Z-CHROMOSOMAL MARKERS DERIVED FROM CHICKEN (GALLUS DOMESTICUS) AND USE THEREOF IN CHROMOSOMAL MAPPING

FIELD OF THE INVENTION

[0002] The invention relates to novel chromosomal markers derived from chicken and use thereof.

BACKGROUND OF THE INVENTION

[0003] Livestock genome maps have progressed very rapidly in the past few years due to the availability of highly polymorphic DNA markers. But in many species, the maps are not dense enough to facilitate a thorough search for quantitative trait loci (QTLs). This is especially true in the case of the chicken. The chicken haploid karyotype consists of 39 chromosomes that are classified into two categories—the macrochromosomes and the microchromosomes. The largest five pairs of macrochromosomes and the Z-chromosome represent about 55 percent of the total DNA content of the chicken genome. The Z-chromosome covers about 210 cM of the estimated 2500-3,000 cM of the chicken genome map (Levin et al. Genomics, 16:224-230 (1993)).

[0004] Knowledge of the genetic composition of the chicken Z-chromosome is limited, in spite of the fact that this chromosome has the most detailed linkage map for this species, largely generated by classical linkage test analyses (Bitgood and Somes, Poultry Breeding and Genetics, 2nd Ed., Crawford R D, ed., Amsterdam: Elsevier, pp. 469-495 (1990)). To date, 19 known loci and 14 genetic markers consisting of 3 chicken middle repetitive sequence element (CRI) markers, 8 random amplified polymorphic DNA (RAPD) markers and 3 microsatellites have been assigned to the chicken Z-chromosome (Bitgood and Somes, (Id.) (1990); Saitoh et al, Chrom. Res., 1: 239-251 (1993); Cheng et al, Poultry Sci., 74: 1855-1874 (1995)).

[0005] The avian sex chromosome constitution differs from that of mammals because females are heterogametic (ZW) and males homogametic (ZZ). It has been observed from comparative linkage analyses that some of the sex linked genes in mammals are autosomal in chicken, while some of the sex linked genes in chicken are autosomal in mammals (Bitgood and Somes, (Id.) (1990)). Accordingly, obtaining farther information concerning the Z-chromosome of chickens would be beneficial in identifying sex-linked genes in chickens and related species.

BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION

[0006] Thus, it is an object of the invention to identify novel chromosomal markers from the Z-chromosome of chicken. It is further an object of the invention to use such markers to construct a Z-chromosome specific DNA map and to use such chromosomal markers to identify Z-chromosome homologs in related avian species, e.g., turkey.

[0007] In order to develop a dense genetic map for chicken, it is important to generate a large number of polymorphic markers per chromosome (Cheng et al, Poultry Sci., 741:1855-1874 (1995)). One way of achieving this goal is to develop chromosome-specific libraries. Chromosome flow-sorting has been the method of choice for the generation of chromosome-specific libraries in humans (Fuscoe et al, Cytogenet Cell Genet, 43:79-86 (1986)) and in swine (Langford et al, Anim. Genet, 24: 261-267 (1993)). Development of flow-sorted chromosomes is technically demanding and frequently yield preparations which have some degree of contamination with other chromosomes (Hozier and Davis, Anal. Biochem, 200: 205-127 (1992)).

[0008] A more effective and direct way of generating chromosome-specific DNA libraries is by chromosome microisolation and microcloning of the chromosome of interest. Chromosome specific libraries generated by chromosome microisolation have been used in swine (Ambady et al, (unpublished data)), cattle (Ponce de León et al, Proc. Natl. Acad. Sci., USA, (in press) 1996)), and chicken (Li et al, Proc. of the 10th Eur. Colloq. on Cytogenetics of Domestic Animals, Utrecht Univ., The Neth., p. 11, August 18-21 (1992)) genetic mapping studies in order to develop maps for particular chromosomes. Generation of polymorphic markers from chromosome-specific libraries for all of the 8 pairs of the chicken macrochromosomes will enable saturation of about 55-70% of the chicken genome. Chromosome-specific DNA can also be used as heterologous chromosome painting probes in closely and distantly related species for comparative genome analysis, study of chromosomal evolution, and for identifying gross chromosomal abnormalities.

