Differences in intestinal size, structure, and function contributing to feed efficiency in broiler chickens reared at geographically distant locations.

The contribution of the intestinal tract to differences in residual feed intake (RFI) has been inconclusively studied in chickens so far. It is also not clear if RFI-related differences in intestinal function are similar in chickens raised in different environments. The objective was to investigate differences in nutrient retention, visceral organ size, intestinal morphology, jejunal permeability and expression of genes related to barrier function, and innate immune response in chickens of diverging RFI raised at 2 locations (L1: Austria; L2: UK). The experimental protocol was similar, and the same dietary formulation was fed at the 2 locations. Individual BW and feed intake (FI) of chickens (Cobb 500FF) were recorded from d 7 of life. At 5 wk of life, chickens (L1, n = 157; L2 = 192) were ranked according to their RFI, and low, medium, and high RFI chickens were selected (n = 9/RFI group, sex, and location). RFI values were similar between locations within the same RFI group and increased by 446 and 464 g from low to high RFI in females and males, respectively. Location, but not RFI rank, affected growth, nutrient retention, size of the intestine, and jejunal disaccharidase activity. Chickens from L2 had lower total body weight gain and mucosal enzyme activity but higher nutrient retention and longer intestines than chickens at L1. Parameters determined only at L1 showed increased crypt depth in the duodenum and jejunum and enhanced paracellular permeability in low vs. high RFI females. Jejunal expression of IL1B was lower in low vs. high RFI females at L2, whereas that of TLR4 at L1 and MCT1 at both locations was higher in low vs. high RFI males. Correlation analysis between intestinal parameters and feed efficiency metrics indicated that feed conversion ratio was more correlated to intestinal size and function than was RFI. In conclusion, the rearing environment greatly affected intestinal size and function, thereby contributing to the variation in chicken RFI observed across locations.

Assessing serum metabolite profiles as predictors for feed efficiency in broiler chickens reared at geographically distant locations.

1. The objective of this study was to investigate differences in growth performance, serum intermediary metabolites, acute-phase proteins and white blood cells in low, medium and high-residual feed intake (RFI) chickens. It was also assessed if the environment affects the feed efficiency (FE) and FE-related performance and serum profiles of chickens. 2. Individual body weight (BW) and feed intake (FI) were recorded from d 7 of life. At 5 weeks of age, female and male broiler chickens (Cobb 500) were selected according to their RFI (L1: Austria; L2: UK; n = 9/RFI group, sex and locatity -45on) and blood samples were collected. 3. Chickens at L1 had similar FI but a 15% higher BW gain compared to chickens at L2. The RFI values of female chickens were -231, 8 and 215 g and those of male chickens -197, 0 and 267 g for low, medium and high RFI, respectively. 4. Location affected serum glucose, urea, cholesterol, non-esterified fatty acids (NEFA) and ovotransferrin in females, and serum glucose and triglycerides in male chickens. Serum uric acid and NEFA linearly increased from low to high RFI in females, whereas in males, cholesterol showed the same linear response from low to high RFI. Serum alpha-1-acid glycoprotein and blood heterophil-to-lymphocyte ratio linearly increased by 35% and 68%, respectively, from low to high RFI but only in male chickens at L1. 5. Regression analysis showed significant positive relationships between RFI and serum uric acid (R2 = 0.49) and cholesterol (R2 = 0.13). 6. It was concluded that RFI-related variation in serum metabolites of chickens was largely similar for the two environments and that serum metabolite patterns could be used to predict RFI in chickens.

Data compression can discriminate broilers by selection line, detect haplotypes, and estimate genetic potential for complex phenotypes.

Accurately establishing the relationships among individuals lays the foundation for genetic analyses such as genome-wide association studies and identification of selection signatures. Of particular interest to the poultry industry are estimates of genetic merit based on molecular data. These estimates can be commercially exploited in marker-assisted breeding programs to accelerate genetic improvement. Here, we test the utility of a new method we have recently developed to estimate animal relatedness and applied it to genetic parameter estimation in commercial broilers. Our approach is based on the concept of data compression from information theory. Using the real-world compressor gzip to estimate normalized compression distance (NCD) we have built compression-based relationship matrices (CRM) for 988 chickens from 4 commercial broiler lines-2 male and 2 female lines. For all pairs of individuals, we found a strong negative relationship between the commonly used genomic relationship matrix (GRM) and NCD. This reflects the fact that "similarity" is the inverse of "distance." The CRM explained more genetic variation than the corresponding GRM in 2 of 3 phenotypes, with corresponding improvements in accuracy of genomic-enabled predictions of breeding value. A sliding-window version of the analysis highlighted haplotype regions of the genome apparently under selection in a line-specific manner. In the male lines, we retrieved high population-specific scores for IGF-1 and a cognate receptor, INSR. For the female lines, we detected an extreme score for a region containing a reproductive hormone receptor (GNRHR). We conclude that our compression-based method is a valid approach to established relationships and identify regions under selective pressure in commercial lines of broiler chickens.

