SELECTION FOR DWARFISM IN POULTRY

Abstract: The invention relates to a gene located on chromosome 1 in chickens, which in birds is involved in so called autosomal dwarfism. The most likely candidate gene has a homolog in mice and men and is called HMGI-c. The sequence of the cDNA and genomic DNA of the gene is provided, as well as uses of said sequence or parts or derivatives thereof, as well as methods of using sequences from this gene or flanking sequences, or microsatellite markers in close vicinity to this gene in methods for selecting for one of the two alleles of this gene. Specifically provided are breeding methods using discrimination between dwarf fenotypes and non dwarf fenotypes, which are a result of the allele variation within this gene.

Description: Title: Selection for dwarfism in poultry Introduction. The present invention relates to the field of agriculture, in particular to animal breeding, more specifically to the breeding of poultry. In breeding poultry selection of animals to breed with is a very important aspect. Traditional selection methods have been applied for centuries.lt is therefore quite difficult to improve breeding stocks of poultry in important phenotypic characteristics using traditional selection methods. Modern biotechnology has provided a number of novel tools for localizing traits on the genetic level, together with means of detecting the different alleles of such traits and thus providing the ability to select animals having the right allele. One of the novel ways of identifying a gene related to a useful trait involves so- called microsatellite markers. Microsatellites are direct repeats of di , tri or tetra nucleotides such as (TG) n # (TA) n (CAC) n or (GGAT) n - where n can vary from 4 to over 30. (Crooijmans et al , 1993) . There repeating DNA elements are found almost random throughout the vertebrate genome. Their number, over 10 4 in a vertebrate genome, and their extreme polymorphism makes them useful for linkage analysis. It is possible to detect different alleles, resulting from variation in the number of repeats, by PCR using locus specific oligo-nucleotides . From 1993, different laboratories have been working on the construction of a chicken genetic map using these microsatellite markers. This genetic map facilitates linking of Quantitative Trait Loci (QTLs) , single gene traits and non-single gene traits, with microsatellite data in order to identify their chromosomal location (s) (Crooijmans et al . , 1993; 1994; 1995; 1996a; Cheng and Crittenden, 1994; Burt et al . , 1995) . Today, the chicken genetic map covers about 90% of the genome. In order to be useful for linkage studies, a genetic map should have a marker at every 20 cM of the linkage map, which is still approximately 1.10 7 basepair. Then a QTL will always be within ten percent recombination from a microsatellite marker, the maximum crossover percentage where usage of markers is still useful (Van der Beek and Van Arendonk, 1993) . In a recent study, Ruyter-Spira et al . (Poultry Science, in press) mapped the Dominant White locus m chicken using microsatellite markers. This gene was successfully mapped on linkage group 22 of the East Lansing International reference family near MCW188 . This study proves that it is now possible to successfully map monogenic traits (i.e. the Dominant White trait) by means of a so-called total genome scan. For this purpose, microsatellite markers of high quality (many alleles, almost evenly spread performance of the PCR product) covering the major part of the chicken genome can be used. Bulked segregant analysis in combination wi th microsatelli te markers In a linkage study, a large number of animals from segregating populations has to be genotyped. When a cross is set up to study a single locus trait with a dominant or a recessive phenotype, it is possible to sample the DNA of the animals with the same phenotype. These two pools will only differ for the region that is closely linked to the phonotype on which the pools were selected, and they will be similar to all other non-lmked regions. Therefore, the distribution of the different alleles of an informative marker will only show a difference between these two pools if the marker is closely linked to the gene causing the different phenotype. Identification of the chromosomal region of interest is possible when a microsatellite map covering the whole genome is available, because linkage between the gene of interest and the microsatellite marker (s) can be detected. Using