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Large scale variation in DNA copy number in chicken breeds

Large scale variation in DNA copy number in chicken breeds Background: Detecting genetic variation is a critical step in elucidating the molecular mechanisms underlying phenotypic diversity. Until recently, such detection has mostly focused on single nucleotide polymorphisms (SNPs) because of the ease in screening complete genomes. Another type of variant, copy number variation (CNV), is emerging as a significant contributor to phenotypic variation in many species. Here we describe a genome-wide CNV study using array comparative genomic hybridization (aCGH) in a wide variety of chicken breeds. Results: We identified 3,154 CNVs, grouped into 1,556 CNV regions (CNVRs). Thirty percent of the CNVs were detected in at least 2 individuals. The average size of the CNVs detected was 46.3 kb with the largest CNV, located on GGAZ, being 4.3 Mb. Approximately 75% of the CNVs are copy number losses relatively to the Red Jungle Fowl reference genome. The genome coverage of CNVRs in this study is 60 Mb, which represents almost 5.4% of the chicken genome. In particular large gene families such as the keratin gene family and the MHC show extensive CNV. Conclusions: A relative large group of the CNVs are line-specific, several of which were previously shown to be related to the causative mutation for a number of phenotypic variants. The chance that inter-specific CNVs fall into CNVRs detected in chicken is related to the evolutionary distance between the species. Our results provide a valuable resource for the study of genetic and phenotypic variation in this phenotypically diverse species. Keywords: Copy number variation, Chicken, aCGH, Line-specific CNVs, Inter-specific CNVs, Genes Background variants (CNVs) lie between these two extremes, ranging The chicken was the first livestock species to have its in size from thousands to millions of bases. genome completely sequenced [1]: a large collection of In human, many CNVs are in linkage disequilibrium chicken single nucleotide polymorphisms (SNPs) has with nearby genetic markers and thus appear to be an- been available for almost a decade [2]. More recently, cient [6]. Others are more recent, such as CNVs af- the number of SNPs has been enlarged to over 7 million fecting olfactory receptor gene diversity [7], or can be [3]. Although numerous studies studying genetic vari- recurrent [8]. Structural variants include a variety of mo- ation have focused on SNPs, there is growing evidence lecular alterations such as duplications, deletions, and for the substantial role of structural polymorphism in inversions [9,10]. A comprehensive map that catalogues phenotypic diversity [4]. Structural variation has been and indexes structural variants - in particular CNVs - recognized as an important mediator of gene and gen- across the genome is a necessary prelude to understand- ome evolution within populations [5]. While the sizes of ing their role in the context of specific phenotypic traits. genetic variants range from a single base to whole chro- Early reports estimated that at least 2% of the human mosomes, historically only the extreme ends of the genome is affected by structural variations [11], but spectrum have been explored. DNA copy number more recent studies suggest that as much as 3.75% of the human genome harbors common CNVs [12]. CNV regions (CNVRs) will be an important comple- * Correspondence: [email protected] ment to SNP-centric genome-wide association studies, Animal Breeding and Genomics Centre, Wageningen University, P.O. box since existing SNP discovery and genotyping methodolo- 338, Wageningen 6700 AH, The Netherlands gies are biased against inclusion of these more complex Full list of author information is available at the end of the article © 2013 Crooijmans et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Crooijmans et al. BMC Genomics 2013, 14:398 Page 2 of 10 http://www.biomedcentral.com/1471-2164/14/398 genetic variants. Furthermore, many of the CNVRs are were detected (Additional file 1). Seventy-five percent of not very well represented and annotated in the genomic these CNVs are losses. Being more conservative, i.e. re- sequence due to biases in chromosome assembly. In quiring the CNV to be observed in at least 2 samples, order to estimate what fraction of the genome is affected 944 CNVs (29.9%) were detected with an average size of by CNV, global studies have been performed in human, 46.1 kb. The real time PCR validation of 12 CNVs ran- chimpanzee, dog, mouse and cattle. In cattle, for ex- ging in being present in 1 to 41 samples did give a suc- ample, 177 high confidence CNVRs were reported as cessful validation of 92%. Only one marker in a potential covering 28.1 Mb, 35 of these CNVRs being apparently CNV which was detected in only one animal failed valid- breed-differential or breed-specific [13]. To determine ation by showing no difference between the reference the full extent of variation and its influence on pheno- sample. These results indicated that the detected CNVs typic variation, the reference genome assembly should have a high chance of being a real CNVs. Furthermore, be near completion and more individual genomes need we could confirm 13 of the 26 high confidence CNVs to be sequenced for the species of interest. Analysis of (50%) identified by Wang et al. (2010) and 23 out of the CNVs in livestock species is of particular interest, not 70 CNVs (23.9%) detected in only one animal in that only because of their economic importance, but also due study. However, we were only able to confirm 21% of to the often- extensive selection pressure applied in the 238 CNVs detected in another study (Wang et al. generating the different lines and varieties. 2012) (Additional file 1). One reason why fewer CNVs Since CNVs potentially affect gene expression [8], were detected in the studies of Wang et al. [18,19] is the CNVs may account for a significant proportion of the use of only 10 and 6 animals respectively from three dif- extensive phenotypic variation observed in this species. ferent breeds in those two studies. Moreover, both stu- Examples of phenotypes associated with a CNV in the dies used a different reference animal and the reference chicken include late feathering on chromosome Z animal was also from the same breed. Also, none of the (GGAZ) [14], pea comb on GGA1 [15], dark brown chicken breeds were in common between our study and plumage color on GGA1 [16] and dermal hyperpigmen- those of Wang [18,19] which may account for the lack tation on GGA20 [17]. Additional CNVs have been of complete validation. detected in the chicken using aCGH [18], but that study The distribution of the gain and loss CNVs over the only examined ten individuals and only identified 96 genome is shown in Figure 1. The average size of the CNVs corresponding to approximately 1.3% of the CNVs detected in this study is 46.3 kb, and the largest chicken genome. Furthermore only 27 of these CNVs CNV, 4.3 Mb (CNV #3126), was observed on GGAZ. were observed in more than one individual. CNV distributions within the different chicken lines are Here we applied an aCGH analysis to different chicken given in Additional file 1: Table S1. Although limited breeds in order to obtain a global CNV map of the sequence information is available for GGA16 (MHC- chicken genome. containing chromosome), the repetitive nature of this chromosome [1] was confirmed by detecting copy num- Results and discussion ber variations on the entire sequenced part of this CNV in chicken chromosome. Another striking case was seen on GGA25 aCGH was carried out using the Agilent 244K chicken where 16 out of the 64 animals showed CNVs in a array with a mean probe spacing of 4000 bp. This array CNVR covering a substantial part of the genome se- is based on the chicken assembly WUSTL 2.1 (Galgal3) quence (from 0 up to 1.85 Mb). Interestingly, GGA25 is and covers chromosomes 1–28, 32 and the sex chromo- one of the more GC-rich chromosomes and it contains a somes Z and W. The virtual chromosome “ChrUn” with relatively large number of minisatellites [20]. concatenated unmapped contigs was not taken into ac- The average number of CNVs per animal was 103, ranging from 61 to 209. The highest number