[0009] This application, in particular, provides a chicken Z-chromosome-specific DNA library, Z-chromosomal markers and use thereof as probes to identify the Z-chromosome homolog in related species, such as turkey.

BRIEF DESCRIPTION OF THE FIGURES

[0010]
FIG. 1 shows amplification of microsatellite markers by PCR and identification of polymorphisims.

[0011]
FIG. 2 shows a genetic map constructed using the identified microsatellite markers.

[0012]
FIG. 3 shows dinucleotide repeats present in the identified microsatellite markers.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Microisolation and Microcloning

[0014] Chicken metaphases were prepared from chicken fibroblast cultures following standard procedures, fixed briefly for 5 minutes each in 9:1, 5:1 and 3:1 methanol: acetic acid and dropped on clean coverslips. Chromosome microisolation and cloning was performed following the procedure described by Ponce de León et al (Proc. Natl. Acad. Sci. USA (in press) (1996)). Briefly, twelve copies of the chicken Z-chromosome were microisolated and transferred to clean siliconized coverslips. Proteinase-K digestion, phenol-chloroform extraction, Sau3AI (50U/μl, New England Biolabs) digestion and ligation to custom prepared Sau3AI adaptors were performed in a nanoliter drop. Ligation products were digested with BgII enzyme (Promega, 10 units/μl) to cleave off the adaptor dimers that form during the ligation process.

[0015] The ligation product was PCR amplified and 10 μl of the amplified product was run on an agarose gel to determine the size of the amplified products. A 2 μl volume of this original amplification was labeled by PCR, using biotin-16-dUTP (Boehringer Mannheim). The purity, specificity and origin of the DNA fragments was determined by FISH on chicken metaphases following the procedure described by Ponce de León et al (Proc. Natl. Acad. Sci. USA (in press) (1996)). The remainder of the PCR product was digested with Sau3AI and passed through a Microcon 30 (Amicon Inc.) spin column to cleave and remove the flanking adaptors respectively.

[0016] In order to produce a chicken Z-chromosome-specific phage library, the digested DNA was cloned in a lambda ZAP Express vector (Stratagene) and packaged using Gigapack II Gold packaging extract (Stratagene). The library was amplified by plate lysate method following the manufacturer's protocol and stored at −70° C. in 7% DMSO and 0.3% chloroform. Average size of library inserts was determined by PCR amplification of 30 randomly picked clones using the T3 and T7 priming sites flanking the insert.

Fluorescent in situ Hybridizations

[0017] The Z-chromosome-specific DNA fragments were fluorescently labeled by PCR with biotin-16-dUTP (3:1 ratio of dTTP:biotin-16-dUTP) and passed through a Sephadex G-50 column to remove unincorporated nucleotides. The protocol described by Ponce de León (Proc. Natl. Acad. Sci., USA (in press) (1996)) was followed. Briefly, 200 nanograms of labeled Z-chromosome specific DNA was mixed with 6 μg of chicken competitor DNA (average size 200-400 bp) and 5.8 μg of salmon sperm DNA (average size 200-400 bp), precipitated and resuspended in 12 μl of hybridization buffer consisting of 50% deionized formamide, 1×SSC and 100% dextran sulphate to achieve a final DNA concentration of 1 μg/μl. The hybridization mix was denatured at 75° C. for 5 minutes and reannealed at 37° C. for 10 minutes and deposited on denatured (70% formamide, 2×SSC at 70° C. for 2 minutes) chicken or turkey metaphases, mounted, sealed with rubber cement and incubated in a humidified chamber at 37° C. for 18 to 20 hours. The slides were washed in 50% formamide/2×SSC at 42° C. for 15 minutes and 0.1×SSC at 60° C. for 15 minutes. Blocking was done using 2% blocking reagent (Boehringer Mannheim) and the signals were detected using avidin-FITC (5 μg/ml, Vector labs) in 1% blocking solution. Slides were washed in 4×SSC/0.1% Tween-20 for 15 minutes at 42° C., stained for 10 minutes in propidium iodide (400 ng/ml in 2×SSC) and rinsed for 5 minutes in 2×SSC/0.01% Tween-20. Slides were mounted in p-phenylenediamine-11 (PPD-11) antifade and observed under a Zeiss Axioskop fluorescent microscope.

