (Diputa- cio ́ n de Huelva, Spain), E. von Eckardt (Sweden), M.T. Par- amio (Universitat Auto` noma de Barcelona, Spain), O. Vidal (Universitat de Girona, Spain), M. Kovac (Slovenia), D. Vidal and C. Gorta ́zar (Instituto de Investigacio ́ n en Re- cursos Cinege ́ticos, Spain), Instituto de Capacitacio ́ n del Oriente (Bolivia), Veterinarios Sin Fronteras (Spain). A.O. and D.G. were recipients of PhD grants from the Min- isterio de Educacio ́ n y Ciencia, (Spain) and Universitat Auto` noma de Barcelona. O.R. received funding from the Molecular Genetics Veterinary Service of the Universitat Auto ` noma of Barcelona (http://svgm.uab.es/svgm). This study was partially funded by projects AGL2002-04271- C03-03 and AGL2007-66707-C02-02 (Ministry of Educa- tion and Science, Spain) to M.A.; AGL2004-0103, AECI/ 5267/06, and BFU2004-02253 (Ministry of Education and Science, Spain) to M.P.E.; AGL2000-1229-C03-03 (Ministry of Education and Science, Spain) to A.S. and National Natural Science Foundation of China (grant 30425045) to L.S.H.

The objective of this work was to analyse the porcine Fatty acid binding protein 2, intestinal (FABP2) gene as a candidate gene for a fatty acid composition quantitative trait loci (QTL) previously described on porcine chromosome 8 in an Iberian by Landrace F2 cross (IBMAP). Re-sequencing of the porcine FABP2 gene in three Iberian and eight Landrace parental animals resulted in the identification of three single-nucleotide polymorphisms, all of them localized in intron 1. The polymorphism FABP2:g.412T>C, localized in intron 1, and two additional microsatellites were genotyped in the IBMAP population in order to perform an association test of the FABP2 gene and to better define the QTL position previously described. Association analyses of the FABP2:g.412T>C with the fatty acid composition traits were not significant in simple association and marker-assisted association tests, suggesting that the FABP2 region sequenced is not responsible for the QTL. However, the addition of three new markers to the pedigree allowed us to define the S0144–SW61 marker interval as the most likely QTL position, facilitating the future study of other candidate genes for this QTL., Work funded by grants from INIA (Acción especial CPE03-010-C3) and MCYT (AGF99-0284-CO2-02). We are indebted to NOVA GENETICA (Lleida, Spain) and SIA ‘Dehesón del Encinar’ (Oropesa, Spain) and their staff for providing the experimental pigs. J. Estellé is recipient of a FPU fellowship from the Spanish Ministerio de Educación y Ciencia. R.N. Pena holds a contractual grant from INIA.

[Background]: Artificial selection has resulted in animal breeds with extreme phenotypes. As an organism is made up of many different tissues and organs, each with its own genetic programme, it is pertinent to ask: How relevant is tissue in terms of total transcriptome variability? Which are the genes most distinctly expressed between tissues? Does breed or sex equally affect the transcriptome across tissues?, [Results]: In order to gain insight on these issues, we conducted microarray expression profiling of 16 different tissues from four animals of two extreme pig breeds, Large White and Iberian, two males and two females. Mixed model analysis and neighbor – joining trees showed that tissues with similar developmental origin clustered closer than those with different embryonic origins. Often a sound biological interpretation was possible for overrepresented gene ontology categories within differentially expressed genes between groups of tissues. For instance, an excess of nervous system or muscle development genes were found among tissues of ectoderm or mesoderm origins, respectively. Tissue accounted for ~11 times more variability than sex or breed. Nevertheless, we were able to confidently identify genes with differential expression across tissues between breeds (33 genes) and between sexes (19 genes). The genes primarily affected by sex were overall different than those affected by breed or tissue. Interaction with tissue can be important for differentially expressed genes between breeds but not so much for genes whose expression differ between sexes., [Conclusion]: Embryonic development leaves an enduring footprint on the transcriptome. The interaction in gene × tissue for differentially expressed genes between breeds suggests that animal breeding has targeted differentially each tissue's transcriptome., ALJF is recipient of a fellowship from National Council of Technological and Scientific Development (CNPq, Brazil), AO is funded by a PhD fellowship (Ministry of Education and Science – MEC – Spain), LTF is supported by a CAPES grant (Ministry of Education, Brazil). Work funded by MEC grants AGL2004-0103/GAN and AGL2007-65563-C02-01/GAN (Spain). The microarray hibridizations were carried out at the Microarray Service, Core Facility of Institute of Research of University Hospital Vall d'Hebron (UCTS IR-HUVH, Barcelona) and subsidized by Fundación Genoma España (www.gen-es.org).

