Description of bovine major histocompatibility complex class IIa haplotypes using parthenogenetic embryo-derived cells.

In this study, we report an approach to characterize individual BoLA haplotypes using cells from parthenogenetic bovine embryos derived from slaughterhouse ovaries. Eight of the 15 parthenogenetic embryos so obtained had not undergone meiotic recombination on the BoLA region and were suitable to describe BoLA haplotypes. Detailed analysis of the BoLA class IIa region identified seven different class IIa haplotypes, including six not previously described and two new alleles of BoLA-DQA and one BoLA-DQB. Our method provided reliable sources of homozygous DNA to describe BoLA haplotypes.

Microsatellite variation in the equine MHC.

Genes within the major histocompatibility complex (MHC) encode proteins involved in innate and adaptive immune responses. Genetic variation in this region can influence the immune response of an individual animal to challenges from a variety of pathogens; however, a complete documentation of genetic variation in the MHC is lacking for most domestic animals, including horses. To provide additional genetic markers for study of the horse MHC, or ELA (equine lymphocyte antigen), we identified 37 polymorphic microsatellite repeats in ELA and used these variations separately and together with published SNPs to investigate linkage disequilibrium (LD) and haplotype structure in a sample of Thoroughbred horses. ELA SNPs alone detected little LD, but microsatellites, either separately or combined with SNPs, revealed substantially more LD. A subset of markers in very high LD across the breadth of ELA may be predictive of structural polymorphisms or linked epistases that are important drivers of haplotype structure in Thoroughbreds.

A high-resolution radiation hybrid map of the river buffalo major histocompatibility complex and comparison with BoLA.

The major histocompatibility complex (MHC) in mammals codes for antigen-presenting proteins. For this reason, the MHC is of great importance for immune function and animal health. Previous studies revealed this gene-dense and polymorphic region in river buffalo to be on the short arm of chromosome 2, which is homologous to cattle chromosome 23. Using cattle-derived STS markers and a river buffalo radiation hybrid (RH) panel (BBURH5000 ), we generated a high-resolution RH map of the river buffalo MHC region. The buffalo MHC RH map (cR5000 ) was aligned with the cattle MHC RH map (cR12000 ) to compare gene order. The buffalo MHC had similar organization to the cattle MHC, with class II genes distributed in two segments, class IIa and class IIb. Class IIa was closely associated with the class I and class III regions, and class IIb was a separate cluster. A total of 53 markers were distributed into two linkage groups based on a two-point LOD score threshold of ≥8. The first linkage group included 32 markers from class IIa, class I and class III. The second linkage group included 21 markers from class IIb. Bacterial artificial chromosome clones for seven loci were mapped by fluorescence in situ hybridization on metaphase chromosomes using single- and double-color hybridizations. The order of cytogenetically mapped markers in the region corroborated the physical order of markers obtained from the RH map and served as anchor points to align and orient the linkage groups.

64×64 pixel smart sensor array for laser Doppler blood flow imaging.

What is believed to be the first fully integrated two-dimensional complementary metal oxide semiconductor (CMOS) imaging array for laser Doppler blood flow imaging is demonstrated. The sensor has 64×64 pixels and includes both analog and digital on-chip processing electronics. This offers several potential advantages over commercial sensors as the processing is tailored to the signals of interest and the data bottleneck that exists between the sensor and processing electronics is overcome. To obtain a space efficient design over 64×64 pixels means that standard processing electronics used off-chip cannot be implemented. Images of both simulated blood flow responses and a blood flow occlusion test demonstrate the capability.

Construction and preliminary characterization of a river buffalo bacterial artificial chromosome library.

River buffalo genome analyses have advanced significantly in the last decade, and the genome sequence of Bubalus bubalis will be available shortly. Nonetheless, large-insert DNA library resources such as bacterial artificial chromosomes (BAC) are still required for validation and accurate assembly of the genome sequence. We constructed a river buffalo BAC library containing 52,224 clones with an average insert size of 97 kb, representing 1.7 × coverage of the genome. This genomic resource for river buffalo will facilitate further studies in this economically important species allowing for instance, whole genome physical mapping and isolation of genes and gene clusters, contributing to the elucidation of gene organization and identification of regulatory elements.

Mapping MHC genes in river buffalo.