of CNVs count in the probe design. To access the chicken CNV landscape, we selected 64 animals from 6 commercial was detected in the commercial White Leghorn line with lines (layer and broiler types), 7 experimental lines (layer an average of 187.5 CNV per animal. The lowest number of CNVs was observed in the Red Jungle Fowl (Aviandiv) and broiler types), Red Jungle Fowls and Silkies. DNA samples were labeled with Cy3 whereas the reference population with an average of 83.8 CNV per animal, DNA sample - derived from UCD001, the Red Jungle which was expected as an inbred Red Jungle Fowl ani- mal was used as a reference in this study. The commer- Fowl animal previously selected to generate the chicken reference genome assembly - was labeled with Cy5. cial broilers showed an average of 128.8 CNVs per We defined conservative parameters for CNV detec- animal. These numbers are considerably higher than those reported by Wang et al. (2012), where the average tion to limit false positive calls (see Methods). Within the 15 lines used in this study, 3,154 CNVs with a diffe- was 40 CNVs per animal. Even when our analysis is re- rent start and/or end location on the chicken genome stricted to the autosomes, as was done in the study of Crooijmans et al. BMC Genomics 2013, 14:398 Page 3 of 10 http://www.biomedcentral.com/1471-2164/14/398 Figure 1 CNV distribution across the chicken genome. Red bars indicate copy-number losses and blue bars copy-number gains. Bar length indicates the number of occurrence for a given CNV, divided into four groups: one contains ≥ 10 animals carrying this CNV; the second is ≥ 5 and <10 animals; the third is ≥ 2 and <5 and the last group has only one animal carrying this CNV. Wang et al. (2012), we still observe many more CNVs (2006). Aggregating CNVs into CNVRs resulted in a (118.8) per individual. A cluster analysis of the samples total of 1,556 non-overlapping regions covering 60 Mb, based on the CNVs detected in each of the animals which represent almost 5.4% of the chicken genome. An results in tight clustering of all individuals from the example of a CNVR in the chicken is given in Figure 3. same line (Figure 2). The largest CVNR detected is located on GGAZ (CNVR CNVRs were determined by aggregating overlapping 1482) and is 4.37 Mb in size. The number of CNVRs in CNVs identified in all samples across the aCGH experi- the chicken is considerably higher than that reported by ments according to the criteria defined by Redon et al. Wang et al. (2010) and Wang et al. (2012), 97 and 130 Crooijmans et al. BMC Genomics 2013, 14:398 Page 4 of 10 http://www.biomedcentral.com/1471-2164/14/398 Figure 2 Heatmap representation of copy number variation between chickens. Unsupervised clustering of CNVs with gains (green) and losses (red) yields a dendrogram that recapitulates features of the known genealogy of these animals within a line or breed. respectively. The number of CNVRs in the chicken is Broiler Mapping population to 68 in the commercial comparable to that reported in human [21] and almost 3 White Layer. The number of line-specific CNVRs varied times higher than in cattle [22]. from 0 for the Broiler Mapping populations to 30 for the The 176- kb CNV linked to the late feathering locus commercial White Layer line. The commercial White [14] was detected in this dataset as CNVR 1508 on Leghorn line was only represented by two individuals, GGAZ between positions 9,971,185 and 10,140,048 with therefore resulting in a higher number of fixed CNVRs a size of 169 kb. The segregation of this CNV is shown (68), of which 30 were line-specific. Of the fixed CNVRs, in Figure 4. As expected, we were unable to identify the the number of line-specific CNVRs was high in the CNV in intron 1 of the SOX5 gene responsible for the experimental lines and the Silkie breed (10 each). pea-comb phenotype in chicken due to the small size of The Silkie breed has a number of striking phenotypic this CNV (3.2 kb), which is below the probe spacing on characteristics such as black skin, white feathers and our array. black bones. We therefore investigated whether there is a relation between some of these CNVRs and some of Line-specific CNVRs these specific phenotypes. For the Silkies, 27 fixed Some CNVs were observed only in a single animal CNVRs were detected of which 10 were breed-specific whereas others seem to be fixed in all individuals of one (Additional file 2). specific line. CNVs that are specific for a line or group Two significant CNVRs are located on GGA20 (CNVR of lines are of particular interest because these are 812 at positions 10,722,231 to 10,844,289 and CNVR potential candidates for genes that affect a phenotype 814 at positions 11,263,937 to 11,435,137) and have specific for that (group of) lines. We therefore identified already been described in detail by Dorshorst et al. those CNVs that were either fixed in at least one line (2011) after fine mapping of the phenotype fibromela- (defined as fixed) or that were fixed in only a single line nosis (FM) in Silkies. The candidate gene involved in or breed (defined as line-specific). pigmentation, the Endothelin 3 gene (NDN3), is located Within the 15 different lines used in this study, we within CNVR 812 [17] and, when up-regulated, is the identified 518 CNVs, comprising a total of 214 CNVRs, primary driver of dermal hyperpigmentation in FM which were line-specific (Additional file 2). The number chickens. The potential function of the other eight line- of CNVRs fixed within a line ranged from 2 in the specific CNVRs in Silkies is not clear. One of the three Crooijmans et al. BMC Genomics 2013, 14:398 Page 5 of 10 http://www.biomedcentral.com/1471-2164/14/398 gains losses Figure 3 Overview of CNVR 209 at GGA1: 144,185,960- 144,403,060. CNVR209 (217.1 kb) consists of 30 individual CNVs (see Additional file 2: Table S2) ranging from 8.8kb to 153.7kb in size across all samples. The frequency of the CNVs within this CNVR varied from 1 to 10 across the study population. Line thickness represents the number of occurrences of the CNV. Gains are indicated with black lines, losses with blue lines. Silkie line-specific CNVR on GGA27 (CNVR 889 at po- activation factor). This candidate gene stimulates B cells sitions 4,128,916 to 4,155193) harbours the gene CCR7, to undergo proliferation and to counter apoptosis and which stimulates melanoma migration, and the gene was examined in more detail. To confirm this CNVR, SMARCA4, a SWI/SNF-related matrix-associated actin- we quantified the relative abundance of the DNA copy dependent regulator of chromatin. Both these genes are number for TNFSF13B using a TaqMan assay. Quantifi- potential candidates for traits related to pigmentation. cation of the ovotranferrin gene, known to be in single Further studies are needed to study the full potential of copy in the chicken genome, was used as an internal ref- these line-specific CNVRs. erence. A primer probe set spanning exon 5 and intron For lines 6 and 6 , we detected 29 and 36 CNVRs 6of TNFSF13B revealed a significant difference in copy 1 3 respectively, which are fixed in these lines, while only a number between line 6 when compared to lines 7 and single CNV and 3 CNVRs respectively are line-specific. N. No difference was detected using a primer probe set One of the line-fixed CNVRs for line 6 (CNVR 209 on spanning intron 1 and exon 1. These results suggest that GGA1 between positions 144,249,310 and 144, 403,060) there is partial duplication of TNFR13B, with exon 5 contains the gene TNFSF13B (tumor necrosis factor duplicated in all lines tested except in lines 6 and 6 . 