Results

[0018] A chicken Z-chromosome specific DNA cocktail was developed by chromosome microisolation, Sau3AI digestion, adaptor ligation and PCR amplification. The amplified DNA fragments ranged in size from 400 bp to 1600 bp with the bulk of the DNA in the 500-1000 bp range. The origin, specificity and purity of the chromosomal DNA fragments was verified by FISH after PCR labeling of a small fraction of the DNA cocktail. The probes showed specific hybridization signal on a medium sized submetacentric chromosome identified as the Z-chromosome based on its morphology and G-banding pattern. After having confirmed the origin and purity of the preparation, the adaptors flanking the inserts were removed by Sau3AI digestion and column purification. Cloning was performed using equimolar ratios of the inserts to the vector ends (lambda ZAP Express, Stratagene). The original library consisted of a total of 8.48×105 plaques representing about 14 chicken Z-chromosome equivalents. The final titer of the amplified library was 1.2×1012 pfU/ml.

[0019] Thirty random plaques were selected and the inserts PCR-amplified using the T3/T7 priming sites flanking the inserts. The average insert size was about 1,000 bp (data not shown). This library was screened to identify microsatellite containing clones to increase the marker density of the chicken Z-chromosome genetic linkage map.

Heterologous Painting of Turkey Metaphase Chromosomes

[0020] The labeled chicken Z-chromosome-specific DNA fragments were used to perform FISH analysis on turkey metaphase chromosomes following the procedure described previously. Washes at the same stringency showed strong hybridization signals on a medium-sized submetacentric chromosome in turkey metaphases (data not shown). This chromosome was identified as the Z-chromosome homolog in the turkey. The obtained results indicate that the chicken and turkey Z-chromosome sequences are highly conserved. The red-legged partridge Z-chromosome has also been shown to be homologous to the chicken Z-chromosome (Dias el al, Proc. of the XXIV Int. Cont. on Anim. Genet., Prague, Czech. p. 133 (Jul. 23-24, 1994)). These results are similar to the FISH results obtained when the bovine X-chromosome painting probes were used on sheep and goat chromosomes (Ponce de Le{acute over (6)}n el al, Proc. Natl. Acad. Sci., USA (in press) (1996)) and with human X-chromosome probes on a wide range of mammalian species (Schertan el al, Nat. Genet., 6:342-347 (1994)) indicating the high degree of sex chromosome conservation among all the mammalian species studied. Solinas-Toldo et al (Genomics, 27: 489-496 (1995)) have previously shown that human chromosome-specific painting probes could identify chromosomal segments in bovine that are homologous to specific human chromosomes. It is expected based on our results that chicken chromosome painting probes can similarly be used in closely and distantly related avian species to identify gross chromosomal rearrangements such as translocations and duplications that have occurred during avian evolution. Since the chicken Z-chromosome sequences are highly conserved in the turkey, the chicken Z-chromosome-specific microsatellite markers should be particularly useful for genetic mapping in turkey.

Conclusions

[0021] Genetic and physical mapping of human and animal genomes has been greatly facilitated by the use of chromosome specific DNA libraries. Mapping with libraries specific to a chromosome or chromosomal region increases marker saturation by reducing the gaps resulting from a purely random shotgun approach. This study was undertaken to construct a genetic and physical map of microsatellites on the chicken Z chromosome. This chromosome is the fifth largest in the chicken genome, comprising about 8% of the total. Notwithstanding its size, very few microsatellites have been assigned to it. DNA originating from the chicken Z chromosome was previously isolated and reported. This was used to construct a small insert library in Lambda ZAP Express, representing 14 chromosome equivalents. This library was screened for microsatellites with an (AC) 12 oligo, and positive clones were isolated. Confirmation of the presence of the microsatellite, as well as its approximate location along the cloned fragment was accomplished by PCR amplification. Clones with adequate flanking regions were sequenced, and primers for 19 microsatellites were constructed. These primers were used to genotype individuals from the East Lansing Poultry Reference Population and a linkage map was constructed. Fourteen markers were scorable and polymorphic in this population. The resulting map contains 12 markers in two linkage groups spanning 90 Cm and two unlinked markers. The physical location of each marker was established by fluorescent in situ hybridization (FISH). Preliminary results with four markers allowed the assignment of one linkage group to the long arm of the Z chromosome, and one to the short arm.

