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Two separate crosses were conducted for this analysis, the first consisting of an F2 intercross between Obese Strain White Leghorn (WL-OS) chickens and a Red Junglefowl (RJF) population derived from a Swedish zoo population and maintained in Götala, Sweden (RJF-Götala). The second intercross was an eighth generation intercross between a line of selected White Leghorn (WL-L13) maintained from the 1960s and a population of RJF originally obtained from Thailand (RJF-T). This F8 advanced intercross line (AIL) is based on an F2 intercross that has been measured for comb mass and a variety of bone morphologies, see [8], [33], [53]. The WL-OS and WL-L13 cross have been separated since approximately 1955, so although they come from the same base population of White Leghorn chickens they have now had over 50 years of separation. Hereafter the abbreviation F2-L13 will be used for the F2 RJF-T/WL-L13 cross, F8-L13 for the F8 RJF-T/WL-L13 AIL, and F2-OS for the WL-OS/RJF-Götala F2 cross. In the case of the F2-OS cross, these were raised in a total of 4 batches at Götala research station of the Swedish University of Agricultural Sciences, Skara, Sweden. Chickens were maintained on standardised conditions and fed ad libitum, under a 12∶12 hour light/dark regime. Pens measuring 3 m×3 m were used for housing, with perches also provided. Individuals were culled at 200 days of age, with their combs surgically removed post-mortem and weighed and both femoral bones extracted. The F8-L13 cross used in this study was generated in 5 batches and reared at the research station of Linköping University, Sweden. Pens for these animals were 2 m×2 m and comprised of three separate levels, perches and food were once again supplied ad libitum. Animals were culled at 212 days of age, with the comb surgically removed post-mortem and weighed, and both femoral bones extracted.
A total of 640 F2 individuals were used for the comb mass analysis, consisting of 308 males and 332 females. Of these, 543 of the individuals were also measured for a variety of bone morphology traits (245 males and 298 females). The WL-OS strain was originally isolated as a model of Hashimoto's thyroiditis, as they suffer from hypothyroidism and therefore require thyroxin to be provided in a food supplement (500 µg/kg food) (Levaxin Tablets, Nycomed AB, Sweden, were administered to all but one batch until 180 days of age). Although this strain tends to be slightly smaller than usual WL strains, they are still substantially larger, and with far greater sized combs, as compared to RJF. In addition, the degree of infiltration that had occurred in the F2 animals thyroid was also measured, and correlated with both comb mass and body weight. No significant correlations were found, and additionally the majority of the QTL discovered in this cross (7 of 11) all go in the expected direction, i.e. alleles of greater effect come from the WL, not RJF, line (with one of the three transgressive QTL also corroborated as transgressive in the other crosses as well). If any comb loci were caused by thyroiditis this would be expected to be the reverse, so we have good cause to consider these QTL are due to generic WL-strain based differences. Animals were culled at 200 days of age. The animal experiments were approved by the ethical committee for animal experiments in Göteborg, Sweden, no. 55-2005 and 233-2006, and by the ethical committee for animal experiments in Uppsala no. 2008-12-19, C321/8.
A total of 447 F8 individuals were assayed for comb measurements (216 males and 231 females). These individuals were generated from a total of 107 families using 122 F7 individuals (63 females and 59 males). Average family size 4.7+/−3.1 (mean, standard deviation). These were the continuation of an inter-cross initially based on 3 WL females and one RJF male, which were then expanded into 811 F2 progeny and then maintained with at a population size of approximately 100 birds per generation until the F7 generation. Of the F8 individuals, a total of 41 males were used as the basis of a qPCR experiment using comb base tissue to check for candidate genes at the major chromosome 3 QTL locus at 15.7 Mb (12 RJF homozygotes, 15 WL homozygotes, 14 heterozygotes). These individuals came from two of the five batches used to produce the F8. Additionally, 20 females were used as the basis for a qPCR experiment using diaphyseal medullary bone (10 of each homozygote class, though one had no metaphyseal CT bone measurements, reducing the correlation with bone density to 19 individuals). The study was approved by the local Ethical Committee of the Swedish National Board for Laboratory Animals.
A total of 352 males and 122 females were used in this analysis, with these QTL already detailed in [8], .