1 3 (ligand) superfamily, member 13b) or BAFF (B cell However, the CNV does not extend as far as exon 1 of Crooijmans et al. BMC Genomics 2013, 14:398 Page 6 of 10 http://www.biomedcentral.com/1471-2164/14/398 would be overlapping with inter-specific CNVs between more distant species. ZZ father ZW mother Gene content of chicken CNVR Within the 1,556 CNVRs, a total of 2,642 unique Ensembl peptides were identified based on chicken build 2.1. To examine whether genes involved in spe- cific pathways or biological processes are more prone k / K k / - to copy number variation, we performed a gene en- 3 copies 1 copy richment analysis for the genes located within the CNVRs. The chicken transcript ids were used as input ZW daughter into DAVID for a gene enrichment and ontology ana- lysis [26]. Terms showing significant enrichments were the GO terms “functional constituent of cytoskeleton”, “nuclear binding”, “cellular response to stress”,and “macromolecule catabolic processes”.The GO term “functional constituent of cytoskeleton” is mainly K / - 2 copies driven by the keratin superfamily. The avian keratin Figure 4 Segregation of CNVR1508 at GGAZ: 9,971,185- genes are over-represented when compared to mam- 10,140,048. A male (ZZ) with k+/K (3 copies) was crossed with a mals [1]. Phylogenetic analysis demonstrated that evo- female k+/− (1 copy) giving a female offspring K/- (2 copies). lution of archosaurian epidermal appendages in the linage leading to birds was accompanied by duplication TNFSF13B, as indicated by the equivalent copy number and divergence of the ancestral ß-keratin gene cluster. across all lines at this region of the gene. Further charac- In the chicken, four subfamilies (claw, feather, feather- terisation of this CNV will be required to identify its like and scale) of the ß-keratin genes have been named boundaries accurately. in accordance with tissue-specific expression and se- One of the three other line-specific CNVRs (CNVR521), quence heterogeneity [27]. These ß-keratin gene sub- detected in lines 6 and 6 and located at GGA18: families are clustered on GGA25 whereas the genes 1 3, 372390–400489, overlaps with the Myosin Heavy Chain fortwo othermonophyleticgroupsoffeather keratins gene 1 (MYH1). are located on GGA27 and GGA2 respectively. We ob- served large CNVRs (CNVRs 863, 873 and 791) within all three regions in the chicken genome up to 2 Mb in Comparative CNV analysis size. Within these CNVRs we observed both CNV We compared the chicken CNVRs to those previously losses and gains. detected in turkey, duck and zebra finch (Additional file 1) [23-25]. From the 16 inter-specific CNVs detected Conclusions between turkey and chicken detected by Griffin et al. In this study we performed aCGH screening of the (2008) using comparative CGH, 10 did not show variation chicken genome to identify CNVs in a comprehensive in the chicken. Within the current study, 15 of these 16 manner. We have identified a large number of genes af- inter-specific CNVs could be verified, whereas the inter- fected by CNV, including genes involved in well-known specific CNV on chrE64 could not be validated because phenotypes such as late feathering and pigmentation in chrE64 was not used for probe design on the array. From Silkies. In particular large gene families such as the kera- the 15 inter-specific regions detected, all (100%) fall into tin gene family and the MHC show extensive variation CNVRs detected in this study. The CNV detected in the in copy number. The CNVs in the chicken overlapping chicken Layer vs. Red Jungle Fowl on GGA2 (position with the inter-specific CNVs (CNVs detected between 25,725,000 to 25,785,000) by Griffin et al. (2008) could not different bird species) are potentially old CNVs. More- be confirmed in our study. When comparing the inter- over, when the evolutionary distance between chicken specific zebra finch / chicken CNVs [25], 9 of the 27 CNVs and the other bird species is enlarged the older (more (33%) did overlap with a chicken CNVR while of the ancient) the CNV is. Many of these CNVs very likely inter-specific CNVs detected between duck and chicken affect traits of economic importance in the chicken and [24], 15 of the 31 (48%) did overlap with a chicken CNVR. our global characterization of CNVRs in the chicken These results indicate that the inter-specific CNVs genome will aid in the identification of structural vari- detected are prone to overlap with a CNVR of the avian ation in the genome underlying important phenotype lineage when more samples are analyzed. Fewer CNVRs differences for qualitative and quantitative traits. Crooijmans et al. BMC Genomics 2013, 14:398 Page 7 of 10 http://www.biomedcentral.com/1471-2164/14/398 Methods the Puregene blood kit or the Qiagen Qiamap DNA Construction of the oligonucleotide microarray blood kit. Blood samples were collected by veterinarians A CGH array for whole genome analysis in chicken according to national legislation. No approval from the (UCSC galGal3 (WUSTL build 2.1, may 20006)) was ethics committee was necessary according to local le- designed and constructed by Agilent Technologies gislation. For the experimental lines from the Institute (http://www.genomics.agilent.com). The chicken genome for Animal Health at Compton, genomic DNA was CGH microarray kit 244A had a median probe spacing extracted from whole blood as previously described [32]. of 4 kb, with probes printed using the Agilent 60-mer We assessed the DNA quality and quantity by OD 260/280 Sure print technology. and OD readings and on 1% agarose gels. The re- 260/230 ference sample (UCD001) used is the same individual Experimental chicken lines that was used to generate the chicken genome reference The experimental lines 6 (line 6 and 6 ), 7 and 15I are sequence [1]. 1 3 2 5 all experimental White Leghorn lines characterized for resistance to viral-induced tumours. Line 6 was been selected for resistance to Marek’s disease (MD) and aCGH data analysis and CNV calling lymphoid leukosis (LL) whereas line 7 is susceptible to Unamplified genomic DNA (1 μg) was labeled with Cy3 MD and 15I is susceptible to MD and LL [28]. Lines 6 , (test samples) or Cy5 (reference sample). The Agilent 5 3 7 , and 15I are kept at the Avian Disease and Oncology Oligonucleotide Array-based CGH for genomic DNA 2 5 Laboratory (ADOL) at East Lansing, MI, USA, while lines Analysis protocol (v4.0:2006) was used for the labeling 6 and 7 are bred at the Pirbright Institute, Compton, of the DNA, Hybridizations, washings, and scanning 1 2 UK. Line N is a control line. Line BrL is a Brown Leghorn of the arrays. Self-self control hybridizations were line selected for resistance to infectious bursal disease performed by labeling the reference sample with Cy3 virus (IBDV) [29-31]. All lines have been maintained by and Cy5. Fluorescence intensities ratios were extracted random mating within the flocks. using Agilent Feature Extraction software (Agilent Tech- nologies). Log2ratio profiles were then normalized using Sample processing aCGH-Spline to remove dye biases and reduce experi- Breeds in this study include Red Jungle Fowl, one com- mental noise [33]. CNV detection was performed using mercial white and two commercial brown layer lines, six a modified version of CNVfinder [32], where we opti- experimental lines (five white and one brown) three mized empirically significance thresholds using replicate commercial broiler and one experimental broiler line, self-self hybridizations. The CNVRs were obtained by and the Silkie breed. In total 64 animals were used merging overlapping CNVs according to similar criteria (Table 1). Genomic DNA was isolated from blood with as described previously [21,34]. Table 1 Sample information Breed Line Breed code Platform # Samples Relation Red Jungle Fowl Red Jungle Fowl aviandiv_101 Aglilent 244K 4 unrelated (F0) Exp.Layer brown Compton_BrL LineBrL Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton_6_sub1 Line6 Aglilent 244K 5 unrelated (F0) Exp. Layer white Eastlansing_6_sub3 Line6 Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton-N LineN Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton_15I Line15I Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton_7_sub2 Line7 Aglilent 244K 5 unrelated (F0) Com. Broiler commercial broiler_A ComBroilA Aglilent 244K 2 unrelated (F0) Com. Broiler commercial broiler_B ComBroilB Aglilent 244K 3 unrelated (F0) Com. Broiler commercial broiler _C ComBroilC Aglilent 244K 4 unrelated (F0) Exp. Broiler broiler_M BroilM Aglilent 244K 8 unrelated (F0) Com.Layer brown commercial layer brown_1 ComBrownL1 Aglilent 244K 4 unrelated (F0) Com.Layer brown commercial layer brown_2 ComBrownL2 Aglilent 244K 4 unrelated (F0) Com.Layer white commercial layer white ComWhiteL Aglilent 244K 2 unrelated (F0) Silkie Silkie Silkie Aglilent 244K 3 unrelated (F0) total 64 Crooijmans et al. BMC Genomics 2013, 14:398 Page 8 of 10 http://www.biomedcentral.com/1471-2164/14/398 Quantitative RT-PCR from different input concentrations, compared to the