The major histocompatibility complex (MHC) contains a set of genes necessary for antigen presentation in the immune system. This gene dense and polymorphic region of the mammalian genome is of considerable interest due to the role of MHC genes in immune function and animal health. Previous cytogenetic studies have indicated that the MHC in river buffalo resides on the short arm of chromosome 2 (BBU2). A 5000-rad radiation hybrid mapping panel was recently generated to enable construction of a whole genome map of river buffalo. To this end, the aims of this project were to elucidate the general organization of the MHC on BBU2, and to compare gene order within this region to the MHC in cattle. PCR primers were selected from the bovine gene map and used with the BBURH5000 panel to map a set of ten MHC class II genes in river buffalo. Analysis indicates that these genes fall into two linkage groups, consistent with organization of the MHC in cattle. This comparison of buffalo and bovine MHC gene order provides the first insight into the organization of the MHC on river buffalo chromosome 2.

Assignment of six genes to bovine chromosomes 5 and 16 by fluorescence in situ hybridization, radiation hybrid mapping and genetic linkage analysis.

Several quantitative trait loci for beef carcass traits have been mapped to bovine chromosome 5. The objective of this study was to map six candidate genes for these traits by fluoresence in situ hybridization, genetic linkage analysis and radiation hybrid mapping. MYF5 and MYF6 were assigned to 5q13, WIF1 to 5q23 and MMP19 to 5q25. A paralog of MYF5 (putatively MYOG) was assigned to 16q12. A novel microsatellite placed MYF5 and MYF6 10.4 cM from BM6026 and 19.1 cM from BL23 on the genetic linkage map. MYF5 (62.6 cR), WNT10B (319.5 cR), WIF1 (500.8 cR) and MMP19 (701.2 cR) were also integrated into the 5000(Rad) radiation hybrid map.

Association of udder traits with single nucleotide polymorphisms in crossbred Bos indicus-Bos taurus cows.

The size, support, and health of udders limit the productive life of beef cows, especially those with background, because, in general, such cows have a reputation for problems with udders. Genomic association studies of bovine udder traits have been conducted in dairy cattle and recently in Continental European beef breeds but not in cows with background. The objective of this study was to determine associations of SNP and udder support scores, teat length, and teat diameter in half (Nellore), half (Angus) cows. Udders of cows ( = 295) born from 2003 to 2007 were evaluated for udder support and teat length and diameter ( = 1,746 records) from 2005 through 2014. These included a subjective score representing udder support (values of 1 indicated poorly supported, pendulous udders and values of 9 indicated very well-supported udders) and lengths and diameters of individual teats in the 4 udder quarters as well as the average. Cows were in full-sibling or half-sibling families. Residuals for each trait were produced from repeated records models with cow age category nested within birth year of cows. Those residuals were averaged to become the dependent variables for genomewide association analyses. Regression analyses of those dependent variables included genotypic values as explanatory variables for 34,980 SNP from a commercially available array and included the genomic relationship matrix. Fifteen SNP loci on BTA 5 were associated (false discovery rate controlled at 0.05) with udder support score. One of those was also detected as associated with average teat diameter. Three of those 15 SNP were located within genes, including one each in (), (), and (). These are notable for their functional role in some aspect of mammary gland formation or health. Other candidate genes for these traits in the vicinity of the SNP loci include () and (). Because these were detected in Nellore-Angus crossbred cows, which typically have very well-formed udders with excellent support across their productive lives, similar efforts in other breeds should be completed, because that may facilitate further refinement of genomic regions responsible for variation in udder traits important in multiple breeds.

Carcass merit between and among family groups of Bos indicus crossbred steers and heifers.

Differences in live and carcass traits attributable to increasing Bos indicus breed influence were compared to the differences between families with similar proportions of B. indicus influence. Families of offspring from 1/2 Angus×1/2 B. indicus mated to Angus, B. indicus, and 1/2 Angus×1/2 B.indicus were raised under similar conditions. Average daily gain, slaughter weight, and dressing percentage were measured in addition to USDA yield and quality grade factors. Breed type did not affect average daily gain, slaughter weight, dressing percentage, carcass weight, adjusted 12th-rib fat thickness, estimated percentage kidney, pelvic, and heart fat, or carcass maturity. Predominately (3/4) Angus progeny produced greater (P<0.05) longissimus muscle areas than 3/4 B. indicus animals. Predominately Angus cattle also had greater (P<0.05) marbling scores and USDA quality grades than predominately B. indicus cattle. Families within breed types differed (P<0.05) with regard to all traits measured. This is interesting in light of the lack of differences between breeds for most traits. In some instances, the differences in marbling score and longissimus muscle area between families within a given breed type were similar or greater in magnitude than the differences observed between predominately Angus and predominately B. indicus breed types. Whereas growth and carcass traits varied between levels of B. indicus breeding, the opportunity does exists to improve these traits by selecting within specific family lines.

