Dan J. Nonneman

Analysis of copy number variations among diverse cattle breeds

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here, we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in modern domesticated cattle using array comparative genomic hybridization (array CGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH). The array CGH panel included 90 animals from 11 Bos taurus, three Bos indicus, and three composite breeds for beef, dairy, or dual purpose. We identified over 200 candidate CNV regions (CNVRs) in total and 177 within known chromosomes, which harbor or are adjacent to gains or losses. These 177 high-confidence CNVRs cover 28.1 megabases or approximately 1.07% of the genome. Over 50% of the CNVRs (89/177) were found in multiple animals or breeds and analysis revealed breed-specific frequency differences and reflected aspects of the known ancestry of these cattle breeds. Selected CNVs were further validated by independent methods using qPCR and FISH. Approximately 67% of the CNVRs (119/177) completely or partially span cattle genes and 61% of the CNVRs (108/177) directly overlap with segmental duplications. The CNVRs span about 400 annotated cattle genes that are significantly enriched for specific biological functions, such as immunity, lactation, reproduction, and rumination. Multiple gene families, including ULBP, have gone through ruminant lineage-specific gene amplification. We detected and confirmed marked differences in their CNV frequencies across diverse breeds, indicating that some cattle CNVs are likely to arise independently in breeds and contribute to breed differences. Our results provide a valuable resource beyond microsatellites and single nucleotide polymorphisms to explore the full dimension of genetic variability for future cattle genomic research.

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.

Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

The purpose of this investigation was to determine the extent to which dystrophin insufficiency caused histomorphological changes in a novel pig model of Becker muscular dystrophy. In our procedures, we used a combination of biochemical approaches, including quantitative PCR and Western blots, along with a histological analysis using standard and immunohistological measures. We found that 8‐wk‐old male affected pigs had a 70% reduction in dystrophin protein abundance in the diaphragm, psoas major, and longissimus lumborum and a 5‐fold increase in serum creatine kinase activity compared with healthy male littermates. Dystrophin insufficiency in the diaphragm and the longissimus resulted in muscle histopathology with disorganized fibrosis that often colocalized with fatty infiltration but not the psoas. Affected animals also had an 80–85% reduction in α‐sarcoglycan localization in these muscles, indicating compromised assembly of the dystrophin glycoprotein complex. Controls used in this study were 4 healthy male littermates, as they are most closely related to the affected animals. We concluded that pigs with insufficient dystrophin protein expression have a phenotype consistent with human dystrophinopathy patients. Given that and their similarity in body size and physiology to humans, we further conclude that this pig line is an appropriate translational model for dystrophinopathies.—Hollinger, K., Yang, C. X., Montz, R. E., Nonneman, D., Ross, J. W., Selsby, J. T. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin‐glycoprotein complex assembly in pig skeletal muscle. FASEB J. 28, 28–1600 (1609). www.fasebj.org

Molecular cloning and characterisation of heparanase mRNA in the porcine placenta throughout gestation

Heparanase (HPSE) is an endoglycosidase that specifically degrades heparan sulfate, which is an abundant glycosaminoglycan of the pig placenta. The aim of the present study was to clone cDNA encoding porcine HPSE and characterise the expression level and localisation of HPSE mRNA in porcine placentas throughout gestation. Placental tissues were collected from litters on Days 25, 45, 65, 85 and 105 of gestation. Three transcript variants similar to HPSE were identified in the pig placenta. In addition, the HPSE gene was mapped to pig chromosome 8 in close proximity to quantitative trait loci for litter size and prenatal survival. Real-time polymerase chain reaction and in situ hybridisation were used to characterise the expression of two HPSE variants, namely HPSE v1 and v2, in the pig placenta throughout gestation. The expression of HPSE v1 and v2 was elevated (P < 0.01) in placentas during very early gestation (Day 25) as well as during late gestation (Days 85 and 105). Finally, HPSE v1 and v2 mRNA were localised to the cuboidal trophoblast cells of the folded bilayer located nearest to the maternal endometrium. These findings illustrate that HPSE likely plays a role in the development and modification of the pig placenta, which has implications for litter size and prenatal survival.