Relative abundance of DNA copy number for candidate mean Ct value of reference sample (UCD001). CNVs was quantified by TaqMan quantitative PCR using an adapted method previously described [35]. Functional gene annotation Primers and probes for TNFSF13B and ovotranferrin Functional gene annotation is performed in DAVID were designed using Primer Express (Applied Biosystems) (gene Functional Classification Tool, DAVID Bioinfor- (Additional file 3). For the PCR we used 1× real-time PCR matics Resources 6.7, NIAID/NIH at http://david.abcc. mix, TNFSF13B forward primer (0.4 μM), and TNFSR13B ncifcrf.gov [26]. Ensemble gene ids within CNVR were reverse primer (0.4 μM), ovo forward primer (0.4 μM), collected (Additional file 5) and used as the input file for and ovo reverse primer (0.4 μM), TNFSF13B FAM probe DAVID. The EASE score and the modified Fisher Exact (0.2 μM), ovo VIC probe (0.2 μM), bovine serum albumin P-Value were given where the smaller, the more -4 (10 μg per reaction). The PCR was performed using the enriched. Cut off P-Value within this study was E10 . TaqMan fast universal PCR master mix reagents (Applied Biosystems, Warrington, UK). Amplification and detection Additional files of specific products was performed using the Applied Biosystems 7500 Fast Real-Time PCR System with the fol- Additional file 1: All CNVs detected within the 64 chicken using aCGH against the Red Jungle Fowl animal used for deriving the lowing thermocycling parameters: 50°C for 2 min, 95°C whole genome sequence. The first column indicated the CNV number for 10 min, followed by 40 cycles of 94°C (15 sec) and whereas column 2 indicates the CNVR number of this CNV. Further 60°C (1 min). Results are expressed in terms of the columns indicate start and end position of the CNV on a particular chromosome followed by the overall occurrence of this CNV in the 64 threshold cycle value (C ), the cycle at which the animals used. Detailed CNV occurrence per line is given in the following change in the reporter dye passes a significance thresh- columns. The last 4 columns give a literature review of overlapping old (ΔR ). n chicken CNV found by others including the inter-specific CNVs detected in other avian species. To account for variation in sampling and DNA prepar- Additional file 2: Presents the specific CNVs either fixed or variable ation, the C values for TNFSF13B-specific product for within a line or over lines. Yellow blocks indicate fixed CNV within a each sample were normalized using the C value of the line, green blocks represent fixed CNVs in a certain group of lines ovotransferrin product for the same sample. Normalized whereas red blocks represent specific CNV fixed within one line. C values were calculated using the formula C +(N ' − t t t Additional file 3: Oligonucleotide primers andprobesusedinreal- time PCR of TNFR13B. C ') × S/S', where N ' is the mean C for ovotransferrin t t t Additional file 4: Marker information for the validation of 12 CNVs among all samples, C ' is the mean C for ovotransferrin in t t by real time PCR. the sample and S and S' are the slopes of the regressions Additional file 5: Genes completely or partial overlapping with the of the standard plots for the test TNFSF13B and CNVRs. For every CNVR the genes are reported with completely or ovotransferrin, respectively. This effectively achieves inter- partial including Ensembl gene id with start en end of this gene. In the last column potential gene name is given (according to Ensembl). polations on the standard plots to obtain the TNFSF13B C values that would have been obtained had all samples Abbreviations had the same (mean) amount of ovotransferrin DNA. CNV: Copy number variation; CNVR: Copy number variation region; Additional validation was performed using a quantita- SNP: Single nucleotide polymorphism; RT-PCR: Reverse transcriptase polymerase tive PCR approach, as described by Weksberg et al. [36], chain reaction; aCGH: Array comparative genomic hybridization; GGA: Gallus gallus; FM: Fibromelanosis; MD: Marek’s disease; LL: Lymphoid leukosis. to investigate the difference CNVs. Copy number was determined for 12 markers in 12 different CNVs. Pri- Competing interests mer3 webtool http://frodo.wi.mit.edu/primer3/ was used The authors have declared that no competing interests exist. to design primers for qPCR validation. Amplicon length Authors’ contributions was limited between (50 bp – 100 bp) and regions with RPMAC, MAMG conceived the study and prepared the manuscript. TF and GC percentage between 30% and 60% were included, RR performed the statistical analysis. MSF and PK performed analysis of the while avoiding runs of identical nucleotides. All other experimental lines including validation. SS performed the validation in the Silkie breed. HHC provided DNA of the reference animal and one of the settings were left at their default. Details of the qPCR experimental lines. MSF, RR, HHC, PK, SS contributed advice and data on primers can be found in Additional file 4: Table S5. biological issues, provided analytical support and contributed to qPCR experiments were conducted using MESA Blue understanding the data. All authors read and approved the final manuscript. qPCR MasterMix Plus for SYBR Assay Low ROX from Acknowledgements Eurogentec, this 2x reaction buffer was used in a total This work was financially supported by European Union grant FOOD-CT-2004- reaction volume of 12.5 μl. All reactions were amplified 506416 (EADGENE), BBSRC Institute Strategic Programme Grants at the Pirbright on 7500 Real Time PCR system (Applied Biosystems Institute and the Roslin Institute, Hendrix Genetics, The Netherlands, Cobb- Vantress Inc, USA and The University of Delaware, College of Agriculture and group). The copy number differences were determined Natural Resources Seed grant ANSC462123. We would like to thank Carl J. by using a standard ΔCt method that compares the Schmidt and Mary E. Delany for their contribution of DNA from some of the mean Ct value of the target CNV fragments, determined lines and Bert Dibbits for the help in performing CNV validation. Crooijmans et al. BMC Genomics 2013, 14:398 Page 9 of 10 http://www.biomedcentral.com/1471-2164/14/398 Supporting data 15. Wright D, Boije H, Meadows JRS, Bed’hom B, Gourichon D, Vieaud A, The aCGH data from this study is submitted to the NCBI Gene Expression Tixier-Boichard M, Rubin C-J, Imsland F, Hallböök F, Andersson L: Copy omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no GSE19866. number variation in intron 1 of SOX5 causes the Pea-comb phenotype in The following supporting data are available with the online version of this chickens. PLoS Genet 2009, 5:e1000512. paper. 16. Gunnarsson U, Kerje S, Bed’hom B, Sahlqvist A-S, Ekwall O, Tixier-Boichard M, Kämpe O, Andersson L: The dark brown plumage color in chickens is Author details caused by an 8.3-kb deletion upstream of SOX10. Pigment Cell Melanoma Animal Breeding and Genomics Centre, Wageningen University, P.O. box Res 2011, 24:268–274. 338, Wageningen 6700 AH, The Netherlands. Genetics and Genomics group, 17. 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BMC Genomics 2013, 14:398 Page 10 of 10 http://www.biomedcentral.com/1471-2164/14/398 34. Graubert TA, Cahan P, Edwin D, Selzer R, Richmond TA, Eis PS, Shannon WD, Li X, McLeod HL, Cheverud JM, Ley TJ: A high-resolution map of segmental DNA copy number variation in the mouse genome. PLoS Genet 2007, 3:e3. doi:10.1371. 35. Baigent SJ, Petherbridge LJ, Howes K, Smith LP, Currie RJ, Nair VK: Absolute quantitation of Marek's disease virus genome copy number in chicken feather and lymphocyte samples using real-time PCR. J Virol Methods 2005, 123(1):53–64. 36. Weksberg R, Hughes S, Moldovan L, Bassett AS, Chow EW, Squire JA: A method for accurate detection of genomic microdeletions using real-time quantitative PCR. BMC Genomics 2005, 6:180. doi:10.1186/1471-2164-14-398 Cite this article as: Crooijmans et al.: Large scale variation in DNA copy number in chicken breeds. BMC Genomics 2013 14:398. 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Large scale variation in DNA copy number in chicken breeds

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Springer Journals
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2013 Crooijmans et al.; licensee BioMed Central Ltd.