Domestic species allow us to study dramatic evolutionary changes at an accelerated rate due to the effectiveness of modern breeding techniques and the availability of breeds that have undergone distinct selection pressures. We present a worldwide survey of haplotype variability around a known causative mutation in porcine gene IGF2, which increases lean content. We genotyped 34 SNPs spanning 27 kb in 237 domestic pigs and 162 wild boars. Although the selective process had wiped out variability for at least 27 kb in the haplotypes carrying the mutation, there was no indication of an overall reduction in genetic variability of international vs. European local breeds; there was also no evidence of a reduction in variability caused by domestication. The haplotype structure and a plot of Tajima's D against the frequency of the causative mutation across breeds suggested a temporal pattern, where each breed corresponded to a different selective stage. This was observed comparing the haplotype neighbor-joining (NJ) trees of breeds that have undergone increasing selection pressures for leanness, e.g., European local breeds vs. Pietrain. These results anticipate that comparing current domestic breeds will decisively help to recover the genetic history of domestication and contemporary selective processes., MassARRAY genotyping was carried out at the Spanish National Genotyping Center (http://www.cegen.org) facilities and was subsidized by Fundación Genoma España. A.O. is the recipient of a Ph.D. grant (Ministerio de Educación y Ciencia, Spain). Work was funded by projects AGL2004-0103, AECI/5267/06 (Spain), and the National Natural Science Foundation of China (grant 30425045).

A total of 598 F2 does from a cross between the high and low lines selected divergently for uterine capacity during 10 generations were used in a candidate gene analysis. The presence of major genes affecting the number of implanted embryos and uterine capacity has been suggested in lines divergently selected for uterine capacity. Uterine capacity is a main component of litter size. The progesterone receptor gene was tested as a candidate gene to determine whether polymorphisms explain differences in litter size and its components. Fragments of the promoter region and exons 1–8 were amplified and sequenced. One SNP was found in the promoter region, 2464G>A, three SNPs in the 5′-UTR exon 1, and a silence SNP in exon 7. The first four SNPs were segregated in two haplotypes. The allele G found in the promoter region was found in 75% of the high-line parental animals and in 29% of the low-line parental animals. The GG genotype had 0.5 kits and 0.5 implanted embryos more than the AA genotype. At 48 hr of gestation, the difference in early embryo survival and embryonic stage of development was small. However, at 72 hr of gestation, the GG genotype had 0.36 embryos more than the AA genotype and also had a more advanced embryonic stage of development, showing a lower percentage of compacted morulae and a higher percentage of blastocysts. The difference in litter size between the GG and GA genotypes was similar to the difference found between homozygote genotypes; however, differences in implanted embryos, early embryo survival, and embryo development were not detected between the GG and GA genotypes., This study was funded by projects by the Comisión Interministerial de Ciencia y Tecnología AGL2001-3068 and AGL2005-07624. M. Merchán acknowledges a Formación de Personal Investigador (FPI) grant from the Ministerio de Ciencia y Tecnología (MCYT) and R. Peiró an FPI grant from Oficina de Ciencia y Tecnología de la Generalitat Valenciana (OCYT).

CDP-diacylglycerol synthase (CDS) catalyzes the conversion of phosphatidic acid to CDP-diacylglycerol, an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We amplified and sequenced 2,053 bp of the pig CDS1 mRNA. The structure of the pig CDS1 gene was determined, being very similar to that of the human, rat, and mouse genes with respect size and organization of the 13 exons. In addition, we identified three polymorphic positions in exons 10 and 11. One of them, the A/C1006, was genotyped in samples belonging to Iberian, Landrace, Large White, Pietrain, and Meishan pig breeds. Expression of this gene was also analyzed by real-time polymerase chain reaction (PCR) in different tissues showing a high CDS1 expression in testis. Moreover, a 1240-bp fragment of the pig CDS2 mRNA was amplified and sequenced. Finally, the CDS1 and CDS2 genes were physically mapped to porcine chromosomes 8 and 17, respectively, by using the INRA, University of Minnesota porcine Radiation Hybrid panel (IMpRH).