Molecular cloning and endometrial expression of porcine high density lipoprotein receptor SR-BI during the estrous cycle and early pregnancy.

During rapid development of the fetus, levels of high density lipoprotein (HDL) are elevated in pregnant women. The receptor for HDL, scavenger receptor class B type I (SR-BI), mediates selective cholesteryl ester uptake and is highly expressed in the human placenta. Because of the rapid growth of uterus during early pregnancy and differences in placentation between swine and humans, we hypothesized that SR-BI may be expressed in porcine endometrium to take up HDL cholesterol. The objectives of this study were to obtain the full coding region for porcine SR-BI, determine endometrial expression of SR-BI mRNA during the estrous cycle and early pregnancy, and map the gene. By iterative screening of a porcine expressed sequence tag library, we obtained the full coding region of SR-BI. Endometrial expression of SR-BI in White composite gilts (n = 3-4 each) was determined by Northern blotting on Days 10, 13, and 15 cyclic gilts and Days 10, 13, 15, 20, 30, and 40 pregnant gilts. In cyclic gilts, endometrial expression of SR-BI did not change between Days 10 and 13, but increased (P < 0.01) between Days 13 and 15. In pregnant gilts, endometrial expression of SR-BI increased (P < 0.01) between Days 10 and 13, remained elevated until Day 30, and decreased (P = 0.015) on Day 40. The SR-BI gene was mapped to 46.3 cM on chromosome 14. These results show that endometrial expression of SR-BI changes during the estrous cycle and early pregnancy, and suggest that SR-BI takes up HDL for endometrial development during early pregnancy.

Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.

Genome-wide association and identification of candidate genes for age at puberty in swine

BackgroundReproductive efficiency has a great impact on the economic success of pork production. Gilts comprise a significant portion of breeding females and gilts that reach puberty earlier tend to stay in the herd longer and be more productive. About 10 to 30xa0% of gilts never farrow a litter and the most common reasons for removal are anestrus and failure to conceive. Puberty in pigs is usually defined as the female’s first estrus in the presence of boar stimulation. Genetic markers associated with age at puberty will allow for selection on age at puberty and traits correlated with sow lifetime productivity.ResultsGilts (nu2009=u2009759) with estrus detection measurements ranging from 140–240 days were genotyped using the Illumina PorcineSNP60 BeadChip and SNP were tested for significant effects with a Bayesian approach using GenSel software. Of the available 8111 five-marker windows, 27 were found to be statistically significant with a comparison-wise error of Pu2009<u20090.01. Ten QTL were highly significant at Pu2009<u20090.005 level. Two QTL, one on SSC12 at 15xa0Mb and the other on SSC7 at 75xa0Mb, explained 16.87xa0% of the total genetic variance. The most compelling candidate genes in these two regions included the growth hormone gene (GH1) on SSC12 and PRKD1 on SSC7. Several loci confirmed associations previously identified for age at puberty in the pig and loci for age at menarche in humans.ConclusionsSeveral of the loci identified in this study have a physiological role for the onset of puberty and a genetic basis for sexual maturation in humans. Understanding the genes involved in regulation of the onset of puberty would allow for the improvement of reproductive efficiency in swine. Because age at puberty is a predictive factor for sow longevity and lifetime productivity, but not routinely measured or selected for in commercial herds, it would be beneficial to be able to use genomic or marker-assisted selection to improve these traits.