[0022] The following nucleic acid sequences are microsatellite markers identified by the above methods. As discussed supra, these markers are useful for genetic mapping and for study of the sex chromosome structure in avian species. Also, such markers should enable the identification of genes encoding desirable traits, e.g., genes involved in growth rates, and for identifying sex-linked genotypes.

EXAMPLE

[0023] The specific Gallus domesticus microsatellite markers identified are set forth below. As noted, these DNA markers will be useful for genetic mapping of domestic chicken as well as related avian species and for studies pertaining to evolution of the sex chromosome in avian species.

1 1gatcactttc cctaatattc ttgtgtttct tgtttgttga cctgtaatgcSEQUENCE 1 (43. Seq) 1agttctgagt tttggaaagg aactaattaa gaccagagga gagataattt101tcttttatca aaaaacaaac aaacaaacaa aaaaacgaat tcttaccact151ttacaaaaat tttccatttt gaaggccagt acagccatag cattcatcta201ctttttgctt tggat 1gatcaggtgg cctgtagtag acaacaacaa caatggggtg ccctttgttgSEQUENCE 2 (71. Seq) 51ccttagtctc taactcgcac ccacacacac tttcaagttg cttgtggcca101ttcttcaggg acagttcttc acaatctatt cctttcctga tgtagaaggc151gtcacctcct cccctcctgc ctcgtttgtc ccttctaaac tgcaggtatt201agtattgata gctaaggtca agtcatggga accatctcac caggtttcag251tgttggcaac tatgttatgc tttcttagga gcatggtggt tccaactctt301ccctgcttat ttcccaagct gtgtgtgatg gtaggatagc attcaagtgg351gaggagccta tcggcttttt ggaggtactc ctaaatccct gatattcccc401tgattcccgt acttcttcct tgccaagggc ccgccaatgc atagttcaat451ttctcatgca gacgctaagg aaaggtggac cc 1gatcgtatgt atttttttac ataggataga aaatggccaa taggaaataaSEQUENCE 3 (80 Seq.) 51gacagtacag ctactaagaa agaaacacaa ttacacacac acacacacac101acacacacac acacatttga aaaacgcgct gcacagcagt gtgggtattt151tttcacaaga gagacacact ctacagtaca cagccagctc tactttgtcg201cacagtctca gtgtgtgttt gccaacagga cgcggttcac agggagatat251tgtcctcttg tgtgtgtgga gacacagaga cagag 1gatcccctgg aggaagggca atggcaaccc actccagtat tcttgcctgaSEQUENCE 4 (81. Seq) 51agaataccat ggtcagtttt gcctcctggg ctatagtcca tggggttgca101aagagtcagg catgactgag cgactctctc tctctctctc tctctctctc151acacacacac acacacacac acacacggcg tctctctctc tctctataca201tataggctgt gtgtctcgct attctcacat gagggaaact catatctagc251acgtggcaca aatattgttt gtggctctca caaaagacat gtgggcgcac301aaaggtcccc ccccggtgga tacancgcct tggtttttta taacccaagc351ctgtg 1gatcacatat gtaaactagg gaattgcata ataagattaa atgtaggtgtSEQUENCE 5 (131 Seq) 51agaacgtggc atgaaggaag gtagaattag gtggtaccta tctcttctga101aacaaactga gaatcctact accaatcaac atattctaca taccacacac151acattttttc tcgagtaaaa tataaactaa tgagaaactt ccctag 1gatcccaagc aacacatagn cagacaatca cacacacaca cacacacacaSEQUENCE 6 (147. Seq) 51cacacacaca cacacacaca cacatcctct ccccacaata