Journal of Heredity Advance Access originally published online on March 29, 2007
Journal of Heredity 2007 98(3):238-242; doi:10.1093/jhered/esm010
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Female-Specific DNA Sequences in the Chicken Genome
From the Robert H. Smith Institute of Plant Sciences and Genetics, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel (Granevitze, Blum, Ben-Ari, and Hillel); the USDA-ARS, Avian Disease and Oncology Laboratory, 3606 E. Mount Hope Road East Lansing, MI 48823 (Cheng); the Laboratoire de Genetique Cellulaire, INRA, Castanet-Tolosan 31326, France (Vignal and Morisson); the Department of Animal Sciences, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel (David); the Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020 (Feldman); and the Institute for Animal Breeding, Federal Agricultural Research Centre, Mariensee, Neustadt 31535, Germany (Weigend)
Address correspondence to Prof. J. Hillel at the address above, or e-mail: hillel{at}agri.huji.ac.il.
Eight in silico W-specific sequences from the WASHUC1 chicken genome assembly gave female-specific PCR products using chicken DNA. Some of these fragments gave female-specific products with turkey and peacock DNA. Sequence analysis of these 8 fragments (3077 bp total) failed to detect any polymorphisms among 10 divergent chickens. In contrast, comparison of the DNA sequences of chicken with those of turkey and peacock revealed a nucleotide difference every 25 and 28 bp, respectively. Radiation hybrid mapping verified that these amplicons exist only on chromosome W. The homology of 6 W-specific fragments with chromo-helicase-DNA–binding gene and expressed sequenced tags from chicken and other species indicate that these fragments may have or have had a biological function. These fragments may be used for early sexing in commercial chicken and turkey flocks.
Chromosome W is the female-specific sex chromosome in avian species; Z is its homologous chromosome. The biological events that resulted in 2 different, though homologous sex chromosomes are still not completely understood. The prevalent opinion is that the 2 sex chromosomes have a common autosomal ancestor, and during evolution, selection and chromosomal aberrations such as inversions prevented effective recombination between some regions of these ancestral chromosomes. As a result, the chromosomes evolved separately (Ellegren and Carmichael 2001; Handley et al. 2004).
In the human genome, it has been demonstrated that recombination occurs between the sex chromosomes X and Y only near the 2 ends of the Y (pseudoautosomal regions), whereas the interior segments of Y do not recombine. Skaletsky et al. (2003) found that the level of sequence homology between human chromosomes X and Y along the nonrecombining regions is distributed in a mosaic pattern. In the avian genome, it is not yet clear where the nonrecombining regions are located on chromosome W. Publications on this subject assume that W-specific regions are likely to be located within nonrecombining regions (Ogawa et al. 1997; O'Neill et al. 2000).
Identifying chromosome W-specific sequences would have several uses. First, gender differentiation genes in chicken, if they exist, are most likely located in W-specific regions (Ogawa et al. 1997). Second, nonrecombining W-specific sequences would be a powerful tool for phylogenetic analyses within and between avian species. Third, polymorphisms in W-specific regions would provide a tool for comparison with polymorphisms in mitochondrial DNA, as both are maternally inherited. Fourth, W-specific sequences may help to improve the current assembly of chromosome W.
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Materials and Methods |
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In Silico Search for W-Specific Fragments
We searched for fragments that exist only on chromosome W and do not have any homologous sequence on chromosome Z or autosomes. For this purpose, we used the published chicken sequence (WASHUC1) at UCSC browser (www.genome.ucsc.edu). We searched within the most proximal 200 kb and the most distal 20 kb of chromosome W. These regions were chosen because published information (Sanger website: www.sanger.ac.uk) predicted that these regions are W specific. In addition, about 10 kb were chosen at regions 1, 2, and 3 million bp from the proximal end of the W chromosome. These sequences were split into fragments of about 5000 bp long. The resulting fragments were compared with the chicken genome using the BLAT algorithm, available at Santa Cruz genome browser (http://genome.ucsc.edu/cgi-bin/hgBlat). Fragments that had matches to non-W regions were discarded. This procedure yielded fragments that can putatively be regarded as W specific.
