Biomedical subjects
Publications and source records attributed to C W Beattie.
Chromosome-specific libraries provide a means to isolate genetic markers from specific chromosomal regions. A small-insert BTA6 library, constructed by microdissection, was screened for dinucleotide repeats (CA)15 and (GA)15. A total of 47 new microsatellite loci were developed and tested for polymorphism and informativeness using the MARC bovine mapping family.
Chromosome-specific libraries aid in the development of genetic maps and focus marker development in areas of the genome with identified quantitative trait loci (QTL). A small-insert BTA29 library constructed by microdissection of a 1:29 Rb-fusion cell line, was screened for dinucleotide repeats (CA)(15) and/or (GA)(15) with the goal of generating new genetic markers for this, the smallest bovine autosome. A total of 90 primer pairs were designed and 82 of these successfully amplified bovine genomic DNA by PCR. In addition to these 82 loci, primer pairs were developed for nine putative genes identified from the sequenced clones by BLAST searches of GenBank. A somatic cell panel was used to test for synteny of the new loci with two previously mapped BTA29 markers located on the MARC bovine linkage map. A total of 75 of the 82 microsatellite (ms) loci were integrated into the MARC bovine linkage map. Linkage analysis placed 69 ms markers on BTA29, five on BTAX and one on BTA1. Combined results of the somatic cell and linkage analyses place 79 new markers (ms and gene-related) on BTA29, six loci on BTAX and two loci on BTA1. The results of this effort significantly increase the marker density on BTA29, expanding the ability to fine map QTL associated with this chromosome.
A chromosome-specific library was developed for Bos taurus autosome 11 by chromosome microdissection and microcloning using a bovine primary fibroblast culture, obtained from a t(X;23) heifer, that spontaneously developed a translocation chromosome involving bovine chromosome 11. The library was screened using (AC)12 oligos, positive clones selected, sequenced and primers developed to generate bovine chromosome 11-specific microsatellite markers. This study suggests that chromosome-specific libraries have great potential for development of microsatellite markers for the construction of marker-saturated linkage maps for each chromosome.
Polymorphic genetic markers are important tools in the construction of comprehensive genetic maps. This paper reports on the isolation and characterization of 90 new bovine microsatellite (ms) loci from enriched genomic libraries. The sequence of one clone (locus MNB-85) showed significant similarity to an intron of the human promyelocytic leukemia zinc finger protein (PLZF) gene. Screening of bovine and porcine somatic cell panels places the bovine PLZF homolog on BTA-15 and the porcine PLZF homolog on SSC-9. The 90 new microsatellite loci increase the number of microsatellites available for cattle by >5%.
This paper reports on the development of a small-insert (approximately 700 bp) total-genomic library for sheep specifically designed for enrichment for microsatellite (ms) loci. Four enriched libraries were prepared by amplification of the primary library with CA15, CA11, TG15 and TG11 oligonucleotide primers. A total of 11,020 clones was recovered, screened for dinucleotide repeats and over 500 positive clones sequenced. Sequence analysis indicated low clone redundancy and yielded 105 new ovine ms loci. Seventy-two percent of the new loci were found to be polymorphic in the sires of the AgResearch International Mapping Flock (IMF). The 105 new microsatellite loci increase the number of microsatellites available for sheep by >7%.
An autosomal scan of the swine genome with 119 polymorphic microsatellite (ms) markers and data from 116 F2 barrows of the University of Illinois Meishan x Yorkshire Swine Resource Families identified genomic regions with effects on variance in carcass composition and meat quality at nominal significance (p-value <0.05). Marker intervals on chromosomes 1, 6, 7, 8 and 12 (SSC1, SSC6, SSC7, SSC8, SSC12) with phenotypic effects on carcass length, 10th rib backfat thickness, average backfat thickness, leaf fat, loin eye area and intramuscular fat content confirm QTL effects identified previously based on genome wide significance (p-value <0.05). Several marker intervals included nominally significant (p-value <0.05) dominance effects on leaf fat, 10th rib backfat thickness, loin eye area, muscle pH and intramuscular fat content.
Explore the source record for details and available documents.
Cross-species amplification of 520 chicken microsatellite markers was tested by polymerase chain reaction with genomic DNA of the turkey (Meleagris gallopavo). Each primer pair was tested at six different combinations of annealing temperature and MgCl2 concentration. A total of 280 (54%) of the primer pairs produced amplification products. The majority of these products were similar, if not identical in size to those expected based on the fragment sizes of the corresponding chicken loci. Structure of the dinucleotide repeat and flanking sequences was examined for 13 turkey fragments (amplified with chicken primers) and 5 chicken fragments (amplified with turkey primers). Sequence analysis found a wide array of mutations between species in addition to differences in repeat length. To estimate the usefulness of the amplified loci for genetic mapping in the turkey, allelic polymorphism was determined for 57 of the 280 amplified loci. A total of 20 of 57 markers (35%) were polymorphic with an average of 1.4 alleles per locus. The results of this study suggest that approximately 20% of the chicken microsatellite markers will be useful for mapping the turkey genome.
