J.J. van der Poel

Two-dimensional screening of the Wageningen chicken BAC library.

Abstract. We have constructed a Bacterial Artificial Chromosome (BAC) library that provides 5.5-fold redundant coverage of the chicken genome. The library was made by cloning partial HindIII-digested high-molecular-weight (HMW) DNA of a female White Leghorn chicken into the HindIII site of the vector pECBAC1. Several modifications of standard protocols were necessary to clone efficiently large partial HindIII DNA fragments. The library consists of 49,920 clones arranged in 130 384-well plates. An average insert size of 134 kb was estimated from the analysis of 152 randomly selected BAC clones. The average number of NotI restriction sites per clone was 0.77. After individual growth, DNA was isolated of the pooled clones of each 384-well plate, and subsequently DNA of each plate was isolated from the individual row and column pools. Screening of the Wageningen chicken BAC library was performed by two-dimensional PCR with 125 microsatellite markers. For 124 markers at least one BAC clone was obtained. FISH experiments of 108 BAC clones revealed chimerism in less than 1%. The number of different BAC clones per marker present in the BAC library was examined for 35 markers which resulted in a total of 167 different BAC clones. Per marker the number of BAC clones varied from 1 to 11, with an average of 4.77. The chicken BAC library constitutes an invaluable tool for positional cloning and for comparative mapping studies.

Whole genome scan for quantitative trait loci affecting body weight in chickens using a three generation design

Abstract An experimental population containing 10 full sib families of a cross between two broiler lines was created. In this population blood samples from 20 full sib animals in generation 1 and 451 full sib animals in generation 2 were used for marker genotyping. Data on body weight at slaughter age (48 days) collected in a feed conversion experiment with 2049 individually housed grandoffspring was analysed. Large differences in mean and variance between male and female body weight were found. To account for these differences, a bivariate analysis treating body weight of males and females as separate traits was used to estimate (co)variance components and breeding values. The model accounted for systematic environmental effects and maternal effects. The estimated heritability of body weight was 0.28 in the males and 0.33 in the females and the genetic correlation between male and female body weight did not significantly deviate from unity. Estimated breeding values, fixed and maternal genetic effects were used to calculate average adjusted progeny trait values for all generation 2 animals adjusted for fixed and maternal genetic effects and for the additive genetic contribution of the other parent. Male and female progeny trait values were combined in one trait value adjusting for sex differences by standardisation for mean and variance. This average adjusted progeny trait value was used for QTL detection. To study presence of QTLs, an across family weighted regression interval mapping approach was used both in half sib as well as a full sib QTL analysis. Genotypes from 368 markers mapped on 24 autosomal linkage groups were available. The most likely position for a QTL affecting body weight was found on chromosome 1 at 240 cM with a test statistic of 2.32. Significance levels were obtained using the permutation test. The chromosomewise significance level of this QTL was 10%, whereas the genomewise significance level was 41%. New aspects of this study are: Genomewide QTL analysis in poultry, full sib analysis in an outbred population structure and correction for heterogeneous variances between sexes.

Across-Line SNP Association Study for Direct and Associative Effects on Feather Damage in Laying Hens

An association study between SNP markers and feather condition score on the back, rump and belly of laying hens was performed. Feather condition score is a measure of feather damage, which has been shown to be closely related to feather pecking behaviour in hens housed in groups. A population of 662 hens was genotyped for 1536 SNPs of which 1022 could be used for the association study. The analysis was conducted across 9 different lines of White Leghorn and Rhode Island Red origin. Across lines linkage disequilibrium is conserved at shorter distances than within lines; therefore, SNPs significantly associated with feather condition score across lines are expected to be closer to the functional mutations. The SNPs that had a significant across-line effect but did not show significant SNP-by-line interaction were identified, to test that the association was consistent across lines. Both the direct effect of the individual’s genotype on its plumage condition, and the associative effect of the genotype of the cage mates on the individual’s plumage condition were analysed. The direct genetic effect can be considered as the susceptibility to be pecked at, whereas the associative genetic effect can be interpreted as the propensity to perform feather pecking. Finally, 11 significant associations between SNPs and behavioural traits were detected in the direct model, and 81 in the associative model. A role of the gene for the serotonin receptor 2C (HTR2C) on chromosome 4 was found. This supports existing evidence of a prominent involvement of the serotonergic system in the modulation of this behavioural disorder in laying hens. The genes for IL9, IL4, CCL4 and NFKB were found to be associated to plumage condition, revealing relationships between the immune system and behaviour.