catcccgaga101ggggggagag acactctctc tccctctcta taggggagac ccggagagct151ggctctgttg tctctctaca ccggacatac agtggagcac atctcacact201tgtgtctttg tctctctaca ccggacatac agtggagcac atctcacact251tgtgtctcta tctctccctg tccctgttga tccatctctc ttcacacatc301tctccagatc ttagcgctag agtctcctgt cttctctctg cgcaatttgt351gtgatagaga cacctgatat gttgtgtggg ggagacatct gtgtgtctct401gtgtcatccc agaggatttt tctctcccac acttagaggc cttctcaaga451gatgggaggt tttaatgggg tgtg 1gatcattctt ctgtttccca ttctaatggg aattctccac acacacacacSEQUENCE 7 (166. Seq) 51acacacacac acacacacat cttcttcccc ttacatggaa aaaaatcctc101cacacccctg gacactgatt actctccctc ttcccagaga gagatc 1gatcccctag agaagggaat ggctactcac tccagtattc ttgcctggagSEQUENCE 8 (196. Seq) 51aattccgtgg tcagaggagc ctggaaggct ataatccata gagtcgcaag101agtcagacag gactgagtga ctaacacaca catgcacaca cacacacaca151cacacacaca cttgctctag ggagaggcat agagatgtaa tctctcctaa201aatgggggtg gcgatggccc ctgcggccaa gtaatcgcca cacatgcgta251ttccccttaa gattgggtta ggcctccctt atgaggagag accagggaga301gaatgggctc tctctctctc tcactcccca accgagtaag tggtaaaaaa351ggttttcctg gattacaatt ttggtgttac agaattggaa aaaaatattt401ttggggctcc cccctcagtt ta 1ctagcaaaaa cacccccaca agttatgaaa acaacggctt aatatagtaaSEQUENCE 9 (199. Seq) 51tgtgtgtgtg tgtgtgtgtg tgttgcacac cacagttttc tctgatactc101aaacctctct ctttctctac aggggccccc cataacacag cggctgagat151gtgtgacggg aaggcgtggc cttttacaca tttgtggtat ggtctgccaa201ggccccctat tgccccccac aactacggag atacactagg ggcgacccgc251aggcgcgcga cccccaggtg gggccccgag 1ctttaggagg ttctctcgag taagcttttt ggatttcttt ggttcccaagSEQUENCE 10 (204. Seq) 51catcacatgg tacaggcagt cacacacaca cacatacaca cacacacaca101cacacacaca cactcctctc cccacaatac ataccgagag gggggagaga151cactctctct ccctctctat agggggagcc ccacagagct ggctctgttg201tctctctcca ccggacatac agtggagcac atctcacact tctgtctcta251tctctccctg cccctgtgac atccatctct cttcacacaa tctcacccag301gatcttagcg ctagagaccc cctgtccttc ttctcctggg gaaatttttt351gtggataaga gacacccgat atattggtgt gggggagaac atcttgtgag401gtctctgttg tgccatccca acaggaattt ttatctcccc cacaattaga451ggcccctcct caagagtgtg tgagggtt 1gatcacagat gtatgtattt ttttacatag gatagaaaat ggacaataggSEQUENCE 11 (235. Seq) 51aaataagaca gtacagctac taagaaagaa cccacattta cacacacaca101cacacacaca cacacacaca agtgtttaat ccgctgcaca gcattgtgga151catttttaca caagagagac acactctaca gtttgcgccc agctctag 1gatcattctt ctgtttccca ttctaatgga attctccaca cacacacacaSEQUENCE 12 (249. Seq.) 51cacacacaca cacacactct tctttctcct gacatggaaa aatctccccc101acaccccggg acactgattt ctctccctct ccccaacact gtgagcaaga151ggagtttatt ttgtgtgtgt cactcttcca gggagagaga gatc 1ctaggcatcg gttgggaggt ggtgagtaat tacttgtctg acattagtccSEQUENCE 13 (258. Seq) 51tgtaacattg ggtgtgtgtg tgtgtgtgtg tgtgtattcc ccttgggaat101tggttttctc