A study of vertebra number in pigs confirms the association of vertnin and reveals additional QTL

BackgroundFormation of the vertebral column is a critical developmental stage in mammals. The strict control of this process has resulted in little variation in number of vertebrae across mammalian species and no variation within most mammalian species. The pig is quite unique as considerable variation exists in number of thoracic vertebrae as well as number of lumbar vertebrae. At least two genes have been identified that affect number of vertebrae in pigs yet considerable genetic variation still exists. Therefore, a genome-wide association (GWA) analysis was conducted to identify additional genomic regions that affect this trait.ResultsA total of 1883 animals were phenotyped for the number of ribs and thoracolumbar vertebrae as well as successfully genotyped with the Illumina Porcine SNP60 BeadChip. After data editing, 41,148 SNP markers were included in the GWA analysis. These animals were also phenotyped for kyphosis. Fifty-three 1xa0Mb windows each explained at least 1.0xa0% of the genomic variation for vertebrae counts while 16 regions were significant for kyphosis. Vertnin genotype significantly affected vertebral counts as well. The region with the largest effect for number of lumbar vertebrae and thoracolumbar vertebrae were located over the Hox B gene cluster and the largest association for thoracic vertebrae number was over the Hox A gene cluster. Genetic markers in significant regions accounted for approximately 50xa0% of the genomic variation. Less genomic variation for kyphosis was described by QTL regions and no region was associated with kyphosis and vertebra counts.ConclusionsThe importance of the Hox gene families in vertebral development was highlighted as significant associations were detected over the A, B and C families. Further evaluation of these regions and characterization of variants within these genes will expand our knowledge on vertebral development using natural genetic variants segregating in commercial swine.

Association of porcine heparanase and hyaluronidase 1 and 2 with reproductive and production traits in a landrace-duroc-yorkshire population.

The ovary and placenta are dynamic structures requiring constant modification both structurally and through cell–cell communication capabilities. The extracellular matrix and basement membranes are primarily composed of a milieu of glycosaminoglycans, including heparan sulfate and hyaluronan. Heparanase (HPSE) and hyaluronidases (HYAL) are responsible for degrading heparan sulfate and hyaluronan, respectively. Therefore, the objective of this study was to evaluate the relationship of SNPs distinct to HPSE, HYAL1, and HYAL2 with measurements of reproduction and production traits in swine. Single trait associations were performed on a Landrace–Duroc–Yorkshire population using SNPs discovered and identified in HPSE, HYAL1, and HYAL2. Analyses were conducted on an extended pedigree and SNPs were found to be associated with reproductive and production traits. Prior to multiple-testing corrections, SNPs within HPSE were weakly associated (Pu2009<u20090.03) having additive effects with age at puberty (−2.5u2009±u20091.08u2009days), ovulation rate (0.5u2009±u20090.24 corpora lutea), and number of piglets born alive (0.9u2009±u20090.44 piglets). A HYAL1 and two HYAL2 SNP were nominally associated (Pu2009≤u20090.0063) with number of piglets born alive after multiple-testing corrections (effects between 1.02 and 1.44 piglets), while one of the same HYAL2 markers maintained a modest association (Pu2009=u20090.0043) having a dominant effect with number of piglets weaned (1.2u2009±u20090.41 piglets) after multiple-testing correction. Functionally, HPSE and HYAL1 and 2 have been shown to participate in events related to ovarian and placental activity. SNPs from these studies could potentially assist with understanding genetic components underlying sow lifetime productivity as measured by piglet survivability based on number born alive and number weaned, thereby contributing to a greater number of pigs/sow/year.

Genome-Wide Copy Number Variations Using SNP Genotyping in a Mixed Breed Swine Population

Copy number variations (CNVs) are increasingly understood to affect phenotypic variation. This study uses SNP genotyping of trios of mixed breed swine to add to the catalog of known genotypic variation in an important agricultural animal. PorcineSNP60 BeadChip genotypes were collected from 1802 pigs that combined to form 1621 trios. These trios were from the crosses of 50 boars with 525 sows producing 1621 piglets. The pigs were part of a population that was a mix of ¼ Duroc, ½ Landrace and ¼ Yorkshire breeds. Merging the overlapping CNVs that were observed in two or more individuals to form CNV regions (CNVRs) yielded 502 CNVRs across the autosomes. The CNVRs intersected genes, as defined by RefSeq, 84% of the time – 420 out of 502. The results of this study are compared and contrasted to other swine studies using similar and different methods of detecting CNVR. While progress is being made in this field, more work needs to be done to improve consistency and confidence in CNVR results.