Genetic lines differ in Toll-like receptor gene expression in spleens of chicks inoculated with Salmonella enterica serovar Enteritidis

Toll-like receptors (TLR) recognize evolutionarily conserved molecular motifs (pathogen-associated molecular patterns) of infectious microbes and initiate innate immune response upon activation with relevant pathogens. This study investigated the acute effect of Salmonella Enteritidis challenge on TLR mRNA expression in cecum and spleen of birds from 3 distinct genetic lines. Chicks from broiler, Leghorn, and Fayoumi lines were inoculated or mock-inoculated with Salmonella Enteritidis. The mRNA expression levels of TLR2, TLR4, and TLR5 genes were assessed by quantitative reverse transcription-PCR of cecum and spleen tissue harvested at 2 or 18 h postinoculation (PI). There were no significant genetic line effects on TLR mRNA expression in spleen or cecum of mock-infected birds, or in the cecum of infected birds. Genetic line effect was significant (P < 0.05) on TLR mRNA expression in the spleen of Salmonella Enteritidis-infected birds. The Fayoumi line had higher TLR2 and TLR4 expression than Leghorn, higher TLR2 mRNA expression than broiler, and the broiler line had higher TLR5 expression than Leghorn and Fayoumi. In Salmonella Enteritidis-infected birds, the TLR2 expression in both cecum and spleen and TLR4 expression in spleen were significantly higher at 18 h PI than 2 h PI. The results demonstrate a significant genetic line effect on TLR expression in the spleen of Salmonella Enteritidis-infected birds, which may partly explain genetic variability in immune response to Salmonella Enteritidis.

Short communication: Genome-wide scan for bovine milk-fat composition. II. Quantitative trait loci for long-chain fatty acids

We present the results of a genome-wide scan to identify quantitative trait loci (QTL) that contribute to genetic variation in long-chain milk fatty acids. Milk-fat composition phenotypes were available on 1,905 Dutch Holstein-Friesian cows. A total of 849 cows and their 7 sires were genotyped for 1,341 single nucleotide polymorphisms across all Bos taurus autosomes (BTA). We detected significant QTL on BTA14, BTA15, and BTA16: for C18:1 cis-9, C18:1 cis-12, C18:2 cis-9,12, CLA cis-9,trans-11, C18:3 cis-9,12,15, the C18 index, the total index, total saturated fatty acids, total unsaturated fatty acids (UFA), and the ratio of saturated fatty acids:unsaturated fatty acids on BTA14; for C18:1 trans fatty acids on BTA15; and for the C18 and CLA indices on BTA16. The QTL explained 3 to 19% of the phenotypic variance. Suggestive QTL were found on 16 other chromosomes. The diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism on BTA14, which is known to influence fatty acid composition, most likely explains the QTL that was detected on BTA14.

Across-line SNP association study of innate and adaptive immune response in laying hens

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in laying hens. For this purpose, the associations between 1022 single nucleotide polymorphism (SNP) markers and immune traits were studied in 583 hens from nine different layer lines. Immune traits were natural antibodies for keyhole limpet haemocyanin (KLH) and lipopolysaccharide (LPS) at 20, 40 and 65 weeks, acquired antibodies to the vaccinal virus of Newcastle disease at 20 weeks, and complement activity measured on sheep and bovine red blood cells at 20, 40 and 65 weeks. We adopted a novel approach based on across-line analysis and testing of the SNP-by-line interaction. Among lines, linkage disequilibrium is conserved at shorter distances than in individual lines; therefore, SNPs significantly associated with immune traits across lines are expected to be near the functional mutations. In the analysis, the SNPs that had a significant across-line effect but did not show significant SNP-by-line interaction were identified to test whether the association was consistent in the individual lines. Ultimately, 59 significant associations between SNPs and immune traits were detected. Our results confirmed some previously identified QTL and identified new QTL potentially involved in the immune function. We found evidence for a role of IL17A (chromosome 3) in natural and acquired antibody titres and in the classical and alternative pathways of complement activation. The major histocompatibility genes on chromosome 16 showed significant association with natural and acquired antibody titres and classical complement activity. The IL12B gene on chromosome 13 was associated with natural antibody titres.