Signal transducers and activators of transcription (STATs) are a family of transcription factors. STAT5A and 5B are two highly related proteins encoded by two distinct genes. Transgenic knockout mice studies have indicated the importance of STAT5 proteins for the regulation of both lactation and growth performance. Moreover, different studies determine the role of STAT5 proteins in the modulation of adipocyte function. In this study, we sequenced one fragment of STAT5B gene from animals of six breeds (Duroc, Iberian, Landrace, Large White, Pie´train and Meishan) to identify genetic variants. A G/A single nucleotide polymorphism in intron 14 creates a polymorphic PstI restriction site and was genotyped by polymerase chain reaction restriction fragment length polymorphism in the six breeds. Allele G was only present in Large White, Pie´train and Meishan populations, detecting only G allele in this last pig breed. The STAT5B gene was located by radiation hybrid mapping to porcine chromosome 12, within the confidence interval for the fatty acid composition quantitative trait loci, previously identified in an Iberian · Landrace cross.

The mitochondrial hypervariable region 1 (HVR1) sequence of 67 goats belonging to the Girgentana, Maltese and Derivata di Siria breeds was partially sequenced in order to present the first phylogenetic characterization of Sicilian goat breeds. These sequences were compared with published sequences of Indian and Pakistani domestic goats and wild goats. Mitochondrial lineage A was observed in most of the Sicilian goats. However, three Girgentana haplotypes were highly divergent from the Capra hircus clade, indicating that a new mtDNA lineage in domestic goats was found.

Selection on uterine capacity has been used in animal breeding as a way to improve the litter size. A divergent selection experiment for uterine capacity was performed in rabbits during ten generations. After the first generations of selection, large differences in number of implanted embryos were obtained between high and low lines. The major part of the differences between lines was due to embryo survival. A segregation analysis suggested the presence of a major gene affecting the reproductive traits. The objective of this work was to test the TIMP-1 gene as a candidate gene for embryo survival in rabbits since it stands up as a target for the investigation of reproductive problems in humans. We have analyzed the parental generation of a F2 cross which consists of 8 and 14 animals from the high and low uterine capacity lines, respectively. The rabbit TIMP-1 gene structure and sequence has been determined, including the proximal promoter region. Despite of the absence of polymorphism between lines in the screened regions (CDS, proximal promoter, exon 1, intron 1, and exon 2), a real-time RT-PCR quantification of the TIMP-1 mRNA in oviduct has shown significant differences between high and low lines at 62 hr of gestation, just when rabbit embryos are located in the oviduct, postulating TIMP-1 as an interesting candidate gene to be involved in the phenotypic differences between the two rabbit lines. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc.

We have characterized and mapped the porcine fatty acid binding protein 5, epidermal (FABP5) gene. According to linkage and RH mapping, this gene is located close to the FABP4 (fatty acid binding protein 4, adipocyte) gene on swine chromosome 4. We resequenced 4.7 kb of the FABP5 gene in the parental population of an Iberian x Landrace cross (IBMAP), identifying seven SNPs arranged in two distinct FABP5 haplotypes. QTL and association analyses in the IBMAP population showed that this gene is strongly associated with fat deposition. QTL and haplotype analysis revealed that both FABP4 and FABP5 (clustered in mammals) are major candidate genes for the FAT1 QTL; the most likely position for the FAT1 QTL is between these two genes. Finally, our results suggest the presence of more than one QTL affecting fatness traits on porcine chromosome 4. © 2006 The Authors.

Long-chain acyl-CoA synthetase (ACSL) catalyses the formation of long-chain acyl-CoA from fatty acid, ATP and CoA, activating fatty acids for subsequent reactions. Long-chain acyl-CoA synthetase thus plays an essential role in both lipid biosynthesis and fatty acid degradation. The ACSL4 gene was evaluated as a positional candidate gene for the quantitative trait loci (QTL) located between SW2456 and SW1943 on chromosome X. We have sequenced 4906 bp of the pig ACSL4 mRNA. Sequence analysis allowed us to identify 10 polymorphisms located in the 3′-UTR region and to elucidate two ACSL4 haplotypes. Furthermore, a QTL and an association study between polymorphisms of the ACSL4 gene and traits of interest were carried out in an Iberian x Landrace cross. We report QTL that have not been previously identified, and we describe an association of the ACSL4 polymorphisms with growth and percentage of oleic fatty acid. Finally, we have determined allelic frequencies in 140 pigs belonging to the Iberian, Landrace, Large White, Meishan, Pietrain, Duroc, Vietnamese, Peccary and Babirusa populations. © 2006 International Society for Animal Genetics.