The right femoral bone of each F2OS and F8L13 individual was measured using a Peripheral Quantitative Computerised Tomography (pQCT) machine (Stratec XCT – Research SA machine, Stratec Medizintechnik, Germany), with two sections taken at 6% (metaphyseal) and two at 50% (diaphyseal) of the total femoral length. Cortical bone was measured using the CORTMODE1 setting, with a density threshold of >1000 mg/cm3. Cortical measures were cortical area, cortical bone content, cortical thickness and cortical density. Medullary measures were recorded using the PEELMODE2 function, using an inner threshold of 1000 mg/cm3 to separate cortical from medullary bone and gives total density, total bone content and total area of the endosteal cavity measures. An additional inner threshold of 150 mg/cm3 in a combination of two PEELMODE2 gives the medullary area, medullary density and medullary bone content.
Two separate fecundity trials were performed. Initially birds were housed individually and eggs were collected daily over a two-week period. The second trial was performed immediately after the first and was identical except birds were given two dummy eggs to incubate and were allowed to keep all eggs laid over a two-week period. At the end of each trial, number of eggs produced, total weight of eggs produced and mean egg weight were recorded. Chickens which are actively brooding (incubating) their eggs will stop producing eggs when a clutch size of around 6–8 eggs has been produced, whilst domestic layers will continually produce eggs and never go into such brooding behaviour. Therefore one method for ascertaining if an individual is brooding will be to deduct the total number of eggs produced in the first trial from the number of eggs produced in the second trial. Negative values therefore indicate that an individual is decreasing egg production when allowed to build up a clutch.
To analyse phenotypic correlations between comb mass and bone characteristics in the F2OS and F8L13 crosses, a GLM was fitted for each individual bone trait measured and included batch and sex fixed effects and weight at slaughter as a covariate. All significantly correlated bone measures were then combined into a global model. The significance values of each measure was then ascertained in this global model.
The clustering test was performed using a permutation test based on the total length of the chicken genome (1.09 Gb), which then had a number of regions equal to the number of QTL detected in the F2OS and F8L13 cross randomly distributed along it. The size of these regions was equal to the average C.I. of QTL from the F2-OS cross (15 Mb) and the F8L13 cross (5 Mb), and tested against the observed number of overlaps between the F2OS and F8L13 (6). The F2L13 cross was not used in this analysis, as 4 of the 5 QTL detected in this cross were strongly replicated in the F8L13 and their inclusion could artificially inflate the degree of replication observed between the two different cross populations. This was repeated 1000 times, with the number of overlaps recorded each time used to generate a significance value. A similar procedure was used to predict the probability of selective sweeps occurring within the overlap regions. In this instance the total number of sweeps detected (133) and the six overlap regions were used, based against the observation that 13 sweeps were observed within the six 6 Mb overlap intervals. When calculating the overlap regions, an extra 1 Mb was added to the region size upstream and downstream, in case using only the overlap between the OS and L13 crosses gave an artificially small region.
DNA preparation for both crosses was performed by Agowa GmbH (Berlin, Germany), using standard salt extraction. A total number of 347 SNPs and 20 microsatellite markers were used for the OS cross, with the marker map generated using Crimap [54], covering ∼2535 cM, with an average marker spacing of ∼7.5 cM. For the F8L13 cross, 652 SNP markers were used to generate a map of length ∼8760 cM, with an average marker spacing of ∼15 cM. QTL analysis was performed using QTL Express (http://qtlcap.ed.ac.uk), qxpak v2.16 [55] and R/Qtl [56] for the standard interval mapping and epistatic analyses. Interval mapping was performed using additive and additive+dominance models. Batch and sex were always included in the model as a fixed effect, whilst bodyweight at slaughter was included as a covariate. To account for a particular QTL varying between the sexes, a sex-interaction analysis was also performed.