this so-called bulked segregant analysis, it is possible to carry out a 'total genome scan' on pooled samples, which greatly reduces the amount of genotypings that has to be carried out. Today, over 600 chicken microsatellite markers are available, covering over 90% of the chicken genome. This makes it possible to make a selection of approximately 150 well- working markers that efficiently can be used for a total genome scan. In a recent experiment, Ruyter-Spira et al . (Poultry Science, in press) successfully mapped the Dominant White locus using pooled samples. A limited number of possibly linked microsatellite markers (4 out of 68 informative markers) was tested on individual samples, finally resulting in the identification of the chromosomal region of the gene. Thus pooling of DNA proved to greatly reduce the amount of PCRs and analyses to perform. In the study of Ruyter-Spira et al . , a dominant trait was successfully mapped on the chicken chromosome. However, many traits are not regulated by single genes. When segregating populations for complex traits are available, it would be theoretically possible to identify the chromosomal regions of interest using a bulked segregant approach. Fuel et al . (1995) used mouse microsatellite markers to identify chromosomal regions endowed with antibody production in Biozzi mice, mice selected for High and Low antibody responses to Sheep Red Blood Cells. They carried out a total genome scan using 90 polymorphic microsatellite markers on 60 individual F2 animals, selected for their extreme phenotype from a total F2 population of 240 animals. However, though it may be possible to localize dominant traits which can be attributed to a single gene to a region of a chromosome, or even regions where different parts of complex traits may be found, it is by no means clear that every complex trait can be localized using this approach, nor that for instance recessive traits can be localized using microsatellite markers. Moreover, even though a trait has been localized to the extent that it has been found associated with some microsatellite markers, this in no way means that a responsible gene for the trait has been identified. Usually the distance between two microsatellite markers is still in the order of about 400- 600 Kb, which is much larger than any usual gene sequence. Furthermore, when contemplating how to localize the regions on one or more chromosomes associated with a certain phenotype, one does not know whether this will be a single gene trait or a complex one. The present invention in one embodiment provides the localization of a trait on a chicken chromosome, as well as a gene associated with said trait. The trait to which the present invention relates is so called autosomal dwarfism. Hereunder a short introduction into dwarfism m chicken is presented for explanatory purposes . Crawford (1990) lists three sex-linked forms and one autosomal form of dwarfism in chicken: dominant sex-linked dwarfism (Z) , recessive sex-linked dwarfism ( rg) , sex- 1inked dwarfism ( dw) and autosomal dwarfism (adw) . Of the first three, the sex-linked dwarfing allele ( dw) is the most studied one. This recessive sex-linked dwarfism is caused by a dysfunctional Growth Hormone receptor (GHR) which causes absence of Growth Hormone (GH) -dependent gene expression in the chicken liver. This defect seems to be the chicken equivalent of Laion dwarfism m humans. despite nigh circulating GH concentrations, plasma Insulm-like Growth Factor-I (IGF-I) levels are extremely low (Huybrechts et al . , 1985; Bowen et al . , 1987; Duπez et al . , 1993) . A point mutation located within the part of the gene encoding the extracellular domain of the GH receptor is responsible for the dysfunctional receptor (Duriez et al . , 1993) . The sexlinked dwarfism chicken is now being used as an animal model for Laron Dwarfism m humans and these animals are also used to study genes regulated by Growth Hormone (Agarwal et al . , 1995; Tanaka et al . , 1996) Recent research revealed that IGF-I mRNA is expressed m the liver m a GH-dependent manner after hatching. In extrahepatic tissues, mRNA expression is independent of GH and GHR before and after hatching, except for the testis, where GH seems to inhibit IGF-I mRNA expression. In dwarf chickens, hepatic IGF-I expression is completely abolished (Tanaka et al . , 1996). In the Cornell K strain White Leghorn chicken, an autosomal recessive gene ( adw) exists causing a 30% reduced size and body weight, later sexual maturity, lower