Crossbred steer temperament as yearlings and whole genome association of steer temperament as yearlings and calf temperament post-weaning.

cattle often have the reputation for a poor or dangerous temperament. Identification of genomic regions that associate with temperament of such cattle may be useful for genetic improvement strategies. The objectives of this study were to evaluate subjective temperament scores (1 to 9; higher scores indicated more unfavorable temperament) for aggressiveness, nervousness, flightiness, gregariousness, and overall temperament of one-half steers in feedlot conditions at 1 yr of age and compare those scores of those steers when evaluated approximately 1 mo postweaning, and conduct whole genome association analyses using SNP markers and the temperament traits of those steers at 1 yr of age and for temperament traits of all calves at weaning. Contemporary groups ( < 0.001) were steers born in the same year and season, and fed in the same feedlot pen. Aggressiveness of steers at 1 yr of age was not associated with aggressiveness at weaning (linear regression coefficient did not differ from 0; = 0.96), but regressions of all other yearling scores of steers on the scores at weaning were positive (coefficients ranged from 0.26 ± 0.04 to 0.32 ± 0.04; < 0.001). Estimates of Pearson correlation coefficients (using unadjusted values and residual values) of the different traits measured at 1 yr of age were large ( > 0.63; < 0.008) except for aggressiveness with nervousness, flightiness, or gregariousness, which did not differ from 0 ( > 0.1). Five SNP on BTA 1, 24, and 29 had suggestive associations (0.17 < [adjusted for FDR] < 0.24) with aggressiveness, nervousness, or flightiness at evaluation postweaning and 13 SNP on 11 chromosomes had suggestive associations (0.07 < [adjusted for FDR] < 0.24) with aggressiveness, nervousness, flightiness, or overall temperament score of steers at 1 yr of age. Genes close to these loci with roles in neural systems of various organisms included synaptotagmin 4 (BTA 24), FAT atypical cadhedrin 3 (BTA 29), tubulin tyrosine ligase-like 1 (BTA 5), spermatogenesis associated 17 (BTA 16), stanniocalcin 2 (BTA 20), and GABA receptor γ 3 (BTA 21).

Adiposity, lipogenesis, and fatty acid composition of subcutaneous and intramuscular adipose tissues of Brahman and Angus crossbred cattle.

The objective of this study was to demonstrate differences in aspects of adipose tissue cellularity, lipid metabolism, and fatty and cholesterol composition in Angus and Brahman crossbred cattle. We hypothesized that in vitro measures of lipogenesis would be greater in three-fourths Angus progeny than in three-fourths Brahman progeny, especially in intramuscular (i.m.) adipose tissue. Progeny ( = 227) were fed a standard, corn-based diet for approximately 150 d before slaughter. Breed was considered to be the effect of interest and was forced into the model. There were 9 breed groups including all 4 kinds of three-fourths Angus calves: Angus bulls Angus-sired F cows ( = 32), Angus bulls Brahman-sired F cows ( = 20), Brahman-sired F bulls Angus cows ( = 24), and Angus-sired F bulls Angus cows ( = 20). There were all 4 kinds of three-fourths Brahman calves: Brahman bulls Brahman-sired F cows ( = 21), Brahman bulls Angus-sired F cows ( = 43), Brahman-sired F bulls Brahman cows ( = 26), and Angus-sired F bulls Brahman cows ( = 13). Additionally, F calves (one-half Brahman and one-half Angus) were produced only from Brahman-sired F bulls Angus-sired F cows ( = 28). Contrasts were calculated when breed was an important fixed effect, using the random effect family(breed) as the error term. Most contrasts were nonsignificant ( > 0.10). Those that were significant ( < 0.05) included cholesterol concentration of subcutaneous (s.c.) adipose tissue (three-fourths Angus > F, three-fourths Brahman > F, and three-fourths crossbred progeny combined > F), s.c. adipocyte volume (three-fourths Angus > F and three-fourths bloods combined > F), lipogenesis from acetate in s.c. adipose tissue (three-fourths Brahman calves from Brahman dams > three-fourths Brahman calves from F dams), and percentage 18:3-3 in s.c. adipose tissue (three-fourths Brahman calves from Brahman-sired F dams < three-fourths Brahman calves from Angus-sired F dams). Intramuscular adipocyte volume ( < 0.001) was less in three-fourths Brahman cattle than in three-fourths Angus cattle. Additionally, several differences were observed in i.m. adipose tissue that were consistent with this being a less-developed adipose tissue in three-fourths Brahman cattle than in three-fourths Angus cattle.