Comparison of growth performance and excreta composition in broiler chickens when ranked according to various feed efficiency metrics.

Several feed efficiency (FE) metrics are currently used in livestock production to select for improved FE. Whether or not different FE metrics similarly estimate physiological characteristics in chickens of diverging FE has not been reported so far. This study aimed to assess potential differences in feed intake (FI), performance, and nutrient excretion in broiler chickens of diverging FE when ranked according to their residual FI (RFI), residual BW gain (RBG), RFI and BW gain (RIG), and G:F between d 7 and 35 of life. The FI was determined daily and BW was recorded once a week. The ranking of chickens into good, medium, and poor FE groups was completed separately for each FE metric. Freshly dropped excreta were collected for pH and DM measurements on d 30 to 32 of life and total excreta for determination of nutrient excretion was collected on d 34 to 36 of life. Relationships among FE metrics were evaluated using regression analysis showing that RFI, RIG, and G:F were more related to each other than to RBG. The FE values greatly varied among chickens for all FE metrics and chickens did not always cluster within the same FE group when using RFI, RIG, RBG, and G:F as the FE metrics because of the calculation approaches. Due to sex-related differences in performance, data of male and female chickens were analyzed separately. The RFI and RIG metrics showed a linear increase ( < 0.01) in total FI from good to poor FE in male and female chickens, whereas G:F showed this effect ( ≤ 0.011) only when BW gain was standardized to 1,500 g. The RBG did not clearly select chickens of enhanced total BW gain and only tended ( < 0.1) to select for greater BW gain from good to poor FE in female chickens. Excreta pH linearly decreased by 0.7 log units and DM content increased in males from good to poor FE when using RFI and RIG, respectively ( < 0.01). In both sexes, RFI ( < 0.05) and RIG metrics ( ≤ 0.06) showed a linear increase in daily nitrogen excretion from good to poor FE. In conclusion, results demonstrate that selection of the metric used to determine the FE of chickens modified the results obtained for comparison of production parameters and nutrient excretion among FE groups. Thereby, the RFI, RIG, and G:F metrics were beneficial in selecting the most feed efficient chickens to reduce feed costs, whereas the use of RFI and RIG may be better to select chickens with improved nitrogen retention and thus reduced excretion of an environmental pollutant.

Evidence for pleiotropism and recent selection in the PLAG1 region in Australian Beef cattle.

A putative functional mutation (rs109231213) near PLAG1 (BTA14) associated with stature was studied in beef cattle. Data from 8199 Bos taurus, Bos indicus and Tropical Composite cattle were used to test the associations between rs109231213 and various phenotypes. Further, 23 496 SNPs located on BTA14 were tested for association with these phenotypes, both independently and fitted together with rs109231213. The C allele of rs109231213 significantly increased hip height, weight, net food intake, age at puberty in males and females and decreased IGF-I concentration in blood and fat depth. When rs109231213 was fitted as a fixed effect in the model, there was an overall reduction in associations between other SNPs and these traits but some SNPs remained associated (P < 10(-4) ). Frequency of the mutant C allele of rs109231213 differed among B. indicus (0.52), B. taurus (0.96) and Tropical Composite (0.68). Most chromosomes carrying the C allele had the same surrounding 10 SNP haplotype, probably because the C allele was introgressed into Brahman from B. taurus cattle. A region of reduced heterozygosity surrounds the C allele; this is small in B. taurus but 20 Mb long in Brahmans, indicating recent and strong selection for the mutant allele. Thus, the C allele appears to mark a mutation that has been selected almost to fixation in the B. taurus breeds studied here and introduced into Brahman cattle during grading up and selected to a frequency of 0.52 despite its negative effects on fertility.