aaccacaagt tcttcttttt tttttttctc cccccttttc151ttctgaaaat aagtacttgg ggggtttccg ccccccccgg taaataaaat 1ctagtggctc ccaagcaaca catagccaga caacacacac acacacacacSEQUENCE 14 (290. Seq) 51acacacacac acacacacac acacacactc ctctccccac aatacatccc101gagagggggg agagacactc tctctccctc tctatagcgg gagccccaca151gagctggctc tgctgtctct ctacaccgga catacagtgg agcacatctc201acattcgtgt ctctatctct ccctgcccct ggtgacatac atctctcttc251acacatctca ccaggtctga gcgctagagt ctcctgtctt ctctctgcgc301aatatttgtg atagagacat ctgatatatt gtgtgtggga gacatcttgt351gagtctctgt gtgcatccca gaggattttt atctccccac actag 1gatccatgaa aactttccga gttgtattgt ctaggtgaaa acacacacaaSEQUENCE 15 (309. Seq) 51acacacacac acacacacac acacaacagg gagatgagtc ttgcaagaga101ataggggaga gttatgtcac caagtctggt gaggtatata gcgtataggg151agccaacatg tcagacatct gatgtgctaa gattaacatt ttattttatt201taatgtgtga gatctcatat agcggctctt cttatatatg acgtctcgca251atgtctcttt atgtgtgtta ttctctgagc ccctgggaga tatctgtcat301cagagagaag agacatacac atacaggggt tatatatttt ctccctgtgt351gtggagatgg agggtatttt ggacaagctc aacactcatt ggctcccaga401gagagaaaag gagcaactgt tgcacccggg gctctgtagc tgggatc 1caattgggta catctacctg gtaccccacc cgggtggaaa atcgcatgggSEQUENCE 16 (341. Seq) 51cccgcggcgg ttctaggaag tactctcgag aagcttttgg gttctttggg101tcccaagcag cacatggaca ggcaatcaca cacacacaca cacacacaca151cacacacaca cacacacaca ctcctctccc cacaatacat cccgagaggg201gggagagtca ctctctctcc ctctctatag ggggcgcccc taagagctgg251ctctgttgtc tatctacacc gcacatacaa tggagcacaa ctcacactag 1gatcaaagca tggaggtcat gccaggcact gaacaaaatg gtagagagtgSEQUENCE 17 (398. Seq) 51attctatgac tgactaagac ctcatgcaac aacaagtgaa gagtcacaac101tgcaaacaga agtacaactt agcaaatcct attttcagga aacactaaac151cgtaatactt gcacgatttt ttctttaata cagtaataat tcttttagaa201tttggatata tcttttaaga tacatatttg tctaaatacc aaggcaggat251atgagcataa aatagctaag gttagctatg gtgttatatt taagaagacc301acagagcaat aggagcatac ttttcttggg gtagaagggg cccttaaagg351tcacctag 1ctagccacat cctataactc cactccacct ttaatcctga tttctgtgtcSEQUENCE 18 (420. Seq) 51tcttctctaa cctctatggc ctttctctaa agttccccaa tatcaacaat101ccttttcccc actgggacct ccagtttatt gattctacca tgtcactatc151catggtcaac cacttgtggt attataggat gtcgcgtgtg tgtgtgtgtg201tgtgtgcatg tgtgtgtgct tgggtgtcag agagttccaa tctgggggac251ctatggtttg taaacaacag gtctcttgcc aaggaagat 1ctagcgctcg tgcccctgca gttcgacact cagtggctcc tccacacacaSEQUENCE 19 (435. Seq) 51cacacacaca cacatcaata tatatataga tagatagata gatagaggag101caatataagt ggcttctcta tttccagcat gttttgaaga gcataaactc151aacagagtat atataaatct gatgtgaccc atgtcatctg ctacagcatg201agagggggta gtgatc

Z-CHROMOSOMAL MARKERS DERIVED FROM CHICKEN (GALLUS DOMESTICUS) AND USE THEREOF IN CHROMOSOMAL MAPPING

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