The leptin receptor gene (LEPR) is a candidate for traits related to growth and body composition, and is located on SSC6 in a region where fatness and meat composition quantitative trait loci (QTL) have previously been detected in several F2 experimental designs. The aims of this work were: (i) to fine map these QTL on a larger sample of animals and generations (F3 and backcross) of an Iberian×Landrace intercross and (ii) to examine the effects of LEPR alleles on body composition traits. Eleven single nucleotide polymorphisms (SNPs) were detected by sequencing LEPR coding regions in Iberian and Landrace pig samples. Three missense polymorphisms were genotyped by pyrosequencing in 33 F0, 70 F1, 418 F2, 86 F3 and 128 individuals coming from the backcross of four F2 males with 24 Landrace females. Thirteen microsatellites and one SNP were also genotyped. Traits analysed were: backfat thickness at different locations (BFT), intramuscular fat percentage (IMFP), eye muscle area (EMA), loin depth (LOD), weight of shoulder (SHW), weight of ribs (RIBW) and weight of belly bacon (BBW). Different statistical models were applied in order to evaluate the number and effects of QTL on chromosome 6 and the possible causality of the LEPR gene variants with respect to the QTL. The results support the presence of two QTL on SSC6. One, at position 60–100 cM, affects BFT and RIBW. The other and more significant maps in a narrow region (130–132 cM) and affects BFT, IMFP, EMA, LOD, SHW, RIBW and BBW. Results also support the association between LEPR alleles and BFT traits. The possible functional implications of the analysed polymorphisms are considered.

A crossed population between Iberian x Landrace pigs consisting of 321 F2, 87 F3, and 85 backcross individuals has been analyzed to refine the number and positions of quantitative trait loci (QTL) affecting shape, growth, fatness, and meat quality traits in SSC4. A multitrait multi-QTL approach has been used. Our results suggest that carcass length and shoulder weight are affected by two loci. The first one, close to the AFABP gene, has a very strong pleiotropic effect on fatness, whereas the second one, in the interval between S0073 and S0214, also affects live weight, although to a lesser extent. This latter QTL would correspond to the FAT1 locus described initially in pigs. It seems that SSC4's loci play an important role in redistributing total weight, and the Landrace allele increases shoulder weight and carcass length much more than ham or total weight. Furthermore, there is also strong evidence of additional loci influencing pH and color in more distant, telomeric positions. © Springer Science+Business Media, Inc. 2005.

During the last two centuries, the Spanish ibex (Capra pyrenaica) has shown a significant demographic decline as a result of the progressive destruction of its natural habitat, disease epidemics, and uncontrolled hunting. Partial sequencing of the class II MHC DRB1 gene revealed that the Spanish ibex has remarkably low levels of genetic variation at this locus, with only six different DRB1 alleles and an observed heterozygosity of 0.429-0.579. The rates of nonsynonymous vs synonymous substitutions were significantly different in the peptide-binding region (dN/dS=5.347, P=0.002), a feature that indicates that the DRB1 gene is under positive selection. A phylogenetic analysis of the Spanish ibex and a set of domestic goat DRB1 alleles revealed that the reported sequences represent four major allelic lineages. The limited allelic repertoire of the DRB1 gene in the Spanish ibex is likely the direct result of the recent history of population bottlenecks and marked demographic decline of this species. A genetic survey of 13 microsatellite loci was consistent with this idea. The Spanish ibex subspecies C. p. hispanica and C. p. victoriae consistently showed considerably lower levels of microsatellite heterozygosity (Ho=0.184-0.231) and allelic diversity (mean number of alleles per locus=2-2.4) than those reported in other wild ruminants. This study demonstrates the significance of both natural selection and the demographic history of populations in determining patterns of genetic variation at MHC loci. In addition, our results emphasize the importance of locally adapted populations for the preservation of genetic diversity.