hatching rate and production of less eggs (reduction of 10%) . The adult body weight is about 1400 g. However, these dwarfs have excellent viability (Crawford, 1990) . This autosomal dwarfism in fowl was already reported many years ago (Cole, 1973) but the nature of the gene causing this type of dwarfism is not known yet. Dwarf animals can be recognised at birth but their phenotype is more apparent at six weeks of age. These animals also have shorter legs (Scanes et al . , 1983; Bowen et al . , 1987) and a striking large skull compared to the rest of the body (Euribrid BV, personal communication) . Phenotypically, heterozygous animals cannot be separated from homozygous animals: chicken which have the dwarf phenotype have the adw/ adw genotype and the chicken which have non-dwarf phenotype carry at least one copy of the dominant allele (Adwl/*) . Leenstra and Pit (1984) therefore suggested the use of the gene in commercial stocks in order to reduce body size and feed requirements. It is not known whether this particular gene is present in any of the bantam breeds (Crawford, 1990) . In a few studies, comparisons have been made between the sex- linked dwarfism (dw) and the autosomal dwarfism ( adw) in chicken. However, information on the autosomal dwarfism is limited. The first comparative study on autosomal recessive dwarfism and sexlinked dwarfism was carried out by Scanes et al . in 1983, ten years before the discovery of the genetic defect by Duriez et al . in 1993. They concluded that neither autosomal dwarfism nor sex- linked dwarfism appeared to be caused by hypopituitarism. Huybrechts et al . (1985) studied plasma concentrations of somatomedin-C (IGF-1) in sex-linked dwarf and autosomal dwarf chicken. Plasma concentrations of IGF-I in sex- linked dwarf chicken were reduced, but were unaffected m autosomal dwarf chicken. GH levels in autosomal dwarf animals were normal. Scanes et al . (1983) found that plasma concentrations of tπiodothyronme (T3) were depressed in sex-linked dwarf chicken, where those of thyroxine (T4) were normal and those of GH were raised. We now know that this must be caused by physiological changes caused by the dysfunctional growth hormone receptor. Lauterio et al (1986) indeed suggested a dependency of T3 levels on GH levels. In autosomal dwarf animals, only a slight decrease m T3 and T4 was observed. The minuteness of these changes suggested that this is not the cause of the dwarf phenotype m these animals. The absence of major differences between the plasma concentrations of T3 and T4 in the autosomal dwarf chicks is thus consistent with the observed situation for GH and IGF-I. It would therefore appear that the lesion in the autosomal dwarfs may not be of an endocrine nature (Huybrechts et al . , 1985) . In other vertebrate animals, such as mice and man, a number of forms of dwarfism have been identified as well. Many of these forms are also related to the Growth-Hormone- Insulin-like Growth Factor pathway. However, m mice a form of dwarfism not related to th s pathway has been found as well . A mouse having such a form of dwarfism is the pygmy mouse, which s an autosomal recessive trait. This form has been attributed to a mutation m a gene called Hmgi -c . This gene is also present m the human genome (located on chromosome 12) but has not been associated with a dwarf syndrome in humans. In chickens or in other birds, such as turkey, duck or goose, a homologue of the Hmgi -c gene has not been found, and, considering the appreciable heterology that exists between mammalian and avian genomic nucleotide sequences, it is generally expected that screening a random library of avian genomic sequences with probes obtained from distantly related mammalian genomic sequences is not the most promising approach. The present invention provides the localization of a gene responsible for autosomal dwarfism m poultry. This gene is localized within 6 Megabases, more likely 2 Megabases of the microsatellite marker LEI146. It very likely lies within 2 centi Morgan of said marker LEI146. The gene can also be defined as having a Lod-score with LEI146 of at least 10, but preferably at least 20, more preferably at least 25 as can be discerned from figure 9 (A reference for the location of the LEI146 marks is R.P.M.A. Crooymans at al . in Animal Genetics 1997, Vol. 28) . The present invention, in a preferred embodiment, provides a solution for the fact that on the one hand, even when a trait has been associated with some microsatellite markers, this in