A genomic scan of 18 swine autosomal chromosomes was constructed with 119 polymorphic microsatellite (ms) markers to identify quantitative trait loci (QTL) for 11 growth traits in the University of Illinois Meishan x Yorkshire Swine Resource Family. A significant QTL effect was found for post-weaning average daily gain (ADG) between 5.5 and 56 kg of body weight that mapped between markers SW373 and SW1301 near the telomere of Chromosome (Chr) 1 q (SSC1). This QTL effect had a nominal (pointwise) p-value of 0.000007, a genome wide p-value of 0.012, and accounted for 26% of the F2 phenotypic variance. The same chromosome region also had significant effects on ADG between birth and 56 kg body weight (p-value =. 000227), and on ADG between 35 and 56 kg (p-value =.00077). These observations suggest that a significant QTL for post-weaning growth resides on SSC1.
RNA fingerprinting by arbitrarily primed (RAP)-PCR was used to identify two bovine genes that were differentially expressed in epithelial cells during an inflammatory response. RNA fingerprints revealed two differentially amplified transcripts when monolayers of Madin-Darby bovine kidney (MDBK) cells were stimulated with Escherichia coli O157:H7 lipopolysaccharide (LPS) in combination with cycloheximide (CX). Sequence analysis showed that both transcripts encoded members of the alpha C-X-C chemokine family; one was interleukin 8 (IL-8), and the other was a protein closely related to bovine growth-regulated protein (GRO)-gamma (89% identical). The latter putative epithelial cell inflammatory protein was designated ECIP-1. IL-8 and ECIP-1 genes were placed on the cattle genetic map with single-nucleotide polymorphism (SNP) markers amplified from genomic DNA. Multi-point linkage analysis indicated that the gene locations were indistinguishable from those of serum albumin (ALB) and vitamin D-binding protein (GC) on bovine Chromosome (BTA) 6. In humans, ALB and GC are located near IL-8, GRO-gamma, and seven other alpha chemokines on Chr 4 (HSA 4q11-4q13), suggesting that this gene cluster has been conserved on BTA6. These results provide a starting point for characterizing allelic variation in chemokine genes and their role in the pathogenesis of bacterial infections in cattle.
Reproductive traits have low heritabilities, are expressed in only one sex, and are not measurable until sexual maturity (Avalos and Smith, Anim Prod 44:153, 1987). Using traditional methods, selection for reproductive traits is relatively less effective than selecting for growth or carcass traits. Traits most affected by a small number of genes with major effects rather than many genes with small effects are most amenable to MAS. As part of our porcine genome scan to identify quantitative trait loci (QTLs) of economic importance in marker-assisted selective (MAS) breeding programs, we examined 8 reproductive and farrowing traits in the University of Illinois (UI) Meishan x Yorkshire Resource Family. Gilts were genotyped with 119 microsatellite markers (MS) with intervals averaging 24 cM over all 18 porcine autosomes. F-ratios supporting QTL location were calculated by the least squares regression method. Results suggestive of linkage at the 5% genome-wide level were observed for the number of stillborn piglets on Chromosome (Chr) 4 (SSC4) (p-value = 0.0001), corpora lutea on SSC8 (p-value = 0.00027), and gestation length on SSC9 (p-value = 0.00019). Results for additional loci relevant to litter size, number of corpora lutea on SSC15 and 7 (p-value = 0.0029 and 0.0028 at 107 and 150 cM, respectively), gestation length on SSC15 and 1 (p-value = 0.0017 and 0.0069 at 96 and 166 cM, respectively), uterine length on SSC7 and 5 (p-value = 0.0044 and 0.0075 at 148 and 1 cM, respectively) and piglets born per litter on SSC6 (p-value = 0.0075 at 102 cM), were not statistically significant at the 5% genome-wide level. Thus, the use of a linked marker to facilitate selection for reproductive traits has considerable potential. By using linked markers, selection can be applied to both sexes before sexual maturity, making genetic selection considerably more efficient and less costly.