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1471-2164
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10.1186/1471-2164-14-398
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Abstract

Background: Detecting genetic variation is a critical step in elucidating the molecular mechanisms underlying phenotypic diversity. Until recently, such detection has mostly focused on single nucleotide polymorphisms (SNPs) because of the ease in screening complete genomes. Another type of variant, copy number variation (CNV), is emerging as a significant contributor to phenotypic variation in many species. Here we describe a genome-wide CNV study using array comparative genomic hybridization (aCGH) in a wide variety of chicken breeds. Results: We identified 3,154 CNVs, grouped into 1,556 CNV regions (CNVRs). Thirty percent of the CNVs were detected in at least 2 individuals. The average size of the CNVs detected was 46.3 kb with the largest CNV, located on GGAZ, being 4.3 Mb. Approximately 75% of the CNVs are copy number losses relatively to the Red Jungle Fowl reference genome. The genome coverage of CNVRs in this study is 60 Mb, which represents almost 5.4% of the chicken genome. In particular large gene families such as the keratin gene family and the MHC show extensive CNV. Conclusions: A relative large group of the CNVs are line-specific, several of which were previously shown to be related to the causative mutation for a number of phenotypic variants. The chance that inter-specific CNVs fall into CNVRs detected in chicken is related to the evolutionary distance between the species. Our results provide a valuable resource for the study of genetic and phenotypic variation in this phenotypically diverse species. Keywords: Copy number variation, Chicken, aCGH, Line-specific CNVs, Inter-specific CNVs, Genes Background variants (CNVs) lie between these two extremes, ranging The chicken was the first livestock species to have its in size from thousands to millions of bases. genome completely sequenced [1]: a large collection of In human, many CNVs are in linkage disequilibrium chicken single nucleotide polymorphisms (SNPs) has with nearby genetic markers and thus appear to be an- been available for almost a decade [2]. More recently, cient [6]. Others are more recent, such as CNVs af- the number of SNPs has been enlarged to over 7 million fecting olfactory receptor gene diversity [7], or can be [3]. Although numerous studies studying genetic vari- recurrent [8]. Structural variants include a variety of mo- ation have focused on SNPs, there is growing evidence lecular alterations such as duplications, deletions, and for the substantial role of structural polymorphism in inversions [9,10]. A comprehensive map that catalogues phenotypic diversity [4]. Structural variation has been and indexes structural variants - in particular CNVs - recognized as an important mediator of gene and gen- across the genome is a necessary prelude to understand- ome evolution within populations [5]. While the sizes of ing their role in the context of specific phenotypic traits. genetic variants range from a single base to whole chro- Early reports estimated that at least 2% of the human mosomes, historically only the extreme ends of the genome is affected by structural variations [11], but spectrum have been explored. DNA copy number more recent studies suggest that as much as 3.75% of the human genome harbors common CNVs [12]. CNV regions (CNVRs) will be an important comple- * Correspondence: [email protected] ment to SNP-centric genome-wide association studies, Animal Breeding and Genomics Centre, Wageningen University, P.O. box since existing SNP discovery and genotyping methodolo- 338, Wageningen 6700 AH, The Netherlands gies are biased against inclusion of these more complex Full list of author information is available at the end of the article © 2013 Crooijmans et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Crooijmans et al. BMC Genomics 2013, 14:398 Page 2 of 10 http://www.biomedcentral.com/1471-2164/14/398 genetic variants. Furthermore, many of the CNVRs are were detected (Additional file 1). Seventy-five percent of not very well represented and annotated in the genomic these CNVs are losses. Being more conservative, i.e. re- sequence due to biases in chromosome assembly. In quiring the CNV to be observed in at least 2 samples, order to estimate what fraction of the genome is affected 944 CNVs (29.9%) were detected with an average size of by CNV, global studies have been performed in human, 46.1 kb. The real time PCR validation of 12 CNVs ran- chimpanzee, dog, mouse and cattle. In cattle, for ex- ging in being present in 1 to 41 samples did give a suc- ample, 177 high confidence CNVRs were reported as cessful validation of 92%. Only one marker in a potential covering 28.1 Mb, 35 of these CNVRs being apparently CNV which was detected in only one animal failed valid- breed-differential or breed-specific [13]. To determine ation by showing no difference between the reference the full extent of variation and its influence on pheno- sample. These results indicated that the detected CNVs typic variation, the reference genome assembly should have a high chance of being a real CNVs. Furthermore, be near completion and more individual genomes need we could confirm 13 of the 26 high confidence CNVs to be sequenced for the species of interest. Analysis of (50%) identified by Wang et al. (2010) and 23 out of the CNVs in livestock species is of particular interest, not 70 CNVs (23.9%) detected in only one animal in that only because of their economic importance, but also due study. However, we were only able to confirm 21% of to the often- extensive selection pressure applied in the 238 CNVs detected in another study (Wang et al. generating the different lines and varieties. 2012) (Additional file 1). One reason why fewer CNVs Since CNVs potentially affect gene expression [8], were detected in the studies of Wang et al. [18,19] is the CNVs may account for a significant proportion of the use of only 10 and 6 animals respectively from three dif- extensive phenotypic variation observed in this species. ferent breeds in those two studies. Moreover, both stu- Examples of phenotypes associated with a CNV in the dies used a different reference animal and the reference chicken include late feathering on chromosome Z animal was also from the same breed. Also, none of the (GGAZ) [14], pea comb on GGA1 [15], dark brown chicken breeds were in common between our study and plumage color on GGA1 [16] and dermal hyperpigmen- those of Wang [18,19] which may account for the lack tation on GGA20 [17]. Additional CNVs have been of complete validation. detected in the chicken using aCGH [18], but that study The distribution of the gain and loss CNVs over the only examined ten individuals and only identified 96 genome is shown in Figure 1. The average size of the CNVs corresponding to approximately 1.3% of the CNVs detected in this study is 46.3 kb, and the largest chicken genome. Furthermore only 27 of these CNVs CNV, 4.3 Mb (CNV #3126), was observed on GGAZ. were observed in more than one individual. CNV distributions within the different chicken lines are Here we applied an aCGH analysis to different chicken given in Additional file 1: Table S1. Although limited breeds in order to obtain a global CNV map of the sequence information is available for GGA16 (MHC- chicken genome. containing chromosome), the repetitive nature of this chromosome [1] was confirmed by detecting copy num- Results and discussion ber variations on the entire sequenced part of this CNV in chicken chromosome. Another striking case was seen on GGA25 aCGH was carried out using the Agilent 244K chicken where 16 out of the 64 animals showed CNVs in a array with a mean probe spacing of 4000 bp. This array CNVR covering a substantial part of