Gene network analyses of first service conception in Brangus heifers: use of genome and trait associations, hypothalamic-transcriptome information, and transcription factors.

Measures of heifer fertility are economically relevant traits for beef production systems and knowledge of candidate genes could be incorporated into future genomic selection strategies. Ten traits related to growth and fertility were measured in 890 Brangus heifers (3/8 Brahman × 5/8 Angus, from 67 sires). These traits were: BW and hip height adjusted to 205 and 365 d of age, postweaning ADG, yearling assessment of carcass traits (i.e., back fat thickness, intramuscular fat, and LM area), as well as heifer pregnancy and first service conception (FSC). These fertility traits were collected from controlled breeding seasons initiated with estrous synchronization and AI targeting heifers to calve by 24 mo of age. The BovineSNP50 BeadChip was used to ascertain 53,692 SNP genotypes for ∼802 heifers. Associations of genotypes and phenotypes were performed and SNP effects were estimated for each trait. Minimally associated SNP (P < 0.05) and their effects across the 10 traits formed the basis for an association weight matrix and its derived gene network related to FSC (57.3% success and heritability = 0.06 ± 0.05). These analyses yielded 1,555 important SNP, which inferred genes linked by 113,873 correlations within a network. Specifically, 1,386 SNP were nodes and the 5,132 strongest correlations (|r| ≥ 0.90) were edges. The network was filtered with genes queried from a transcriptome resource created from deep sequencing of RNA (i.e., RNA-Seq) from the hypothalamus of a prepubertal and a postpubertal Brangus heifer. The remaining hypothalamic-influenced network contained 978 genes connected by 2,560 edges or predicted gene interactions. This hypothalamic gene network was enriched with genes involved in axon guidance, which is a pathway known to influence pulsatile release of LHRH. There were 5 transcription factors with 21 or more connections: ZMAT3, STAT6, RFX4, PLAGL1, and NR6A1 for FSC. The SNP that identified these genes were intragenic and were on chromosomes 1, 5, 9, and 11. Chromosome 5 harbored both STAT6 and RFX4. The large number of interactions and genes observed with network analyses of multiple sources of genomic data (i.e., GWAS and RNA-Seq) support the concept of FSC being a polygenic trait.

Genome-wide association studies of female reproduction in tropically adapted beef cattle.

The genetics of reproduction is poorly understood because the heritabilities of traits currently recorded are low. To elucidate the genetics underlying reproduction in beef cattle, we performed a genome-wide association study using the bovine SNP50 chip in 2 tropically adapted beef cattle breeds, Brahman and Tropical Composite. Here we present the results for 3 female reproduction traits: 1) age at puberty, defined as age in days at first observed corpus luteum (CL) after frequent ovarian ultrasound scans (AGECL); 2) the postpartum anestrous interval, measured as the number of days from calving to first ovulation postpartum (first rebreeding interval, PPAI); and 3) the occurrence of the first postpartum ovulation before weaning in the first rebreeding period (PW), defined from PPAI. In addition, correlated traits such as BW, height, serum IGF1 concentration, condition score, and fatness were also examined. In the Brahman and Tropical Composite cattle, 169 [false positive rate (FPR) = 0.262] and 84 (FPR = 0.581) SNP, respectively, were significant (P < 0.001) for AGECL. In Brahman, 41% of these significant markers mapped to a single chromosomal region on BTA14. In Tropical Composites, 16% of these significant markers were located on BTA5. For PPAI, 66 (FPR = 0.67) and 113 (FPR = 0.432) SNP were significant (P < 0.001) in Brahman and Tropical Composite, respectively, whereas for PW, 68 (FPR = 0.64) and 113 (FPR = 0.432) SNP were significant (P < 0.01). In Tropical Composites, the largest concentration of PPAI markers were located on BTA5 [19% (PPAI) and 23% (PW)], and BTA16 [17% (PPAI) and 18% (PW)]. In Brahman cattle, the largest concentration of markers for postpartum anestrus was located on BTA3 (14% for PPAI and PW) and BTA14 (17% PPAI). Very few of the significant markers for female reproduction traits for the Brahman and Tropical Composite breeds were located in the same chromosomal regions. However, fatness and BW traits as well as serum IGF1 concentration were found to be associated with similar genome regions within and between breeds. Clusters of SNP associated with multiple traits were located on BTA14 in Brahman and BTA5 in Tropical Composites.