PCR and Bird Types
Primers were designed using the Primer3 software package (Rozen and Skaletsky 2000) for each of 21 fragments that were found to be W-specific in silico. PCR was performed with a final volume of 20 µl with 2.5 mM MgCl2, 0.09 mM dNTP, 1x Taq buffer, and 0.05 U/µl Taq polymerase. DNA was extracted as described in Atzmon et al. (2002). The DNA templates were extracted from individuals randomly selected from the following types of chickens (origin in brackets): Red Jungle fowl (Wild), H'mong (Vietnam), Gushi (China), Marans (France—Half Asian), Cochin (Germany—Asian), Orlov (Germany—Half Asian), Asil and Malay (Germany, Game), Brakel (NW-European), Malawi (African), Baladi (Sudan), White egg layer, Broiler dam line, Broiler sire line, and Brown egg layer (all are commercial purebred lines). Variation among these types of chickens is discussed in Hillel et al. (2007). In addition, DNA was extracted from blood taken from randomly sampled individuals in turkey, duck, and peacock fancy flocks in Israel.
Sequencing
PCR amplifications of the 8 gender-specific fragments were performed using the primers listed in Table 1. The primers were tailed with M13 universal sequences (standard primer sequence M13uni and M13rev for forward and reverse primers, respectively; MWG Biotech AG, Ebersberg, Germany). PCR was performed using HotStar Taq Master Mix Kit (Qiagen GmbH, Hilden, Germany). Unincorporated primers and dNTPs were removed prior to sequencing using ExoSAP-IT (USB Corporation, Cleveland, Ohio) following the manufacturer's recommendations. Sequencing of both strands was done using the Thermo Sequenase Cycle Sequencing Kit (Amersham Bioscience, Freiburg, Germany) and one of 2 universal primers labeled with fluorescent dye. Sequencing products were visualized by 8% PAGE on a LI-COR 4200–automated DNA sequencer (LI-COR Biosciences, Lincoln, NE). For each fragment, forward and reverse sequences obtained from different individuals were aligned using AlignIR software (LI-COR Bioscience Lincoln, Nebraska). To search for nucleotide variation in these fragments, 10 females one from each of 10 divergent chicken types: Red Jungle fowl, H'mong, Gushi, Broiler dam line, Cochin, Asil, Malay, Brakel, Malawi, and Baladi were sequenced and aligned.
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Radiation Hybrid Mapping
Radiation Hybrid mapping was performed by genotyping on the ChickRH6 panel (Morisson et al. 2002). PCR conditions were as described in Cheng and Crittenden (1994). Primary 2-point mapping of the markers was performed by using the ChickRH web server (http://chickrh.toulouse.inra.fr) after which multipoint RH map calculation with the positive markers was performed using the Carthagene program (de Givry et al. 2005).
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Results |
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Twenty-one sequences, each several hundred base pairs in length, were identified in silico with no homology to chromosome Z or autosomes. Primer pairs were used for PCR with template DNA extracted from 8 males and 8 females randomly chosen from each of several commercial and experimental chicken types. All sets of primers yielded PCR products, 8 of which were seen only in females (Table 1, Figure 1). One female no. 8 did not yield the expected HUR0418 PCR fragment. This was subsequently considered as a technical error because it did not repeat anymore (Figure 1B). The DNA sequences of the 8 fragments were found to be identical to the sequences from the published chicken genome (WASHUC1).
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Nonchicken Avian Species
The 8 primer sets that gave female-specific amplicons in chicken were used in PCR with DNA templates from 3 other avian species: turkey (Meleagris gallopavo), duck (Cairina moschata), and Peacock (Pavo cristatus). Fragments HUR0418 and HUR0421 are generated only from female birds when the template DNA was from turkeys. Fragment HUR0420 is produced only in females when template DNA was from peacocks. Fragments HUR0417 and HUR0421 were generated from both sexes of duck DNA. Fragment HUR0423 amplified PCR products in both sexes when the template DNA was from peacocks.