Identification of QTLs Involved in Open-Field Behavior in Young and Adult Laying Hens

Line differences for open-field behavior in chickens have been observed, and it has been shown that this behavior has a genetic component. The aim of this study was to detect quantitative trait loci (QTL) involved in open-field behavior. For this purpose, open-field behavior was studied at 5 and 29 weeks of age in F2 hens coming from an intercross between two commercial White Leghorn laying lines selected for egg production traits. Latencies, durations, and frequencies of general activity (sitting, standing, walking, and stepping), defecation, and vocalizations were recorded individually for each bird, and a factor score was calculated. All animals (F0, F1, and F2) were screened with 180 microsatellite markers. Regression interval mapping was applied using both a paternal half-sib analysis and a line-cross analysis method. For general activity at 5 weeks of age, a significant QTL was detected on GGA4 and a suggestive QTL on GGA2 under the line-cross model. For general activity at 29 weeks of age, a significant QTL was detected on GGA4 and two suggestive QTLs were detected on GGA1 and on GGA10, respectively, also using the line-cross analysis. The QTL on GGA4 at 5 weeks of age did not overlap with the QTL on GGA4 at 29 weeks of age. The current study indicated that open-field behavior in young chickens was regulated by QTL that differ from the QTL for open-field behavior in adult chickens.

Natural antibody isotypes as predictors of survival in laying hens

To identify possible relationships between survival and titers of natural antibody (NAb) isotypes in serum of laying hens, birds from 12 purebred layer lines of 2 commercial breeds, Rhode Island Red (n = 524) and White Leghorn (n = 538), were monitored for survival during one laying period (from 20 until 70 wk of age). Titers of NAb isotype IgM- and IgG-binding keyhole limpet hemocyanin (KLH) in serum were measured at 20, 40, and 65 wk of age, respectively. Overall, the titers of IgM and IgG binding KLH decreased with aging. At the same age, lines within breed showed significantly different titers of isotypes (P < 0.0001). Multivariable logistic regression analysis showed that NAb isotype IgM and IgG titers at 20 wk of age were associated with survival at 20 to 40 wk of age. In the R breed, odds ratios of 0.56 (P < 0.0001) for IgM and 0.72 (P = 0.02) for IgG were estimated; in the W breed, these were 0.74 (P < 0.01) and 0.99 (P = 0.95) for IgM and IgG, respectively. We conclude that titers of Nab isotypes, especially the IgM-binding KLH at 20 wk of age, are indicative for survival during the laying period. The higher the titers of NAb isotypes, the higher the probability of layers to survive.

QTL Mapping in chicken using a three generation full sib family structure of an extreme broiler X broiler cross

Abstract A three generation population has been created for mapping both production and health traits in chicken. The Fl and F2 population were genotyped while phenotypes were collected on the F3 animals. The population consisted of 10 full‐sib families with a total of 476 individuals (Fl and F2), and an F3 generation consisting of over 18,000 animals. In total, 264 microsatellites were analyzed on all Fl and F2 animals, and an additional 120 microsatellites were analyzed on only 4 of the 10 families (196 animals). A linkage map of the chicken genome containing 384 microsatellite markers has been constructed by analyzing the segregation of these markers in this population. Preliminary analysis indicate a QTL for body weight at 48 days on chromosome 1. Body weight was measured on 2100 F3 animals housed in cages, and the data was analyzed by a regression interval mapping approach. Higher F‐values were obtained by using a bivariate approach showing that differences in mean and variance of a trait measured on...

Integration of chicken genomic resources to enable whole-genome sequencing

Different genomic resources in chicken were integrated through the Wageningen chicken BAC library. First, a BAC anchor map was created by screening this library with two sets of markers: microsatellite markers from the consensus linkage map and markers created from BAC end sequencing in chromosome walking experiments. Second, HindIII digestion fingerprints were created for all BACs of the Wageningen chicken BAC library. Third, cytogenetic positions of BACs were assigned by FISH. These integrated resources will facilitate further chromosome-walking experiments and whole-genome sequencing.