A single nucleotide polymorphism-derived regulatory gene network underlying puberty in 2 tropical breeds of beef cattle.

Harsh tropical environments impose serious challenges on poorly adapted species. In beef cattle, tropical adaptation in the form of temperature and disease resistance, coupled with acclimatization to seasonal and limited forage, comes at a cost to production efficiency. Prominent among these costs is delayed onset of puberty, a challenging phenotype to manipulate through traditional breeding mechanisms. Recently, system biology approaches, including gene networks, have been applied to the genetic dissection of complex phenotypes. We aimed at developing and studying gene networks underlying cattle puberty. Our starting material comprises the association results of ~50,000 SNP on 22 traits, including age at puberty, and 2 cattle breed populations: Brahman (n = 843) and Tropical Composite (n = 866). We defined age at puberty as the age at first corpus luteum (AGECL). By capturing the genes harboring mutations minimally associated (P < 0.05) to AGECL or to a set of traits related with AGECL, we derived a gene network for each breed separately and a third network for the combined data set. At the intersection of the 3 networks, we identified candidate genes and pathways that were common to both breeds. Resulting from these analyses, we identified an enrichment of genes involved in axon guidance, cell adhesion, ErbB signaling, and glutamate activity, pathways that are known to affect pulsatile release of GnRH, which is necessary for the onset of puberty. Furthermore, we employed network connectivity and centrality parameters along with a regulatory impact factor metric to identify the key transcription factors (TF) responsible for the molecular regulation of puberty. As a novel finding, we report 5 TF (HIVEP3, TOX, EYA1, NCOA2, and ZFHX4) located in the network intersecting both breeds and interacting with other TF, forming a regulatory network that harmonizes with the recent literature of puberty. Finally, we support our network predictions with evidence derived from gene expression in hypothalamic tissue of adult cows.

Detection of chromosome segments of zebu and taurine origin and their effect on beef production and growth.

Cattle in breeds formed by recent crossing of Bos taurus (Bt) and Bos indicus (Bi) subspecies should contain chromosomes that are a composite of Bt and Bt segments. Using data from a 50K SNP chip, we were able to identify whether a chromosome segment of 11 SNP in a composite animal descended from a Bt or a Bi ancestor. When the method was tested in purebred Bt or Brahman cattle, about 94% of segments were assigned correctly. About 10% of the genome in Australian Brahman cattle appears to be of Bt origin, as might be expected from their history. We then examined the effect of the origin of each chromosome segment on BW in a population of 515 Bt × Bi composite cattle and found 67 chromosome segments with a significant (P<0.01) effect. We confirmed these effects by examining these 67 segments in a population of Brahman cattle and in a population of mixed breeds including composite breeds such as Santa Gertrudis and Brahman cattle. About 66% of the 67 segments had an effect in the same direction in the confirmation analyses as in the discovery population. However, the effect on BW and other traits of chromosome segment origin is small, indicating that we had low power to detect these effects with the number of animals available. Consequently, when chromosome segment origin was used in genomic selection to predict BW, the accuracy was low (0.08). Chromosome segments that had a positive effect on BW tend to be at greater frequency in composite breeds than chromosome segments with a negative effect on BW.

Predicting genetic merit for mastitis and fertility in dairy cattle using genome wide selection and high density SNP screens.

Two novel methods for genome wide selection (GWS) were examined for predicting the genetic merit of animals using SNP information alone. A panel of 1,546 dairy bulls with reliable EBVs was genotyped for 15,380 SNPs that spanned the whole bovine genome. Two complexity reduction methods were used, partial least squares (PLS) and regression using a genetic algorithm (GAR), to find optimal solutions of EBVs against SNP information. Extensive internal cross-validation was used tofind the best predictive models followed by external validation (without direct use of the pedigree or SNP location). Both PLS and GAR provided both accurate fit to the training data set for somatic cell count (SCC) (max r = 0.83) and fertility (max r = 0.88) and showed an accuracy of prediction of r = 0.47 for SCC, and r = 0.72 for fertility. This is the first empirical demonstration that genome wide selection can account for a very high proportion of additive genetic variation in fitness traits whilst exploiting only a small percentage of available SNP information, without use of pedigree or QTL mapping. PLS was computationally more efficient than GAR.

Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle.

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.

Genome-wide genetic diversity of Holstein Friesian cattle reveals new insights into Australian and global population variability, including impact of selection.

