G. A. Rohrer

Coordinated international action to accelerate genome-to-phenome with FAANG, the Functional Annotation of Animal Genomes project

We describe the organization of a nascent international effort, the Functional Annotation of Animal Genomes (FAANG) project, whose aim is to produce comprehensive maps of functional elements in the genomes of domesticated animal species.

Swine Genome Sequencing Consortium (SGSC): a strategic roadmap for sequencing the pig genome.

The Swine Genome Sequencing Consortium (SGSC) was formed in September 2003 by academic, government and industry representatives to provide international coordination for sequencing the pig genome. The SGSC’s mission is to advance biomedical research for animal production and health by the development of DNAbased tools and products resulting from the sequencing of the swine genome. During the past 2 years, the SGSC has met bi-annually to develop a strategic roadmap for creating the required scientific resources, to integrate existing physical maps, and to create a sequencing strategy that captured international participation and a broad funding base. During the past year, SGSC members have integrated their respective physical mapping data with the goal of creating a minimal tiling path (MTP) that will be used as the sequencing template. During the recent Plant and Animal Genome meeting (January 16, 2005 San Diego, CA), presentations demonstrated that a human–pig comparative map has been completed, BAC fingerprint contigs (FPC) for each of the autosomes and X chromosome have been constructed and that BAC end-sequencing has permitted, through BLAST analysis and RH-mapping, anchoring of the contigs. Thus, significant progress has been made towards the creation of a MTP. In addition, whole-genome (WG) shotgun libraries have been constructed and are currently being sequenced in various laboratories around the globe. Thus, a hybrid sequencing approach in which 3x coverage of BACs comprising the MTP and 3x of the WG-shotgun libraries will be used to develop a draft 6x coverage of the pig genome.

Characterization of Conserved and Nonconserved Imprinted Genes in Swine

To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE, and SLC38A4, tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.

Differential expression of cyclooxygenase-2 around the time of elongation in the pig conceptus.

Alterations in uterine luminal fluid composition as a result of conceptus estradiol-17beta production are believed to play a significant role in the loss of 30-40% of potential pig conceptuses. Shortly after the initiation of conceptus estradiol-17beta synthesis and secretion, the conceptuses are transformed from 1cm spheres to 2-5cm tubular forms and finally to filamentous threads of variable length via a process known as elongation. We have attempted to characterize gene products whose expression is either initiated or terminated as the conceptus elongates. Using RNA fingerprinting, we determined that the inducible form of the rate-limiting enzyme in prostaglandin synthesis, cyclooxygenase-2, is expressed in the filamentous pig conceptus, but not in either the spherical or transitional morphologies. Furthermore, increased expression of cyclooxygenase-2 by the filamentous conceptus was associated with increases in the content of prostaglandins (particularly prostaglandin E(2)) found in uterine luminal fluid.

Use of direct in situ single-copy (DISC) PCR to physically map five porcine microsatellites

Direct in situ single-copy polymerase chain reaction (DISC-PCR) was used to assign and orient a linkage group to pig chromosome 1. Five microsatellites were analyzed, and all five were successfully localized using this procedure. Physical data were used to orient the linkage group with respect to the centromere and estimate the amount of coverage of chromosome 1. There was excellent concordance between the physical and linkage maps. The linear order of the microsatellites was identical, and relative distances were similar. All markers were located on the long arm of chromosome 1. Coverage was estimated at about 32%. Thus, DISC-PCR rapidly and easily assigned and ordered microsatellite markers for which large genomic clones do not exist.

Porcine SINE-associated microsatellite markers: evidence for new artiodactyl SINEs

Approximately 24% (170/710) of porcine (dG-dT)n·(dC-dA)n microsatellites isolated in our laboratory are associated with a previously described porcine Short Interdispersed Element (SINE) termed PRE-1 SINE. Another 5.6% (40/710) of the microsatellites were adjacent to two previously unidentified SINE sequences, which we have designated ARE-1P (Artiodactyl Repetitive Element-1 Porcine) and ARE-2P. The ARE repeats were also found in bovine microsatellite and genomic sequences in the GenBank database. Genotypic information was obtained from 68.9% of primers where at least one primer sequence was obtained from the PRE-1 SINE and 66.6% of primer pairs designed from the ARE SINEs. The use of primers derived from SINEs significantly increases the number of primer pairs available for genetic linkage studies in swine.