A comparative map of bovine chromosome 19 based on a combination of mapping on a bacterial artificial chromosome scaffold map, a whole genome radiation hybrid panel and the human draft sequence.

We have constructed a medium density physical map of bovine chromosome 19 using a combination of mapping loci on both a bovine bacterial artificial chromosome (BAC) scaffold map and a whole genome radiation hybrid (WGRH) panel. The resulting map contains 70 loci spanning the length of bovine chromosome 19. Three contiguous groups of BACs were identified on the basis of multiple loci mapping to individual BAC clones. Bovine chromosome 19 was found in this study to be comprised almost entirely from regions of human chromosome 17, with a small region putatively assigned to human chromosome 10. Fourteen breakpoints between the bovine and human chromosomes were detected, with a possibility of five more based on ordering of the WGRH map.

alpha-Adrenergic agents. 3. Behavioral effects of 2-aminotetralins.

Studies with 5-substituted-8-methoxy-2-amino-tetralin compounds suggest that some are alpha 1-adrenoceptor agonists, which readily penetrate the blood-brain barrier. They potentiate the locomotor activity that is induced by apomorphine (AP) in reserpinized mice, an effect that has been suggested to result from activation of central alpha-receptors. This effect is selectively blocked by the preferential alpha 1-antagonist phenoxybenzamine, but not by drugs that block other types of receptors. The effect is also produced by the centrally administered alpha 1-agonists phenylephrine and methoxamine, but not by various types of standard CNS stimulants. When administered in high doses, some of the aminotetralin compounds induce locomotor activity in reserpinized mice without AP, an effect also found with high doses of centrally administered phenylephrine and methoxamine. This effect is blocked by a series of drugs at doses that correspond to their alpha 1-antagonist potencies.

Genetic evaluation of aspects of temperament in Nellore-Angus calves.

The objective of this work was to estimate heritability of each of 5 subjectively measured aspects of temperament of cattle and the genetic correlations of pairs of those traits. From 2003 to 2013, Nellore-Angus F2 and F3 calves (n = 1,816) were evaluated for aspects of temperament at an average 259 d of age, which was approximately 2 mo after weaning. Calves were separated from a group and subjectively scored from 1 (calm, good temperament) to 9 (wild, poor temperament) for aggressiveness (willingness to hit an evaluator), nervousness, flightiness, gregariousness (willingness to separate from the group), and a distinct overall score by 4 evaluators. Data were analyzed using threshold and linear models with additive genetic random effects. Two-trait animal models (nonthreshold) included the additive genetic covariance for pairs of traits and were used to estimate additive genetic correlations. Contemporary groups (n = 104) represented calves penned together for evaluation on given evaluation days. Heifers had greater (worse) means for all traits than steers (P < 0.05). The regression of score on age in days was included in final models for flightiness (P = 0.05; -0.006 ± 0.003) and gregariousness (P = 0.025; -0.007 ± 0.003). Estimates of heritability were large (0.51, 0.4, 0.45, 0.49, and 0.47 for aggressiveness, nervousness, flightiness, gregariousness, and overall temperament, respectively; SE = 0.07 for each). The ability to use this methodology to distinctly separate different aspects of calf temperament appeared to be limited, as estimates of additive genetic correlations were near unity for all pairs of traits; estimates of phenotypic correlation ranged from 0.88 ± 0.01 to 0.99 ± 0.002 for pairs of traits. Distinct subsequent analyses indicated a significant negative relationship of 4 of the various temperament scores with weight at weaning (regression coefficients ranged from -0.008 ± 0.002 for nervousness, flightiness, and gregariousness to -0.003 ± 0.002 for aggressiveness). In subsequent analyses, the regression of temperament trait on sequence of evaluation within a pen was highly significant and solutions ranged from 0.05 ± 0.007 for aggressiveness to 0.08 ± 0.007 for all other traits. The apparent large additive genetic variance for any one of these traits may be useful in identification of genes responsible for differences in cattle temperament.