Past breeding strategies for dairy cattle have been very effective in producing rapid genetic gain to achieve industry targets and raise profitability. Such gains have been largely facilitated by intense selection of sires combined with the use of artificial insemination. However, this practice can potentially limit the level of genetic diversity through inbreeding and selection plateaus. The rate of inbreeding in Australia is increasing, primarily as a result of semen importation from a small number of prominent bulls from the USA. The effect of this genetic influx in the Australian dairy cattle population is poorly understood both in terms of diversity and local adaptation/divergence. This study uses 845 genome-wide SNP genetic markers and 431 bulls to characterize the level of genetic diversity and genetic divergence within the Australian and international Holstein Friesian dairy population. No significant differences in genetic diversity (as measured by heterozygosity [H(o)] and allelic richness [A]) were observed over the 25-year time period (1975-1999) for bulls used in Australia. The importation of foreign semen into Australia has increased the effective population size until it was in effect a sub-sample of the global population. Our data indicate that most individuals are equally closely related to one another, regardless of country of origin and year of birth. In effect, the global population can be considered as one single population unit. These results indicate that inbreeding, genetic drift and selection has had little effect at reducing genetic diversity and differentiating the Australian Holstein Friesian population at a genome-wide level.

Technical note: Whole-genome amplification of DNA extracted from cattle semen samples.

The bovine genome sequence project and the discovery of many thousands of bovine single nucleotide polymorphisms has opened the door for large-scale genotyping studies to identify genes that contribute to economically important traits with relevance to the beef and dairy industries. Large amounts of DNA will be required for these research projects. This study reports the use of the whole-genome amplification (WGA) method to create an unlimited supply of DNA for use in genotyping studies and long-term storage for future gene discovery projects. Two commercial WGA kits (GenomiPhi, Amersham Biosciences, Sydney, Australia, and REPLI-g, Qiagen, Doncaster, Australia) were used to amplify DNA from straws of bull semen, resulting in an average of 7.2 and 67 microg of DNA per reaction, respectively. The comparison of 3.5 kb of sequences from the amplified and unamplified DNA indicated no detectable DNA differences. Similarly, gene marker analysis conducted on genomic DNA and DNA after WGA indicated no difference in marker amplification or clarity and accuracy of scoring for approximately 10,000 single nucleotide polymorphism markers when compared with WGA samples genotyped in duplicate. These results illustrate that WGA is a suitable method for the amplification and recovery of DNA from bull semen samples for routine genomic investigations.

Nucleotide diversity on the ovine Y chromosome.

To investigate the impact of male-mediated introgression during the evolution of sheep breeds, a sequencing approach was used to identify single nucleotide polymorphisms (SNPs) from the male-specific region of the ovine Y chromosome (MSY). A total of 4380 bp, which comprised nine fragments from five MSY genes was sequenced within a panel of 14 males from seven breeds. Sequence alignment identified a single segregating site, an A/G SNP located approximately 1685 bp upstream of the ovine SRY gene. The resulting estimation of nucleotide diversity (piY = 0.90 +/- 0.50 x 10(-4)) falls towards the lower end of estimates from other species. This was compared with the nucleotide diversity estimated from the autosomal component of the genome. Sequence analysis of 2933 bp amplified from eight autosomal genes revealed a nucleotide diversity (piA = 2.15 +/- 0.27 x 10(-3)) higher than previously reported for sheep. Following adjustment for the contrasting influence of effective population size and a male biased mutation rate, comparison revealed that approximately 10% of the expected nucleotide diversity is present on the ovine Y chromosome.

Generation and exploration of a dense genetic map in a region of a QTL affecting corpora lutea in a Meishan x Yorkshire cross.

Previously genomic scans revealed quantitative trait loci (QTL) on porcine Chromosome 8 (SSC8) as significantly affecting the number of corpora lutea (CL) in swine. In one study, statistical evidence for the putative QTL was found in the chromosomal region defined by the microsatellites (MS) SW205, SW444, SW206, and SW29. A Yeast Artificial Chromosome library was screened by using the corresponding primers for clones containing these MS by PCR. From five positive YAC clones, 10 additional MS were isolated and mapped to SSC8 with the INRA-University of Minnesota porcine Radiation Hybrid (IMpRH) panel. The genetic map position of the QTL has been refined by addition of these 10 markers. The QTL evaluation included pedigrees of F2-intercross Meishan x Yorkshire design, with phenotypic data of 108 F2 female offspring and genotypic data for 29 MS markers on SSC8. The analysis was performed by using the least squares regression method. The calculated QTL effect for CL obtained by the multilocus least squares method showed a maximum test statistic (F value = 13.98) at position 99 cM between three MS derived from YACs containing SW205 and SW1843 spanning an interval of 7.1 cM. The point-wise (nominal) P-value was 5.21 x 10-6 corresponding to a genome-wide P-value of 0.009. The additive QTL effect explained 17.4% of the phenotypic variance.