no way means that a responsible gene for the trait has been identified, whereas on the other hand screening a random library of avian genomic sequences with probes obtained from distantly related mammalian genomic sequences will not lead to the proper identification of the wanted gene. The invention provides a combination of the bulked segregant analysis, where a trait is linked to microsatellite markers with the screening of a thus selected genomic library with probes derived from the supposedly heterologous gene . An important aspect of the present invention is that the combination of first mapping the gene for autosomal dwarfism to a certain location on chromosome 1 using microsatellite-markers , thereby enabling identification of the homologous chromosome segment m man and mice, through the presence of earlier mapped genes on said location at chromosome 1 (in particular Ly2 and especially Igf-1) , provided two candidate genes for autosomal dwarfism, of which Hmgi -c was identified as a very likely candidate by means of cleverly selected probes. Specifically, the invention provides the localisation of the Adw/adw gene between two microsatellite markers, MCW43 (LGAL4 gene) and MCW18 , close to markers UMA364 and LEI146, and m addition shows that a chicken gene hybridising w th the murine Hmgi -c can be found at the same location. Thus the Adw/adw dwarfism trait is associated with the Hmgi -c gene. The thus identified gene was cloned and cDNA was obtained with the sequence as shown in Fig 1. The invention thus provides a recombinant nucleic acid having a sequence comprising at least a functional part of a gene responsible for autosomal dwarfism, particularly the Hmgi -c gene or a functional derivative thereof of an avian species. Since avian genomes will be more homologous among each other than when compared with a mammalian genome, the invention also provides the recombinant nucleic acid having a sequence comprising at least a functional part of a gene responsible for autosomal dwarfism, particularly the Hmgi -c gene or a functional derivative thereof of such avian species as chickens, turkeys, ducks or geese. In particular, the invention provides the alleles of genes which relate to autosomal dwarfism found in birds, which in the recessive homozygous form lead to the phenotype dwarf . The average skilled expert will now be able to design or construct a nucleic acid probe capable of distinguishing the 'dwarf' allele of the gene responsible for autosomal dwarfism, particularly Hmgi -c gene from the non-dwarf allele and method for detecting alleles of a gene responsible for autosomal dwarfism, particularly the H gi -c gene comprising hybridization of at least one nucleic acid probe to a sample comprising at least a functional part of such a gene, possibly involving amplification involving said gene. Amplification methods are widely known in the art. With such probes and methods a testkit for distinguishing between alleles of a gene responsible for autosomal dwarfism, particularly the Hmgi -c gene, for instance comprising at least one nucleic acid probe and a hybridization buffer, and possibly in addition a nucleic acid polymerase, can be developed. Using such a method or testkit can be applied for selecting birds, such us chicken, turkeys, ducks or geese which are homozygous for the ' dwarf ' -allele of the gene responsible for autosomal dwarfism, particularly the Hmgi -c gene, can be selected which is method for making a breeding line consisting of 1 dwarf ' -birds, comprising selecting birds homozygous for the dwarf ' allele of the gene responsible for autosomal dwarfism, particularly the Hmgi -c gene. Such birds can be employed in methods for breeding birds comprising crossing a bird from a breeding line with non-dwarf birds. In particular, broiler birds (which are used for meat production) such as broiler chickens can then be inexpensively produced by employing one parent line that is homozygous for the dwarf allele and another that is homozygous for the non-dwarf allele. The resulting heterozygous progeny will all be not dwarf. Experimental Part Animals for microsatelli te analysis A segregating population for the autosomal dwarf locus was obtained by crossing White Leghorn cocks, homozygous recessive on the putative autosomal dwarfism locus (adw/adw) with Cornish hens not carrying the recessive allele (Adw/Adw) . The Fi (Adw/adw, ) cocks (26) and hens (50) were crossed and a F2 generation was bred in five progeny batches per parental cross. After birth, animals