A recessive insertional mutation was identified in one line of transgenic mice for the caprine beta-lactoglobulin (betaLG) gene. High mortality after birth, a significant reduction in postnatal growth and adult body size, changes in the morphometric features of the head, and infertility are the most prominent phenotypic traits of the mutant animals. Molecular cloning and sequencing of the transgene insertion site showed that 22 copies of the betaLG transgene are inserted in an intronic region of the phospholipase C-beta1 (PLC-beta1) gene, which plays a pivotal role in modulating different cellular functions. As a result of the insertional mutation (PLC-beta1(betaLG) mutation), a hybrid messenger RNA (mRNA) between the mouse PLC-beta1 and the goat betaLG genes is transcribed. The tissue-specific pattern of expression of this hybrid mRNA in PLC-beta1(betaLG) homozygotes is equivalent to that of the endogenous PLC-beta1 mRNA in nontransgenic animals, which is reported for the first time in this species, but expression levels are significantly reduced. Although the hybrid PLCbeta1-betaLG mRNA contains all the essential information to produce a PLCbeta1 protein that could be activated, this protein was not detected by Western blot. The PLC-beta1(betaLG) mouse model described here represents a useful tool to investigate the role of the PLC-beta1 gene in the molecular mechanisms underlying growth and fertility.

We present a QTL genome scan for fatty acid composition in pigs. An F 2 cross between Iberian x Landrace pigs and a regression approach fitting the carcass weight as a covariate for QTL identification was used. Chromosomes (Chrs) 4, 6, 8, 10, and 12 showed highly significant effects. The Chr 4 QTL influenced the linoleic content and both the fatty acid double-bond index and peroxidability index. In Chr 6 we found significant associations with the double-bond index and the unsaturated index of fatty acids. Chr 8 showed clear effects on the percentages of palmitic and palmitoleic fatty acids as well as the average chain length of fatty acids. In Chr 10 we detected a significant QTL for the percentage of myristic fatty acid, with an F value that was slightly above the genomewide threshold. The percentage of linolenic fatty acid was affected by a region on Chr 12. A nearly significant QTL for the content of gadoleic fatty acid was also detected in Chr 12. We also analyzed the genomic QTL distribution by a regression model that fits the backfat thickness as a covariate. Some of the QTL that were detected in our analysis could not be detected when the data were corrected by backfat thickness. This work shows how critical the selection of a covariate can be in the interpretation of results. This is the first report of a genome scan detection of QTL directly affecting fatty acid composition in pigs.

A new polymorphism has been identified in the goat kappa-casein gene by evaluating genomic DNA from the Montefalcone breed in Italy. The polymorphic site consists of a single nucleotide substitution A to G at position 242 of the exon 4 and produces an amino acid substitution Asp/Gly. A polymerase chain reaction-restriction fragment length polymorphism protocol for rapid genotyping of the variant has been developed, using the HaeIII enzyme. Animals from Italian, Spanish, and French breeds have been analyzed to investigate the occurrence of the allele in other populations. The allele appears to be exclusive to the Montefalcone breed.

Polymorphism in the goat kappa casein gene was studied using the base excision sequence scanning (BESS) method and sequencing. Seven polymorphic sites, corresponding to single nucleotide transversions were detected. Three of these were silent mutations while the other four produced amino acid substitutions. The association between these polymorphic sites was investigated, which resulted in the identification of three goat kappa casein alleles, designated A, B, and C. Protocols for rapid genotyping of the C variant were developed by polymerase chain reaction–restriction fragment length polymorphism using Alw44I and BseNI restriction endonucleases. The occurrence of this allele was found to be very low in Spanish breeds but more frequent in the French Saanen goat. Further studies among different goat populations are necessary to establish the distribution of these alleles and their effects on the quality and functional properties of milk.

Two new variants have been detected and characterized for the goat β-lactoglobulin gene at the cDNA level and confirmed at the genomic level. The two polymorphisms are located on exon 7 of the gene. One of the polymorphic sites is produced by a single nucleotide substitution in position +4601, allowing a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) genotyping procedure to be developed using SacII restriction enzyme. The other polymorphic position contains a 10 bp long insertion at position +4641 that can be detected by capillary electrophoresis of the PCR product amplified with a fluorescent primer. The association of these two polymorphisms was also investigated, resulting in the description of two new alleles. Both of these contained the point mutation at the SacII site, with or without the 10 bp insertion at position +4641. The distribution of these new polymorphisms was studied in a population of males of four different goat breeds. The gene frequencies for these variants were similar in Spanish and French breeds.