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Directed isolation and mapping of microsatellites from swine Chromosome 1q telomeric region through microdissection and RH mapping.

Several quantitative trait loci (QTLs) (vertebrate number, birth weight, age at puberty, growth rate, gestation length, and backfat depth) have been independently mapped to the distal region of swine Chromosome (SSC) 1q in several resource populations. In order to improve the map resolution and refine these QTLs more precisely on SSC1q, we have isolated and mapped additional microsatellites (ms), using chromosome microdissection and radiation hybrid (RH) mapping. Five copies of the telomeric region of SSC1q were microdissected from metaphase spreads and pooled. The chromosomal fragment DNA was randomly amplified by using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), enriched for ms, and subcloned into a PCR vector. Screening of subsequent clones with ms probes identified 23 unique ms sequences. Fifteen of these (65%) were subjected to radiation hybrid (RH) mapping by using the INRA-University of Minnesota porcine RH panel (IMpRH); and the remaining eight were not suited for the RH mapping. Twelve microsatellites were assigned to SSC1q telomeric region of IMpRH map (LOD >6), and three remain unlinked (LOD <6). Out of the 15 microsatellite markers, 9 were polymorphic in NIAI reference population based on the Meishan and Göttingen miniature pig. In summary, we have used microdissection and radiation hybrid mapping to clone and map 12 new microsatellites to the swine gene map to increase the resolution of SSC1q in the region of known QTLs.

Generation and mapping of expressed sequence tags from virus-infected swine macrophages.

In an effort to identify genes that have a major effect on macrophage function during viral infection, we employed differential display reverse transcription (DDRT)-PCR to capture expressed sequence tags (ESTs) of swine alveolar macrophages infected by the porcine reproductive and respiratory syndrome virus (PRRSV). Sequence analyses showed that approximately 60% of these ESTs had significant similarity (> or =93%) to known pig ESTs or genes or matched sequences from other species with homology > or =80%. To determine chromosomal localization, PCR-based mapping was performed across either swine somatic cell hybrid or radiation hybrid panels. A total of 48 porcine viral response ESTs were mapped via the swine somatic cell panel or the INRA-Minnesota porcine Radiation Hybrid (IMpRH) panel (LOD > 6.0). Northern blot analyses confirmed PRRSV-induced altered transcript expression for several ESTs, including a 2'-5' oligoadenylate synthetase and a putative dual-specificity phosphatase. These virus-response ESTs represent good candidate genes for understanding PRRSV pathogenesis and for dissecting host genes which may have major effect on disease resistance.

An RNA helicase, RHIV -1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13.

The impact of porcine reproductive and respiratory syndrome virus (PRRSV) infection on porcine alveolar macrophages (Mo) was examined by differential display reverse transcription PCR (DDRT-PCR). A PRRSV-induced expressed gene tag (EST) was used to isolate and identify a single cDNA clone from a library prepared from porcine peripheral blood. Rapid amplification of cDNA ends (RACE) was employed to clone a 1.5 kb fragment at the 5' end of the mRNA. DNA sequencing identified an open reading frame (ORF) of 2820 bp. Deduced amino acid sequence revealed the eight conserved domains characteristic of the DEAD/H box protein superfamily. The putative porcine RNA helicase induced by virus (RHIV -1) showed 84% amino acid similarity to human retinoic acid-induced gene (RIG-I). Porcine RHIV -1 transcripts were ubiquitously expressed in various pig tissues, while in PRRSV-infected pigs, higher expression was observed in several tissues persistent for PRRSV. These data indicate the association of PRRSV genome replication with enhaced host cell RNA helicase gene expression. Finally, the RHIV -1 gene was localized on porcine chromosome 10q13 between markers SSC25A02 and SWR334 via somatic cell panel and radiation hybrid (RH) mapping strategies.