Evaluation of reciprocal differences in Bos indicus x Bos taurus backcross calves produced through embryo transfer: I. Birth and weaning traits.

Angus (A) and Bos indicus (B; Brahman or Nellore) reciprocal backcross, embryo transfer calves, belonging to 28 full-sib families, were evaluated for differences in birth weight, gestation length, and weaning weight. Two methods were investigated; method I made no distinction between how the F(1) parents were produced, whereas method II distinguished between the 2 types of F(1) parents (AB vs. BA corresponding to A x B vs. B x A, respectively). Bos indicus backcross calves had a 4.3 d longer (P < 0.05) gestation length but did not differ in their average birth weight from A backcrosses. Among B backcrosses, B x F(1) calves had a 5.2 d longer (P = 0.01) gestation length than F(1) x B calves (290.5 vs. 285.3, respectively). Under method II analysis, there was a consistent trend for gestation length, in which BA F(1) parents produced calves that ranked greater than calves from AB F(1) parents, as sires and dams. Crosses with a greater proportion of B in the sire in relation to the amount in the dam had a heavier (P < 0.05) birth weight (F(1) x A and B x F(1); 38.1 and 38.4 kg, respectively) than their respective reciprocal crosses (A x F(1) and F(1) x B; 34.3 and 33.5 kg, respectively). The F(1) x A and B x F(1) crosses showed a large difference in birth weight between males and females (5.3 and 4.1 kg, respectively), whereas A x F(1) and F(1) x B crosses showed a small difference (P > 0.10) in birth weight between males and females (1.5 and 1.1 kg, respectively). Further examination within each sex showed a difference between male reciprocals that was generally much larger than that between female reciprocals. Calves with a greater percentage of B in the sire compared with the proportion in the dam ranked heavier for weaning weight as for birth weight, though these differences were not significant. In breeding systems involving B x Bos taurus crosses, even when using embryo transfer, not only does the breed composition of the calves affect their preweaning performance, but the particular cross that produces the calves also should be considered in making breeding decisions.

Evaluation of reciprocal differences in Bos indicus x Bos taurus backcross calves produced through embryo transfer: II. Postweaning, carcass, and meat traits.

Angus (A) x Bos indicus (B; Brahman or Nellore) reciprocal backcross, embryo transfer calves belonging to 28 full-sib families were evaluated for differences in feedyard initial BW, feedyard final BW, carcass weight, LM area, adjusted fat thickness, intramuscular fat, and Warner-Bratzler shear force. Two methods of analysis were investigated; method I made no distinction between how the F(1) parents were produced, whereas method II distinguished the 2 types of F(1) parents (AB vs. BA, corresponding to A x B vs. B x A, respectively). No significant reciprocal differences for these weight and carcass traits were detected under method I analyses, although the same trend existed for subsequent BW rankings as for birth weight and weaning weight. For each weight phase, the cross that involved a larger proportion of B in the sire in relation to the amount in the dam (F(1) x A and B x F(1)) ranked heavier than the respective reciprocal cross (A x F(1) and F(1) x B). As a whole, A backcross calves had larger (P < 0.001) LM area, more (P < 0.001) marbling, and lower (P < 0.001) Warner-Bratzler shear force than B back-cross calves, but no consistent trends were detected between reciprocal crosses for any of these traits, in contrast with the trends observed for the weight traits. Furthermore, males were heavier than females entering (P < 0.001) and leaving (P < 0.001) the feedyard, produced a heavier carcass (P < 0.001), and had larger LM area (P < 0.05) with less adjusted fat (P < 0.001). No difference existed between the sexes for Warner-Bratzler shear force or marbling. No interactions involving sex, sire type, and dam type were observed for any of these traits. The results were similar under methods I and II analyses, with the exception that a significant sire type x dam type interaction was observed for initial feedyard BW. Results from this study suggest that for weight-related traits, both the breed constitution of the embryo transfer calf and the cross that produces the calf play an important role in its ultimate performance for B crossbred calves. For body composition and meat-related traits, it appears that the breed makeup of the embryo transfer calf itself is more important to animal performance than the specific cross used to produce the calf.