The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes the characterisation of PCV-2 isolates from animals affected with PMWS from Germany, Spain and France. We could demonstrate the presence of circovirus by electron microscope, in situ hybridisation and PCR. PCR revealed incidence of PCV-2 in many tissues of one infected animal with the exception of heart and nervous system. The phylogenetic analysis of all PCV-2 isolates yet published in the database, showed relationship of isolates from Spain, Germany and France, with three sequences from Canada determined recently and two isolates from Taiwan, while other North American sequences display a separate cluster. PCR screening of randomly collected organ samples from German pigs not affected with PMWS, revealed a rate of infection with PCV-1 of 5% and with PCV-2 of 26.8%, while blood samples showed a lower incidence.

In this article, we describe the use of a caprine beta-lactoglobulin (betaLG) expression cassette previously obtained by our group to target the expression of the human follicle-stimulating hormone (hFSH) to the mammary gland of transgenic mice. The hFSH is a pituitary glycoprotein composed of two subunits (alpha and beta). At present, this protein is obtained from mammalian cellular fermentors, and it is extensively used in the treatment of human infertility. Four lines of double (hFSHalpha/beta) transgenic mice that stably transmitted the transgenes were obtained, and hFSHalpha and hFSHbeta mRNA was detected by reverse transcriptase-polymerase chain reaction in the mammary gland of lactating females from all four transgenic lines. The hFSH protein was present in the mammary gland of the lactating females, but could not be detected in the milk by Western blot, probably as a result of low levels of transgene expression.

Beta-lactoglobulin (betaLG) is a whey protein found in the milk of most mammals, except those of humans and rodents. In ruminants, only one type of betaLG is expressed in milk, although the presence of pseudogenes has been reported. The milk of other mammals (dog, horse, dolphins) contains two types of betaLG: type I is related to ruminant betaLG, while type II has a strong similarity to the ruminant pseudogenes. The presence of three types of betaLG has been described only in cat milk. For the first time, we have cloned, sequenced, and characterized the three types of feline betaLG cDNAs from a mammary gland sample of a lactating cat. Since no specific probe could be easily used to differentiate them by Northern Blotting, we developed a new technique named Fluorescent-Ratio PCR (FR-PCR) in order to assess their level of expression during lactation. The relative amounts of each feline betaLG cDNA was quantified in two cats by capillary electrophoresis of the restricted RT-PCR product, labeled with fluorescent primers. We observed the same percentage of expression of the three betaLG genes in two cats. The differences of expression could be due to changes in the promoter and 3' non-coding region affecting the level of transcription and the mRNA stability. The FR-PCR technique was shown to be reproducible and accurate even for small percentage differences, being very useful when a small amount of sample is available.

Different levels of the major milk protein beta-lactoglobulin are found in evolutionarily related ruminant species: with sheep milk containing as much as three times the concentration in goat milk. In an attempt to understand why these differences exist, we have characterised, using DNaseI as a probe of structure, the chromatin surrounding the goat beta-lactoglobulin promoter and compared it to that of the sheep homologue. The goat gene displays a mammary-specific chromatin pattern, which is reformed on expressing goat beta-lactoglobulin transgenes. This implies that this chromatin structure is sequence dependent and suggests that it plays a role in regulating beta-lactoglobulin gene expression. This pattern differs from that seen on the ovine beta-lactoglobulin gene in lactating sheep mammary chromatin. Thus, even between highly related species, the transcriptional mechanisms regulating activity of a gene can differ.