Bioinformatic Analysis
To search for known functions associated with the 8 female-specific fragments of the chicken, these DNA sequences were compared with available National Center for Biotechnology Information databases using BLAST algorithm. The comparisons were made against EST databases separately from all other available databases because homology with ESTs raises the possibility that these are or were transcribing sequences. A match was accepted when the length of the matching sequence was >50 bp and when the E-value was less than 0.0001 (the probability to obtain such sequence homology by chance). Fragment HUR0417 had 20 matches with ESTs extracted from various tissues and organs of chickens, among them: testis, brain, activated immune cells, and lymphocytes. Fragment HUR0423 had 12 matches with ESTs extracted from chicken, mouse, rat, and pig. Fragment HUR0419 had 2 matches with unrecognized ESTs extracted from chicken. No matches were detected for the other 5 fragments with ESTs databases. BLAST against other available databases showed that fragments HUR0420, HUR0421, and HUR0424 had high homology with introns 11, 12, and 24, respectively, of the chromo-helicase-DNA–binding (CHD1) gene of several poultry species (accession numbers for example: AY298960, AY628486, and AY628489). Fragment HUR0423 had 29 matches with intron C of CHD1 gene and to delta-crystalline enhancer–binding protein from several species.
RH Mapping
The 8 W-specific fragments were mapped by RH using the INRA ChickRH6 Chicken RH panel. As the RH maps for GGAW are still under development, only few markers are available for this chromosome in the database. Among the previously identified W-linked markers, the only significant linkage was detected for MADH4 that was found to be linked at 49.3 cR, with a Lod score of 5.5, to fragment HUR0417. MADH4 had not previously been linked to any other marker by RH. HUR0419 was independent and finally, the remaining fragments HUR0418, HUR0420, HUR0421, HUR0422, HUR0423, and HUR0424 formed a linkage group 4.1 cR long (Figure 2).
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Single Nucleotide Polymorphisms
We searched sequence differences among diverse chicken populations and between chicken and 2 other avian species, turkey and peacock. DNA samples of 10 female chickens taken from 10 divergent populations were sequenced at the 8 W-specific fragments. In a total length of 3077 bp for the 8 fragments, multiple alignments revealed no polymorphism. On the other hand, when sequences were amplified from DNA of nonchicken species and aligned to homologous sequences of chicken, a high frequency of nucleotide differences was detected. Comparison of fragments HUR0418 and HUR0421 between a turkey and a chicken revealed a nucleotide difference every 34 and 16 bp, respectively. Similarly, the comparison of fragments HUR0420 and HUR0423 between a peacock and a chicken revealed a nucleotide difference every 31 and 26 bp, respectively. Small indels (1–8 bp) were detected in sequence comparison of chicken with turkey and peacock.
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Discussion |
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Eight fragments are reported here as W-specific DNA sequences that were detected by in silico analysis and verified by female-specific PCR amplifications. The identification of these fragments detected within 250 kb in this study, and other female-specific fragments detected in similar studies, indicates that such fragments are quite widespread in the W chromosome (Ogawa et al. 1997; Hori et al. 2000; O'Neill et al. 2000). The physical mapping results based on the RH panel revealed that these fragments span at least 4 different regions of the W chromosome. This suggests that these regions are located in a mosaic pattern similar to the nonrecombining regions in the human Y chromosome (Skaletsky et al. 2003).
Physical Location and Chromosomal Structure
The reliable RH map of the 8 fragments should assist in improving the assembly of chromosome W (Figure 2). Fragments HUR0420, HUR0421, HUR0423, and HUR0424 form a cluster of markers on the RH map. This cluster is found 4 cR away from another cluster composed of HUR0418 and HUR0422 but the 2 clusters are more than 4 mb away on the sequence assembly of GGAW. Given the ratios of 46 kb/cR or 52 kb/cR found for other maps (Morisson et al. 2005), the predicted distance should be about 200 kb between the 2 clusters of markers. Similarly, the fragments HUR0419 and HUR0417 that are 
135 kb away on the sequence assembly of GGAW were found independent on the RH map. In addition, using the same conversion ratio, the distance between HUR0417 and MADH4 should be around 2.5 mb. Although still imprecise, these results lay the foundations for future RH and physical maps of GGAW by presenting for the first time estimated physical distances between markers based on RH mapping.