Validation of commercial DNA tests for quantitative beef quality traits.

Associations between 3 commercially available genetic marker panels (GeneSTAR Quality Grade, GeneSTAR Tenderness, and Igenity Tender-GENE) and quantitative beef traits were validated by the US National Beef Cattle Evaluation Consortium. Validation was interpreted to be the independent confirmation of the associations between genetic tests and phenotypes, as claimed by the commercial genotyping companies. Validation of the quality grade test (GeneSTAR Quality Grade) was carried out on 400 Charolais x Angus crossbred cattle, and validation of the tenderness tests (GeneSTAR Tenderness and Igenity Tender-GENE) was carried out on over 1,000 Bos taurus and Bos indicus cattle. The GeneSTAR Quality Grade marker panel is composed of 2 markers (TG5, a SNP upstream from the start of the first exon of thyroglobulin, and QG2, an anonymous SNP) and is being marketed as a test associated with marbling and quality grade. In this validation study, the genotype results from this test were not associated with marbling score; however, the association of substituting favorable alleles of the marker panel with increased quality grade (percentage of cattle grading Choice or Prime) approached significance (P < or = 0.06), mainly due to the effect of 1 of the 2 markers. The GeneSTAR Tenderness and Igenity TenderGENE marker panels are being marketed as tests associated with meat tenderness, as assessed by Warner-Bratzler shear force. These marker panels share 2 common mu-calpain SNP, but each has a different calpastatin SNP. In both panels, there were highly significant (P < 0.001) associations of the calpastatin marker and the mu-calpain haplotype with tenderness. The genotypic effects of the 2 tenderness panels were similar to each other, with a 1 kg difference in Warner-Bratzler shear force being observed between the most and least tender genotypes. Unbiased and independent validation studies are important to help build confidence in marker technology and also as a potential source of data required to enable the integration of marker data into genetic evaluations. As DNA tests associated with more beef production traits enter the marketplace, it will become increasingly important, and likely more difficult, to find independent populations with suitable phenotypes for validation studies.

Construction of bovine whole-genome radiation hybrid and linkage maps using high-throughput genotyping.

High-density whole-genome maps are essential for ordering genes or markers and aid in the assembly of genome sequence. To increase the density of markers on the bovine radiation hybrid map, and hence contribute to the assembly of the bovine genome sequence, an Illumina BeadStation was used to simultaneously type large numbers of markers on the Roslin-Cambridge 3000 rad bovine-hamster whole-genome radiation hybrid panel (WGRH3000). In five multiplex reactions, 6738 sequence tagged site (STS) markers were successfully typed on the WGRH3000 panel DNA. These STSs harboured SNPs that were developed as a result of the bovine genome sequencing initiative. Typically, the most time consuming and expensive part of creating high-density radiation hybrid (RH) maps is genotyping the markers on the RH panel with conventional approaches. Using the method described in this article, we have developed a high-density whole-genome RH map with 4690 loci and a linkage map with 2701 loci, with direct comparison to the bovine whole-genome sequence assembly (Btau_2.0) in a fraction of the time it would have taken with conventional typing and genotyping methods.

A 2.5-Mb contig constructed from Angus, Longhorn and horned Hereford DNA spanning the polled interval on bovine chromosome 1.

The polled locus has been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. As an intermediate step in our efforts to identify the polled locus and the underlying causative mutation for the polled phenotype, we have constructed a BAC-based physical map of the interval containing the polled locus. Clones containing genes and markers in the critical interval were isolated from the TAMBT (constructed from Angus and Longhorn genomic DNA) and CHORI-240 (constructed from horned Hereford genomic DNA) BAC libraries and ordered based on fingerprinting and the presence or absence of 80 STS markers. A single contig spanning 2.5 Mb was assembled. Comparison of the physical order of STSs to the corresponding region of human chromosome 21 revealed the same order of genes within the polled critical interval. This contig of overlapping BAC clones from horned and polled breeds is a useful resource for SNP discovery and characterization of positional candidate genes.