The leptin (LEP) and its receptor (LEPR) regulate food intake and energy balance through hypothalamic signaling. However, the LEP-LEPR axis seems to be more complex and its expression regulation has not been well described. In pigs, LEP and LEPR genes have been widely studied due to their relevance. Previous studies reported significant effects of SNPs located in both genes on growth and fatness traits. The aim of this study was to determine the expression profiles of LEP and LEPR across hypothalamic, adipose, hepatic and muscle tissues in Iberian x Landrace backcrossed pigs and to analyze the effects of gene variants on transcript abundance. To our knowledge, non porcine LEPR isoforms have been described rather than LEPRb. A short porcine LEPR isoform (LEPRa), that encodes a protein lacking the intracellular residues responsible of signal transduction, has been identified for the first time. The LEPRb isoform was only quantifiable in hypothalamus while LEPRa appeared widely expressed across tissues, but at higher levels in liver, suggesting that both isoforms would develop different roles. The unique LEP transcript showed expression in backfat and muscle. The effects of gene variants on transcript expression revealed interesting results. The LEPRc.1987C>T polymorphism showed opposite effects on LEPRb and LEPRa hypothalamic expression. In addition, one out of the 16 polymorphisms identified in the LEPR promoter region revealed high differential expression in hepatic LEPRa. These results suggest a LEPR isoform-specific regulation at tissue level. Conversely, non-differential expression of LEP conditional on the analyzed polymorphisms could be detected, indicating that its regulation is likely affected by other mechanisms rather than gene sequence variants. The present study has allowed a transcriptional characterization of LEP and LEPR isoforms on a range of tissues. Their expression patterns seem to indicate that both molecules develop peripheral roles apart from their known hypothalamic signal transduction function., This work was funded by Ministerio de Ciencia e Innovación (MICINN) projects AGL2008-04818-C03 and AGL2011-29821-C02-02. D. Pérez-Montarelo was funded by a Formación de Personal Investigador (FPI) PhD grant from the Spanish Ministerio de Ciencia e Innovación (BES-2009-025417).

Background Porcine fatty acid composition is a key factor for quality and nutritive value of pork. Several QTLs for fatty acid composition have been reported in diverse fat tissues. The results obtained so far seem to point out different genetic control of fatty acid composition conditional on the fat deposits. Those studies have been conducted using simple approaches and most of them focused on one single tissue. The first objective of the present study was to identify tissue-specific and tissue-consistent QTLs for fatty acid composition in backfat and intramuscular fat, combining linkage mapping and GWAS approaches and conducted under single and multitrait models. A second aim was to identify powerful candidate genes for these tissue-consistent QTLs, using microarray gene expression data and following a targeted genetical genomics approach. Results The single model analyses, linkage and GWAS, revealed over 30 and 20 chromosomal regions, 24 of them identified here for the first time, specifically associated to the content of diverse fatty acids in BF and IMF, respectively. The analyses with multitrait models allowed identifying for the first time with a formal statistical approach seven different regions with pleiotropic effects on particular fatty acids in both fat deposits. The most relevant were found on SSC8 for C16:0 and C16:1(n-7) fatty acids, detected by both linkage and GWAS approaches. Other detected pleiotropic regions included one on SSC1 for C16:0, two on SSC4 for C16:0 and C18:2, one on SSC11 for C20:3 and the last one on SSC17 for C16:0. Finally, a targeted eQTL scan focused on regions showing tissue-consistent effects was conducted with Longissimus and fat gene expression data. Some powerful candidate genes and regions were identified such as the PBX1, RGS4, TRIB3 and a transcription regulatory element close to ELOVL6 gene to be further studied. Conclusions Complementary genome scans have confirmed several chromosome regions previously associated to fatty acid composition in backfat and intramuscular fat, but even more, to identify new ones. Although most of the detected regions were tissue-specific, supporting the hypothesis that the major part of genes affecting fatty acid composition differs among tissues, seven chromosomal regions showed tissue-consistent effects. Additional gene expression analyses have revealed powerful target regions to carry the mutation responsible for the pleiotropic effects., This work was funded by the MICINN project AGL2011-29821-C02 (Ministerio de Economía y Competitividad). We thank to Fabian Garcia, Anna Mercadé and Carmen Barragán for their assistance in DNA preparation and SNP genotyping.

A beta-lactoglobulin (beta-LG) pseudogene has been isolated and sequenced (7634 bp) in goat. Its structure is remarkably similar to that of the beta-LG gene with all seven exons placed in the same relative position. The pseudogene seems to have originated by gene duplication, but a non uniform distribution of similarities along the sequence suggests that events of gene conversion have also occurred during its evolution. The comparison of the predicted ancestral protein encoded by the pseudogene shows its evolutionary relationship to the monomeric beta-LG II forms of the beta-LG reported in some non-ruminant species. Southern-blot analysis shows that similar pseudogenes are also found in the genome of sheep and cow. The pseudogene has been mapped by fluorescent in situ hybridization (FISH) to sheep chromosome 3p28 and cattle and goat chromosomes 11q28, in the same mapped position as that found for the beta-LG gene in all these species.