Polymorphism on Female-Specific Fragments
Polymorphism in sex-specific (nonrecombining) regions is a powerful tool for reconstructing ancient evolutionary events as has been demonstrated clearly for the Y chromosome of human populations (Behar et al. 2003). Such polymorphism was not observed across the 8 W-specific fragments (3077 bp). This extremely low single nucleotide polymorphism (SNP) frequency is in agreement with Berlin and Ellegren (2004), who reported a single SNP within 7643 bp, and contrasts with autosomes and chromosome Z, where the SNP frequency is much higher: on average, 1 SNP every 350 bp (http://www.ncbi.nlm.nih.gov).
In our view, the low frequency of SNPs in the W chromosome of chickens is very important phenomenon that requires further study and verification. Meanwhile, the following 3 biological phenomena might contribute to our findings: 1) chromosome W is of smaller effective population size than Z (one third) or autosomes (one quarter); 2) W chromosomes have fewer opportunities for mutations because female gametes have less cell divisions than male gametes; and 3) the heterogametic chromosome is not protected against recessive deleterious mutations as it is hemizygous (Berlin and Ellegren 2004).
In contrast to the very low frequency of SNPs in the W-specific regions of chickens, when comparing chicken sequences with sequences of turkey and peacock, we found an average of one nucleotide difference every 24 and 31 bp, respectively. This finding, if shown to be more general, enables phylogenetic analysis of distant avian species because its polymorphism origin is very ancient.
Gender-Specific PCR Products
In previous studies, sequences from avian genomes have been reported to amplify female-specific PCR products (Ogawa et al. 1997; D'costa and Petitte 1998; Hori et al. 2000; O'Neill et al. 2000; Duan and Fuerst 2001). Female-specific fragments can be used for sex determination and/or early gender identification. However, further study is needed to verify whether this female specificity of our fragments spans the entire PCR product or is restricted only to the primer sequences. Southern blot hybridization of one of the 8 fragments (HUR0423) revealed that the whole fragment is W specific (unpublished data).
PCR amplification using the 8 W-specific fragments with DNA templates of birds from nonchicken species revealed several homologies between taxa. Deeper analysis of these fragments among taxa may help elucidate the evolutionary stages that led to the development of sex chromosomes and sex determination mechanism in avian species.
Homology of the W-Specific Fragments with Functional Regions
Homology of female-specific fragments with ESTs raises the possibility that they have or have had biological function. Being W specific, they may be associated with gender determination. This speculation is supported by the finding that fragments HUR0420, HUR0421, and HUR0424 are homologous to several introns in the CHD1W gene. This gene is involved in chromatin condensation and is believed to activate transcription by preventing the repressive effects of chromatin structure (Delmas et al. 1993).
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Acknowledgments |
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We thank Dr. Yehiam Salts, Sara Shabtai, Annett Weigend, and Anke Floerke for their advice and technical assistance. We thank Prof. Avigdor Cahaner and Mrs Shelly Druyan for their assistance in sample collection. The DNA samples of diverse chicken breeds used in this study were taken from the DNA bank established during the EC-funded project AVIANDIV (AVIANDIV EC Contract No. BIO4-CT98-0342 [1998–2000]; S.W. [Coordinator], M. A. M. Groenen, M. Tixier-Boichard, A.V., J.H., K. Wimmers, T. Burke, and A. Mäki-Tanila [http://w3.tzv.fal.de/aviandiv]) and on-going cooperation projects with Ngo Thi Kim Cuc from Rare Animal and Biodiversity Department, National Institute of Animal Husbandry, Hanoi, Vietnam, G. H. Chen from College of Animal Science and Technology, Yangzhou University, Yangzhou, P.R. China, and R. Shamseldin from Sudan University of Science and Technology, College of Veterinary Medicine and Animal Production, Khartoum, Sudan. This work is the subject of a US Patent Application.
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Footnotes |
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Corresponding Editor: Susan Lamont
Received August 8, 2006
Accepted January 31, 2007
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