Significance thresholds for both crosses were calculated using permutation tests [57], [58], however these values differed greatly between the two study populations due to the massively increased number of recombinations in the F8L13 population (as reflected in the inflated map size) and also to account for possible family effects in this population. A suggestive significance level of a genome-wide 20% threshold was used (mainly due to being more conservative than the standard suggestive threshold suggested by Lander and Kruglyak [59]). For the F2OS cross, additive+dominance effects had a suggestive threshold of around ∼3.0 and a significant threshold of ∼3.9, depending on the trait, whilst additive+dominance sex interaction models had a suggestive threshold of ∼3.8 LOD and a significant threshold of ∼4.6 LOD. In the case of the comb analysis, a single point threshold was used where a QTL had been indicated in the F8L13 or F2L13 mapping populations at a locus, but where a significant or suggestive QTL in the F2OS population was not immediately apparent. 5% single point threshold for additive only model was LOD ∼1.4, whilst for an additive+dominance model this was LOD ∼2.0. Confidence intervals (C.I.) for QTL were calculated using a 1.8 LOD drop method (i.e. where the LOD score on either side of the peak decreases by 1.8 LOD). The nearest marker to this 1.8 LOD decrease is then used to give the C.I. in megabases (the use of the physical location of markers allows a direct comparison between the different crosses). The use of a 1.8 LOD drop has been shown to most reliably give an accurate 95% confidence interval for an intercross type population [60]. Thresholds for an AIL are potentially problematic, as the family structure can cause inflated LOD scores, resulting in false positive results. To avoid this initially, we used a large number of families (107) to generate the total number of individuals, to break down this sub-structure as much as possible. For instance, if only one offspring were used per family, there would be no structure and the population would function exactly as recombinant inbred lines [61]. To check if family was a significant factor on the comb trait, a GLM consisting of sex, batch, family and weight was used to predict comb weight. In this instance, only one family out of 107 was significant (p = 10−6), and consisted of two large males. Furthermore to generate the significance thresholds we also used a family factor included in the permutation model, which resulted in greater thresholds. The threshold without family as a factor was LOD 3.4 for a suggestive threshold and LOD 4.15 for a significant threshold. With family included as a cofactor, a suggestive threshold was 4.55 and a significant threshold was 5.4 was obtained.
Comb base RNA was isolated with Ambion TRI reagent (Applied Biosystems, Carlsbad, CA, USA), following the manufacturer's protocol. After removal of the comb, a part of the forehead was frozen in liquid nitrogen. Frozen comb base tissue was removed with a razor and disrupted with a hammer and bag. Samples were homogenized on a FastPrep-24 instrument (MP Biomedicals, Solon, OH, USA) in tubes with TRI and ceramic beads (Lysing matrix D, MP Biomedicals). First strand cDNA for qPCR was made with Fermentas (St. Leon-Rot, Germany) RevertAid Reverse Transcriptase, using 10 mM dNTPs, RiboLock nuclease inhibitor, and oligo(dT)18 primer (Thermo Fisher Scientific, Freemont, CA, USA), according to the manufacturer's protocol. qPCR was performed with Maxima SYBR Green qPCR mastermix (Thermo Fischer Scientific) in 15 µl reactions with 0.3 M of each oligonucleotide primer on a Rotor-Gene 6000 real-time cycler (Corbett Research, Cambridge, UK). The PCR program consised of a 10 min activation step at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 minute at 60°C. After cycling, products were melted by ramping the temperature from 72°C to 95°C. qPCR data was analysed with the comparative ΔΔCt method [62]. Target gene threshold cycle values were normalized by subtracting the geometric mean value of three reference genes, β2 microglobulin, TATA box binding protein, and RNA polymerase II subunit C1. A batch effect (due to individuals coming from two of the five batches comprising the cross) was included in the General Linear Model analysis, as well as weight at slaughter for all individuals, when comb mass was included in the model.
To ascertain the effects of differential gene expression from the two candidate genes in the QTL interval on the phenotypic trait, the gene expression levels (described above) were used in a linear model. The initial model to test gene expression was y (comb mass) = mean + batch + weight at slaughter + expression level (called either HAO1 or BMP2 in the model column in Tables 1 and 2). To test the effect of this on genotype an additional QTL genotype factor was added (model HAO1 + QTL and BMP2 + QTL in Tables 1 and 2). One measure of causality is that the additional of a candidate gene expression covariate should decrease the QTL genotype effect in this model, therefore these models were then checked against the model y = mean + batch + weight at slaughter + QTL genotype (QTL only model in Tables 1 and 2), to observe the drop in QTL significance. Additionally, two further models were also checked. These were y = mean + batch + weight at slaughter + BMP2 expression + HAO1 expression, and y = mean + batch + weight at slaughter + BMP2 expression + HAO1 expression+QTL genotype. These were fitted to check for the potential of gene interactions between both candidates affecting the trait. Linear models were also fitted to test the association between HAO1 and BMP2 levels with the other comb mass QTL identified. Initially, a basic model of y (gene expression level) = mean + batch + weight at slaughter + QTL genotype was fitted for each QTL separately. All significant QTL were then combined into a single model (y = mean + batch + QTL1 + QTL2, etc) to test for the global combined significance. This global model was then compared to the base model with only the chromosome 3 QTL genotype included. Linear models were also used to test the effects of HAO1 and BMP2 expression levels on other related fecundity and bone morphometric traits. In this case, a model of y (trait of interest) = mean + weight at slaughter + QTL genotype (chromosome 3) + HAO1/BMP2 expression level was fitted.
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