the genome se- is based on the chicken assembly WUSTL 2.1 (Galgal3) quence (from 0 up to 1.85 Mb). Interestingly, GGA25 is and covers chromosomes 1–28, 32 and the sex chromo- one of the more GC-rich chromosomes and it contains a somes Z and W. The virtual chromosome “ChrUn” with relatively large number of minisatellites [20]. concatenated unmapped contigs was not taken into ac- The average number of CNVs per animal was 103, ranging from 61 to 209. The highest number of CNVs count in the probe design. To access the chicken CNV landscape, we selected 64 animals from 6 commercial was detected in the commercial White Leghorn line with lines (layer and broiler types), 7 experimental lines (layer an average of 187.5 CNV per animal. The lowest number of CNVs was observed in the Red Jungle Fowl (Aviandiv) and broiler types), Red Jungle Fowls and Silkies. DNA samples were labeled with Cy3 whereas the reference population with an average of 83.8 CNV per animal, DNA sample - derived from UCD001, the Red Jungle which was expected as an inbred Red Jungle Fowl ani- mal was used as a reference in this study. The commer- Fowl animal previously selected to generate the chicken reference genome assembly - was labeled with Cy5. cial broilers showed an average of 128.8 CNVs per We defined conservative parameters for CNV detec- animal. These numbers are considerably higher than those reported by Wang et al. (2012), where the average tion to limit false positive calls (see Methods). Within the 15 lines used in this study, 3,154 CNVs with a diffe- was 40 CNVs per animal. Even when our analysis is re- rent start and/or end location on the chicken genome stricted to the autosomes, as was done in the study of Crooijmans et al. BMC Genomics 2013, 14:398 Page 3 of 10 http://www.biomedcentral.com/1471-2164/14/398 Figure 1 CNV distribution across the chicken genome. Red bars indicate copy-number losses and blue bars copy-number gains. Bar length indicates the number of occurrence for a given CNV, divided into four groups: one contains ≥ 10 animals carrying this CNV; the second is ≥ 5 and <10 animals; the third is ≥ 2 and <5 and the last group has only one animal carrying this CNV. Wang et al. (2012), we still observe many more CNVs (2006). Aggregating CNVs into CNVRs resulted in a (118.8) per individual. A cluster analysis of the samples total of 1,556 non-overlapping regions covering 60 Mb, based on the CNVs detected in each of the animals which represent almost 5.4% of the chicken genome. An results in tight clustering of all individuals from the example of a CNVR in the chicken is given in Figure 3. same line (Figure 2). The largest CVNR detected is located on GGAZ (CNVR CNVRs were determined by aggregating overlapping 1482) and is 4.37 Mb in size. The number of CNVRs in CNVs identified in all samples across the aCGH experi- the chicken is considerably higher than that reported by ments according to the criteria defined by Redon et al. Wang et al. (2010) and Wang et al. (2012), 97 and 130 Crooijmans et al. BMC Genomics 2013, 14:398 Page 4 of 10 http://www.biomedcentral.com/1471-2164/14/398 Figure 2 Heatmap representation of copy number variation between chickens. Unsupervised clustering of CNVs with gains (green) and losses (red) yields a dendrogram that recapitulates features of the known genealogy of these animals within a line or breed. respectively. The number of CNVRs in the chicken is Broiler Mapping population to 68 in the commercial comparable to that reported in human [21] and almost 3 White Layer. The number of line-specific CNVRs varied times higher than in cattle [22]. from 0 for the Broiler Mapping populations to 30 for the The 176- kb CNV linked to the late feathering locus commercial White Layer line. The commercial White [14] was detected in this dataset as CNVR 1508 on Leghorn line was only represented by two individuals, GGAZ between positions 9,971,185 and 10,140,048 with therefore resulting in a higher number of fixed CNVRs a size of 169 kb. The segregation of this CNV is shown (68), of which 30 were line-specific. Of the fixed CNVRs, in Figure 4. As expected, we were unable to identify the the number of line-specific CNVRs was high in the CNV in intron 1 of the SOX5 gene responsible for the experimental lines and the Silkie breed (10 each). pea-comb phenotype in chicken due to the small size of The Silkie breed has a number of striking phenotypic this CNV (3.2 kb), which is below the probe spacing on characteristics such as black skin, white feathers and our array. black bones. We therefore investigated whether there is a relation between some of these CNVRs and some of Line-specific CNVRs these specific phenotypes. For the Silkies, 27 fixed Some CNVs were observed only in a single animal CNVRs were detected of which 10 were breed-specific whereas others seem to be fixed in all individuals of one (Additional file 2). specific line. CNVs that are specific for a line or group Two significant CNVRs are located on GGA20 (CNVR of lines are of particular interest because these are 812 at positions 10,722,231 to 10,844,289 and CNVR potential candidates for genes that affect a phenotype 814 at positions 11,263,937 to 11,435,137) and have specific for that (group of) lines. We therefore identified already been described in detail by Dorshorst et al. those CNVs that were either fixed in at least one line (2011) after fine mapping of the phenotype fibromela- (defined as fixed) or that were fixed in only a single line nosis (FM) in Silkies. The candidate gene involved in or breed (defined as line-specific). pigmentation, the Endothelin 3 gene (NDN3), is located Within the 15 different lines used in this study, we within CNVR 812 [17] and, when up-regulated, is the identified 518 CNVs, comprising a total of 214 CNVRs, primary driver of dermal hyperpigmentation in FM which were line-specific (Additional file 2). The number chickens. The potential function of the other eight line- of CNVRs fixed within a line ranged from 2 in the specific CNVRs in Silkies is not clear. One of the three Crooijmans et al. BMC Genomics 2013, 14:398 Page 5 of 10 http://www.biomedcentral.com/1471-2164/14/398 gains losses Figure 3 Overview of CNVR 209 at GGA1: 144,185,960- 144,403,060. CNVR209 (217.1 kb) consists of 30 individual CNVs (see Additional file 2: Table S2) ranging from 8.8kb to 153.7kb in size across all samples. The frequency of the CNVs within this CNVR varied from 1 to 10 across the study population. Line thickness represents the number of occurrences of the CNV. Gains are indicated with black lines, losses with blue lines. Silkie line-specific CNVR on GGA27 (CNVR 889 at po- activation factor). This candidate gene stimulates B cells sitions 4,128,916 to 4,155193) harbours the gene CCR7, to undergo proliferation and to counter apoptosis and which stimulates melanoma migration, and the gene was examined in more detail. To confirm this CNVR, SMARCA4, a SWI/SNF-related matrix-associated actin- we quantified the relative abundance of the DNA copy dependent regulator of chromatin. Both these genes are number for TNFSF13B using a TaqMan assay. Quantifi- potential candidates for traits related to pigmentation. cation of the ovotranferrin gene, known to be in single Further studies are needed to study the full potential of copy in the chicken genome, was used as an internal ref- these line-specific CNVRs. erence. A primer probe set spanning exon 5 and intron For lines 6 and 6 , we detected 29 and 36 CNVRs 6of TNFSF13B revealed a significant difference in copy 1 3 respectively, which are fixed in these lines, while only a number between line 6 when compared to lines 7 and single CNV and 3 CNVRs respectively are line-specific. N. No difference was detected using a primer probe set One of the line-fixed CNVRs for line 6 (CNVR 209 on spanning intron 1 and exon 1. These results suggest that GGA1 between positions 144,249,310 and 144, 403,060) there is partial duplication of TNFR13B, with exon 5 contains the gene TNFSF13B (tumor necrosis factor duplicated in all lines tested except in lines 6 and 6 . 