Cytogenetic assignment of 53 microsatellites from the USDA-MARC porcine genetic map

This study provides 53 new fluorescent in situ hybridization cytogenetic assignments for microsatellite markers linked on the swine genetic map. Forty microsatellites are physically assigned for the first time. The chromosomal locations of eight markers were either confirmed or refined, while five loci were assigned to locations different from those given in previous reports. Markers were selected to provide physical anchors based on their presumed proximity to centromeres or telomeres and at approximately 30 cM intervals across the genetic map. The number of physical anchors for pig (SSC) chromosomes 8, 15, and 18 linkage groups was significantly improved. Centromeric regions were localized to areas less than 10 cM for SSC 1, 2, 3, 6, 7, 8, and 9. Although the recombination rate was generally higher across small biarmed chromosomes and lowest for large acrocentric chromosomes, two regions with particularly low (1q2.1→q2.9 and 13q2.3→q4.1) and three regions with extremely high (5p1.5→p1.2, 6p1.4→p1.3, and 12p1.5→p1.4) rates of recombination were detected. These assignments represent an overall 10% increase in the number of physically assigned markers in Sus scrofa and more than a 20% increase in the number of Type II loci assigned to the pig cytogenetic map.

Molecular Characterization and Expression of Porcine Bone Morphogenetic Protein Receptor-IB in the Uterus of Cyclic and Pregnant Gilts

Abstract Previous gene mapping analyses revealed a quantitative trait locus for uterine capacity on chromosome 8. Comparison of porcine and human genetic maps suggests that the bone morphogenetic protein receptor IB (BMPR-IB) gene may be located near this region. The objectives of this study were to 1) clone the full coding region for BMPR-IB, 2) examine BMPR-IB gene expression by the endometrium and its cellular localization in cyclic and pregnant gilts, and 3) map the BMPR-IB gene. By iterative screening of an expressed sequence tag library, we obtained a 3559-base pair cDNA clone including the full coding region of BMPR-IB. Endometrial BMPR-IB mRNA expression of White composite gilts was determined by Northern blotting in Days 10, 13, and 15 cyclic and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial BMPR-IB mRNA expression was elevated on Days 13 and 15 (P < 0.01) compared with Day 10. Expression of BMPR-IB mRNA was localized in both luminal and glandular epithelium on Day 15. However, in pregnant gilts, BMPR-IB mRNA expression was not significantly different in the endometrium from Day 10 to Day 20, and it was significantly decreased on Days 30 and 40 (P = 0.011). The BMPR-IB gene was mapped to 108 cM on chromosome 8. These findings show that BMPR-IB mRNA expression is regulated differently in cyclic and pregnant gilts; this pattern of gene expression may be important for endometrial function during the luteal phase of the estrous cycle as compared with early pregnancy.

Linkage assignment of eleven genes to the porcine genome

We report comparative linkage mapping of eleven genes in the swine genome by RFLP analysis. These genes include: Acid phosphatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Factor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2 (ITGβ2), Opioid Receptor Mu-1 (OPRM1), Prohormone Converter (PCI/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). The CCKBR and ITGβ2 loci define the ends of the linkage groups on Chromosomes (Chro) (SSC) 9p and 13qter, respectively.

Mapping genes to swine X chromosome provides reference loci for comparative mapping.

High-resolution, microsatellite-based genetic maps of swine (Rohrer et al. 1996) provide a useful tool in mapping economically important loci and marker-assisted selection (MAS). The merging of the swine genetic and cytogenetic maps has also relied chiefly on microsatellites (Alexander et al. 1996). However, positional candidate cloning of genes accounting for significant genetic variation of a QTL ultimately requires alignment of the human and swine maps. At present, the swine linkage map incorporates only a limited number of comparative markers. The X Chromosome (Chr) has no genes with linkage data. We studied three genes assigned to the X Chr in humans and cloned in pigs to initiate the comparative linkage map for the X Chr. In this study, we linked hypoxanthine phosphoribosyltransferase (HPRT1) assigned to HSA Xq 26.1 (Pai et al. 1980), coagulation factor IX (F9), responsible for X-linked hemophilia in man assigned to HSA Xq 26.3–27.1 (Nguyen et al. 1989); and the vitamin D-dependent, calcium-binding protein (CALB3), or calbindin-D9k, assigned to HSA Xp 22.2 (Biancalana et al. 1994) to the porcine X Chr. These results provide the first linked, comparative reference loci for the X Chr in swine. A total of 94 individuals from eight 2 generation divergent, intraspecific backcross families were used in this study from the USDA-MARC Swine Reference Population (Rohrer et al., 1994). Genotypic data from informative markers displaying Mendelian inheritance were entered and stored in the MARC genome database (Keele et al. 1994).