A whole-genome radiation hybrid (WG-RH) panel was used to generate a first-generation radiation map of the porcine (Sus scrofa) genome. Over 900 Type I and II markers were used to amplify the INRA-University of Minnesota porcine Radiation Hybrid panel (IMpRH) comprised of 118 hybrid clones. Average marker retention frequency of 29.3% was calculated with 757 scorable markers. The RHMAP program established 128 linkage groups covering each chromosome (n = 19) at a lod >/= 4.8. Fewer than 10% of the markers (59) could not be placed within any linkage group at a lod score >/=4.8. Linkage group order for each chromosome was determined by incorporating linkage data from the swine genetic map as well as physical assignments. The current map has an estimated ratio of approximately 70 kb/cR and a maximum theoretical resolution of 145 kb. This initial map forms a template for establishing accurate YAC and BAC contigs and eventual positional cloning of genes associated with complex traits.
We report the identification and radiation hybrid mapping of members of the proteasome/ubiquitin system in pigs that, so far, have only been identified in humans and cattle. Expressed sequence tags (ESTs) were constructed from ten oligo(dT)-primed individually tagged, directionally cloned and normalized cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN) and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic infection model. The ESTs mapped using the 7000 rad IMpRH panel (Hawken et al., 1999) were ubiquitin fusion-degradation 1 like protein (UFD1L), ubiquitin activating enzyme E1 and ubiquitin-S27a fusion protein which mapped to porcine chromosomes 14, 7 and X, respectively.
As part of the University of Minnesota's initiative to map the turkey genome, we are currently evaluating chicken microsatellite loci for use in mapping the turkey genome. To date, 141 primer pairs have been tested for amplification at six different combinations of temperature and MgCl2 concentration. Microsatellite primer pairs from the Chicken Comprehensive Mapping Kit #2, and additional unpublished chromosome 1 and 2 primers were screened. Analyzable PCR products were produced from 78 of the 141 (55%) primer combinations. In the majority of cases (68%), PCR fragments obtained from the turkey were similar in size to respective chicken loci. The presence of dinucleotide repeats (CA/TG repeats) was determined by Southern hybridization with a (TG)15, oligonucleotide probe. Five of 12 (41.63%) turkey fragments hybridized under low stringency conditions. The length of the dinucleotide repeats in the turkey, relative to the chicken sequences, were found to correspond directly with hybridization intensity. Amplification of homologous loci was confirmed by direct sequencing and subsequent alignment of the turkey and chicken sequences. The results of this study indicate that the use of chicken-specific microsatellite primers will rapidly and significantly enhance construction of a genetic map for the turkey.
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.
We report the use of microsatellites (MS) to track the recent evolution of swine. Allelic frequencies for nine MS loci linked on swine chromosome 6 (SSC6) representing four western and one Chinese swine breeds were used to estimate genetic distances and times of breed divergence. A phylogenetic tree was constructed which partitioned into western and Meishan breed branches. Yorkshire and Hampshire breeds exhibited the most recent divergence with a calculated distance of 391 years. The oldest divergence, of 2,227 years, was between Meishan and Hampshire swine. Estimates of breed divergence are consistent with historical records. Additional analysis suggests that polymorphic MS linked on a single chromosome are sufficient to determine evolutionary relationships within a single species.
Previous studies suggest the median allele length of microsatellites is longest in the species from which the markers were derived, suggesting that an ascertainment bias was operating. We have examined whether the size distribution of microsatellite alleles between sheep and cattle is source dependent using a set of 472 microsatellites that can be amplified in both species. For those markers that were polymorphic in both species we report a significantly greater number of markers (P < 0.001) with longer median allele sizes in sheep, regardless of microsatellite origin. This finding suggests that any ascertainment bias operating during microsatellite selection is only a minor contributor to the variation observed.
A genetic map of Ovis aries (haploid n = 27) was developed with 519 markers (504 microsatellites) spanning approximately 3063 cM in 26 autosomal linkage groups and 127 cM (female specific) of the X Chromosome (Chr). Genotypic data were merged from the IMF flock (Crawford et al., Genetics 140, 703, 1995) and the USDA mapping flock. Seventy-three percent (370/504) of the microsatellite markers on the map are common to the USDA-ARS MARC cattle linkage map, with 27 of the common markers derived from sheep. The number of common markers per homologous linkage group ranges from 5 to 22 and spans a total of 2866 cM (sex average) in sheep and 2817 cM in cattle. Marker order within a linkage group was consistent between the two species with limited exceptions. The reported translocation between the telomeric end of bovine Chr 9 (BTA 9) and BTA 14 to form ovine Chr 9 is represented by a 15-cM region containing 5 common markers. The significant genomic conservation of marker order will allow use of linkage maps in both species to facilitate the search for quantitative trait loci (QTLs) in cattle and sheep.