1 3 (ligand) superfamily, member 13b) or BAFF (B cell However, the CNV does not extend as far as exon 1 of Crooijmans et al. BMC Genomics 2013, 14:398 Page 6 of 10 http://www.biomedcentral.com/1471-2164/14/398 would be overlapping with inter-specific CNVs between more distant species. ZZ father ZW mother Gene content of chicken CNVR Within the 1,556 CNVRs, a total of 2,642 unique Ensembl peptides were identified based on chicken build 2.1. To examine whether genes involved in spe- cific pathways or biological processes are more prone k / K k / - to copy number variation, we performed a gene en- 3 copies 1 copy richment analysis for the genes located within the CNVRs. The chicken transcript ids were used as input ZW daughter into DAVID for a gene enrichment and ontology ana- lysis [26]. Terms showing significant enrichments were the GO terms “functional constituent of cytoskeleton”, “nuclear binding”, “cellular response to stress”,and “macromolecule catabolic processes”.The GO term “functional constituent of cytoskeleton” is mainly K / - 2 copies driven by the keratin superfamily. The avian keratin Figure 4 Segregation of CNVR1508 at GGAZ: 9,971,185- genes are over-represented when compared to mam- 10,140,048. A male (ZZ) with k+/K (3 copies) was crossed with a mals [1]. Phylogenetic analysis demonstrated that evo- female k+/− (1 copy) giving a female offspring K/- (2 copies). lution of archosaurian epidermal appendages in the linage leading to birds was accompanied by duplication TNFSF13B, as indicated by the equivalent copy number and divergence of the ancestral ß-keratin gene cluster. across all lines at this region of the gene. Further charac- In the chicken, four subfamilies (claw, feather, feather- terisation of this CNV will be required to identify its like and scale) of the ß-keratin genes have been named boundaries accurately. in accordance with tissue-specific expression and se- One of the three other line-specific CNVRs (CNVR521), quence heterogeneity [27]. These ß-keratin gene sub- detected in lines 6 and 6 and located at GGA18: families are clustered on GGA25 whereas the genes 1 3, 372390–400489, overlaps with the Myosin Heavy Chain fortwo othermonophyleticgroupsoffeather keratins gene 1 (MYH1). are located on GGA27 and GGA2 respectively. We ob- served large CNVRs (CNVRs 863, 873 and 791) within all three regions in the chicken genome up to 2 Mb in Comparative CNV analysis size. Within these CNVRs we observed both CNV We compared the chicken CNVRs to those previously losses and gains. detected in turkey, duck and zebra finch (Additional file 1) [23-25]. From the 16 inter-specific CNVs detected Conclusions between turkey and chicken detected by Griffin et al. In this study we performed aCGH screening of the (2008) using comparative CGH, 10 did not show variation chicken genome to identify CNVs in a comprehensive in the chicken. Within the current study, 15 of these 16 manner. We have identified a large number of genes af- inter-specific CNVs could be verified, whereas the inter- fected by CNV, including genes involved in well-known specific CNV on chrE64 could not be validated because phenotypes such as late feathering and pigmentation in chrE64 was not used for probe design on the array. From Silkies. In particular large gene families such as the kera- the 15 inter-specific regions detected, all (100%) fall into tin gene family and the MHC show extensive variation CNVRs detected in this study. The CNV detected in the in copy number. The CNVs in the chicken overlapping chicken Layer vs. Red Jungle Fowl on GGA2 (position with the inter-specific CNVs (CNVs detected between 25,725,000 to 25,785,000) by Griffin et al. (2008) could not different bird species) are potentially old CNVs. More- be confirmed in our study. When comparing the inter- over, when the evolutionary distance between chicken specific zebra finch / chicken CNVs [25], 9 of the 27 CNVs and the other bird species is enlarged the older (more (33%) did overlap with a chicken CNVR while of the ancient) the CNV is. Many of these CNVs very likely inter-specific CNVs detected between duck and chicken affect traits of economic importance in the chicken and [24], 15 of the 31 (48%) did overlap with a chicken CNVR. our global characterization of CNVRs in the chicken These results indicate that the inter-specific CNVs genome will aid in the identification of structural vari- detected are prone to overlap with a CNVR of the avian ation in the genome underlying important phenotype lineage when more samples are analyzed. Fewer CNVRs differences for qualitative and quantitative traits. Crooijmans et al. BMC Genomics 2013, 14:398 Page 7 of 10 http://www.biomedcentral.com/1471-2164/14/398 Methods the Puregene blood kit or the Qiagen Qiamap DNA Construction of the oligonucleotide microarray blood kit. Blood samples were collected by veterinarians A CGH array for whole genome analysis in chicken according to national legislation. No approval from the (UCSC galGal3 (WUSTL build 2.1, may 20006)) was ethics committee was necessary according to local le- designed and constructed by Agilent Technologies gislation. For the experimental lines from the Institute (http://www.genomics.agilent.com). The chicken genome for Animal Health at Compton, genomic DNA was CGH microarray kit 244A had a median probe spacing extracted from whole blood as previously described [32]. of 4 kb, with probes printed using the Agilent 60-mer We assessed the DNA quality and quantity by OD 260/280 Sure print technology. and OD readings and on 1% agarose gels. The re- 260/230 ference sample (UCD001) used is the same individual Experimental chicken lines that was used to generate the chicken genome reference The experimental lines 6 (line 6 and 6 ), 7 and 15I are sequence [1]. 1 3 2 5 all experimental White Leghorn lines characterized for resistance to viral-induced tumours. Line 6 was been selected for resistance to Marek’s disease (MD) and aCGH data analysis and CNV calling lymphoid leukosis (LL) whereas line 7 is susceptible to Unamplified genomic DNA (1 μg) was labeled with Cy3 MD and 15I is susceptible to MD and LL [28]. Lines 6 , (test samples) or Cy5 (reference sample). The Agilent 5 3 7 , and 15I are kept at the Avian Disease and Oncology Oligonucleotide Array-based CGH for genomic DNA 2 5 Laboratory (ADOL) at East Lansing, MI, USA, while lines Analysis protocol (v4.0:2006) was used for the labeling 6 and 7 are bred at the Pirbright Institute, Compton, of the DNA, Hybridizations, washings, and scanning 1 2 UK. Line N is a control line. Line BrL is a Brown Leghorn of the arrays. Self-self control hybridizations were line selected for resistance to infectious bursal disease performed by labeling the reference sample with Cy3 virus (IBDV) [29-31]. All lines have been maintained by and Cy5. Fluorescence intensities ratios were extracted random mating within the flocks. using Agilent Feature Extraction software (Agilent Tech- nologies). Log2ratio profiles were then normalized using Sample processing aCGH-Spline to remove dye biases and reduce experi- Breeds in this study include Red Jungle Fowl, one com- mental noise [33]. CNV detection was performed using mercial white and two commercial brown layer lines, six a modified version of CNVfinder [32], where we opti- experimental lines (five white and one brown) three mized empirically significance thresholds using replicate commercial broiler and one experimental broiler line, self-self hybridizations. The CNVRs were obtained by and the Silkie breed. In total 64 animals were used merging overlapping CNVs according to similar criteria (Table 1). Genomic DNA was isolated from blood with as described previously [21,34]. Table 1 Sample information Breed Line Breed code Platform # Samples Relation Red Jungle Fowl Red Jungle Fowl aviandiv_101 Aglilent 244K 4 unrelated (F0) Exp.Layer brown Compton_BrL LineBrL Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton_6_sub1 Line6 Aglilent 244K 5 unrelated (F0) Exp. Layer white Eastlansing_6_sub3 Line6 Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton-N LineN Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton_15I Line15I Aglilent 244K 5 unrelated (F0) Exp. Layer white Compton_7_sub2 Line7 Aglilent 244K 5 unrelated (F0) Com. Broiler commercial broiler_A ComBroilA Aglilent 244K 2 unrelated (F0) Com. Broiler commercial broiler_B ComBroilB Aglilent 244K 3 unrelated (F0) Com. Broiler commercial broiler _C ComBroilC Aglilent 244K 4 unrelated (F0) Exp. Broiler broiler_M BroilM Aglilent 244K 8 unrelated (F0) Com.Layer brown commercial layer brown_1 ComBrownL1 Aglilent 244K 4 unrelated (F0) Com.Layer brown commercial layer brown_2 ComBrownL2 Aglilent 244K 4 unrelated (F0) Com.Layer white commercial layer white ComWhiteL Aglilent 244K 2 unrelated (F0) Silkie Silkie Silkie Aglilent 244K 3 unrelated (F0) total 64 Crooijmans et al. BMC Genomics 2013, 14:398 Page 8 of 10 http://www.biomedcentral.com/1471-2164/14/398 Quantitative RT-PCR from different input concentrations, compared to the Relative abundance of DNA copy number for candidate mean Ct value of reference sample (UCD001). CNVs was quantified by TaqMan quantitative PCR using an adapted method previously described [35]. Functional gene annotation Primers and probes for TNFSF13B and ovotranferrin Functional gene annotation is performed in DAVID were designed using Primer Express (Applied Biosystems) (gene Functional Classification Tool, DAVID Bioinfor- (Additional file 3). For the PCR we used 1× real-time PCR matics Resources 6.7, NIAID/NIH at http://david.abcc. mix, TNFSF13B forward primer (0.4 μM), and TNFSR13B ncifcrf.gov [26]. Ensemble gene ids within CNVR were reverse primer (0.4 μM), ovo forward primer (0.4 μM), collected (Additional file 5) and used as the input file for and ovo reverse primer (0.4 μM), TNFSF13B FAM probe DAVID. The EASE score and the modified Fisher Exact (0.2 μM), ovo VIC probe (0.2 μM), bovine serum albumin P-Value were given where the smaller, the more -4 (10 μg per reaction). The PCR was performed using the enriched. Cut off P-Value within this study was E10 . TaqMan fast universal PCR master mix reagents (Applied Biosystems, Warrington, UK). Amplification and detection Additional files of specific products was performed using the Applied Biosystems 7500 Fast Real-Time PCR System with the fol- Additional file 1: All CNVs detected within the 64 chicken using aCGH against the Red Jungle Fowl animal used for deriving the lowing thermocycling parameters: 50°C for 2 min, 95°C whole genome sequence. The first column indicated the CNV number for 10 min, followed by 40 cycles of 94°C (15 sec) and whereas column 2 indicates the CNVR number of this CNV. Further 60°C (1 min). Results are expressed in terms of the columns indicate start and end position of the CNV on a particular chromosome followed by the overall occurrence of this CNV in the 64 threshold cycle value (C ), the cycle at which the animals used. Detailed CNV occurrence per line is given in the following change in the reporter dye passes a significance thresh- columns. The last 4 columns give a literature review of overlapping old (ΔR ). n chicken CNV found by others including the inter-specific CNVs detected in other avian species. To account for variation in sampling and DNA prepar- Additional file 2: Presents the specific CNVs either fixed or variable ation, the C values for TNFSF13B-specific product for within a line or over lines. Yellow blocks indicate fixed CNV within a each sample were normalized using the C value of the line, green blocks represent fixed CNVs in a certain group of lines ovotransferrin product for the same sample. Normalized whereas red blocks represent specific CNV fixed within one line. C values were calculated using the formula C +(N ' − t t t Additional file 3: Oligonucleotide primers andprobesusedinreal- time PCR of TNFR13B. C ') × S/S', where N ' is the mean C for ovotransferrin t t t Additional file 4: Marker information for the validation of 12 CNVs among all samples, C ' is the mean C for ovotransferrin in t t by real time PCR. the sample and S and S' are the slopes of the regressions Additional file 5: Genes completely or partial overlapping with the of the standard plots for the test TNFSF13B and CNVRs. For every CNVR the genes are reported with completely or ovotransferrin, respectively. This effectively achieves inter- partial including Ensembl gene id with start en end of this gene. In the last column potential gene name is given (according to Ensembl). polations on the standard plots to obtain the TNFSF13B C values that would have been obtained had all samples Abbreviations had the same (mean) amount of ovotransferrin DNA. CNV: Copy number variation; CNVR: Copy number variation region; Additional validation was performed using a quantita- SNP: Single nucleotide polymorphism; RT-PCR: Reverse transcriptase polymerase tive PCR approach, as described by Weksberg et al. [36], chain reaction; aCGH: Array comparative genomic hybridization; GGA: Gallus gallus; FM: Fibromelanosis; MD: Marek’s disease; LL: Lymphoid leukosis. to investigate the difference CNVs. Copy number was determined for 12 markers in 12 different CNVs. Pri- Competing interests mer3 webtool http://frodo.wi.mit.edu/primer3/ was used The authors have declared that no competing interests exist. to design primers for qPCR validation. Amplicon length Authors’ contributions was limited between (50 bp – 100 bp) and regions with RPMAC, MAMG conceived the study and prepared the manuscript. TF and GC percentage between 30% and 60% were included, RR performed the statistical analysis. MSF and PK performed analysis of the while avoiding runs of identical nucleotides. All other experimental lines including validation. SS performed the validation in the Silkie breed. HHC provided DNA of the reference animal and one of the settings were left at their default. Details of the qPCR experimental lines. MSF, RR, HHC, PK, SS contributed advice and data on primers can be found in Additional file 4: Table S5. biological issues, provided analytical support and contributed to qPCR experiments were conducted using MESA Blue understanding the data. All authors read and approved the final manuscript. qPCR MasterMix Plus for SYBR Assay Low ROX from Acknowledgements Eurogentec, this 2x reaction buffer was used in a total This work was financially supported by European Union grant FOOD-CT-2004- reaction volume of 12.5 μl. All reactions were amplified 506416 (EADGENE), BBSRC Institute Strategic Programme Grants at the Pirbright on 7500 Real Time PCR system (Applied Biosystems Institute and the Roslin Institute, Hendrix Genetics, The Netherlands, Cobb- Vantress Inc, USA and The University of Delaware, College of Agriculture and group). The copy number differences were determined Natural Resources Seed grant ANSC462123. We would like to thank Carl J. by using a standard ΔCt method that compares the Schmidt and Mary E. Delany for their contribution of DNA from some of the mean Ct value of the target CNV fragments, determined lines and Bert Dibbits for the help in performing CNV validation. Crooijmans et al. BMC Genomics 2013, 14:398 Page 9 of 10 http://www.biomedcentral.com/1471-2164/14/398 Supporting data 15. 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Journal

BMC Genomics – Springer Journals

Published: Dec 1, 2013

Keywords: life sciences, general; microarrays; proteomics; animal genetics and genomics; microbial genetics and genomics; plant genetics and genomics

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