were phenotyped: 'sure dwarf, 'probably dwarf' and 'non- dwarf ' . Blood samples from the first four batches F2 chickens were collected at approximately six weeks of age and animals were weighed and phenotyped again at this time point. Now animals were separated in three groups: 'sure dwarf', 'probably dwarf/probably non-dwarf ' and 'sure non- dwarf ' . Because phenotyping at six weeks of age is far more reliable than phenotyping at birth, these groups were used for analysis. Of these groups, the 'sure dwarf' and ' sure non-dwarf ' animals of five crosses were used for the bulked segregant microsatellite analysis, with a total progeny of 83 animals (36 'sure dwarf') and 10 parents (none of the parents used twice) . 2 . 2 Blood samples Blood samples m EDTA were taken from chickens of all F2 families and their F]_, parents and frozen at -80° C. until analysis. DNA from the blood samples was purified using a Puregene DNA isolation kit (Gentra Systems Inc., NC, USA) according to the manufacturers recommendations. DNA was isolated from individual blood samples and from pooled blood samples: Blood pools from 'sure dwarf' animals and from 'sure not dwarf' animals from five families were made (Crooijmans et al . , 1996b). DNA concentration from each sample was measured using a GeneQuant RNA/DNA calculator (Pharmacia, Uppsala, Sweden) and diluted in Tris-EDTA (pH = 8.0) to a final concentration of 10 μg/ml (OD26O) • PCR and Gel electrophoresis The PCR reactions were performed using a PTC-100-96V Programmable Thermal Controller (MJ Research, Watertown, NM USA) . The reactions were performed in a total volume of 12 μl containing approximately 50 to 100 ng genomic DNA, 1.5 mM MgCl 2 , 50 mM KC1, 10 mM Tris-HCl (pH = 9.0), 1 mM tetramethylammoniumchloride (TMAC1) , 0.1% Triton X-100, 0.01% gelatin, 200 μM DNTP, 0.25 Unit Goldstar polymerase (Eurogentec S.A., Ougree, Belgium) and 30 ng of each microsatellite primer. The PCR reactions started with 5 minutes at 95°C. and continued with 35 cycles of 30 sec. at 95°C, 45 sec. at 50°-55°C. (depending on microsatellite) and 90 sec. at 72°C, followed by a final elongation of five minutes at 72 °C. The amplification products were separated on a 6% denaturing polyacrylamide gel (Sequagel-6, National Diagnostics, Atlanta, G, USA) using a Pharmacia Automated Laser Fluorescent (ALF) DNA sequencer. Sizing of the fragments was performed using DNA fragments of known sizes (Pharmacia, Uppsala Sweden) as internal standards and Fragment Manager V 1.2 software was used to analyse the data (Pharmacia) . Bulged segregant analysis and individual analysis Initially, 111 microsatellite markers were selected to perform a total genome scan (supplement VI) . PCR products of each marker tested on the five pooled DNA samples of the progeny (dwarf/not dwarf) and their parents were analysed and allelic frequencies in the progeny were calculated according to the peak area of the allele. When a parent is heterozygous for a microsatellite marker, it often occurs that the peak area of the one allele is different from the other. Different alleles sometimes have different amplification in PCR. Therefore, peak areas in the progeny have to be adjusted for these different peak areas. Individual progeny samples were tested for markers showing a marked difference in allelic frequencies to determine the exact frequencies of the alleles and to facilitate linkage analysis. Linkage analysis Linkage analysis was performed using CRI-MAP software version 2.4 (Green et al . , 1990) . A minimum LOD-score (Log 10 of odds (Ott, 1995)) of three was taken to indicate significant linkage. From a certain number of observations on two microsatellites on a group of animals and their parents, one can count the number of recombinants (i.e. the recombination traction) . The LOD-score resembles the Log 10 likelihood of these two markers being linked divided by the likelihood of no fmk-age. A LOD-score higher than 3 is usually regarded as a threshold for significance. Phenotypic differences between animals Of all families used m the analysis, both dwarfs and non- dwarf animals were genotyped for the different microsatellites. However, body weight data were available of all progeny of these families. This facilitated analysis of body weight data m combination with microsatellite data. Body weight differences between the 'sure dwarf' and 'probably dwarf/probably non-dwarf ' / 'sure non-dwarf ' animals were determined using the General Linear Model (GLM) procedure of the SAS 6.11 software (SAS. Cary, NC, USA) . Isolation of (putative) chicken Hmgi -c clones from a genomic library The mouse Hmgi -c cDNA, subcloned in the Bluescπpt KS+ plasmid (Stratagene, La Jolla, CA, USA) , was obtained from Dr. G. Manfioletti, Dipartimento di Biochemica, Biofisica e Chimica delle Macromolecole, Universita di Trieste, Italia) . The plasmid contains a ±1.8 kb insert coding for the Hmgi -c and also the Amp r gene. The insert can be cut out using EcoRI (5' end) and HindiII (3' end). XLl-Blue E. Coli bacteria were transformed with the plasmid and grown overnight on agar plates containing Amp. Positive clones were isolated and grown overnight in 2 ml LC medium containing Amp. DNA was isolated using the Easyprep system (Pharmacia, Uppsala, Sweden) and after testing the DNA on a 0.8% agarose gel, the Hmgi -c cDNA was cut out using HindiII and EcoRI . Fragments were separated on a 0.8% agarose gel and the cDNA was isolated from the agarose gel. The gel slice was transferred into a punctured sterile eppendorf tube that had been plugged with approximately 2-3 mm of siliconised sterile glass wool. This tube was put into another eppendorf tube and centrifuged for 10 minutes at 6,000 rpm m a microfuge. The eluate containing the DNA fragment was thus collected m the bottom tube. After precipitation and determination of cDNA concentration using λ fragments with known concentration the fragment was ready for labeling using the Ready-to-go DNA labeling kit (α- 32 P-dCTP) (Pharmacia, Uppsala, Sweden) . A commercial chicken genomic library was screened with the mouse H gi -c probe m order to find the chicken homologue of this murine gene. A genomic library of a male adult Leghorn Liver, cloned m the EMBL3 phage (Clontech cat. #CL10123 , Clontech Inc., Palo Alto, CA, USA), was used. The library contained 5.9 * 10 6 independent clones, with an average insert size of 15 kb (8-22 kb) . The exact titre of the library was determined using UB406 E. Coli indicator ceus and appeared to be 5 * 10 9 pfu/ml . Now the library was plated out on big petri dishes. To be sure that each genomic clone is represented on the plates, the total coverage should be at least three times the total genome of the chicken (1,2 x 10 9 bp) . Nitro-cellulose filters were drawn and hybridised overnight with the 32 P radioactive labeled mouse HMGI-C cDNA. Hybridising plaques were detected by autoradiography. Isolation of putative chicken HMGI-C clones from a chicken embryo cDNA library The mouse Hmgi -c cDNA probe described above was used to screen a cDNA library derived from a 5 day old chicken embryo cloned into the UNI -ZAP vector (Stratagene, La Jolla, CA, USA). Approximately 100,000 clones were plated out as described above, and nitro-cellulose filters were hybridised with 32 P radioactive labeled mouse Hmgi -c cDNA. After two more rounds of screening, pure hybridising plaques were used to recover the pBluescript plasmid containing the cDNA insert by using the in v vo excision protocol described by the supplier (Stratagene) . Eventually, two different cDNA clones (1400 and 2200 bp insert sizes) were partially sequenced using an automated sequencer (ALF, Pharmacia) . FISH procedure Metaphase chromosomes were obtained from cultures of chicken embryo fibrablasts after treatment with a hypotonic solution and fixed m methanol-acetic acid (3:1) . The spreads were stained with quinachnn, analyzed by fluorescence microscopy and photographed using a cooled CCD camera. For fluorescence in situ hybridization the LEI146 probe was labeled with bιotm-16-dUTP and the Hmgi -c probe was labeled with either bιotin-16-dUTP or digoxιgenin-11-dUTP. The biotinylated probe was detected by subsequent incubation with AvFITC, BioGAA and again AvFITC, and the digoxigenin labeled probe was detected by subsequent incubation with MAdig, RAMTRITC and SWARTRITC. the DNA was counterstamed with DAPI . In the pre- photographed spreads the signals were detected by fluorescence microscopy and pictures were taken by a cooled CCD camera. As a result a specific signal for both probes was found on chromosome lp near the centromere. Isolation of LEI 146 posi tive clones from a genomic library Apart from the screening with the radioactive mouse Hmgi -c cDNA, a screening was performed using the LEI146 microsatellite marker. The PCR product was precipitated and labeled using the Ready-to-go DNA labeling kit (α- 32 P- dCTP) (Pharmacia, Uppsala, Swec an) . Hybridisation with the genomic library was carried out as described above. 3. Results Bulϊed segregant analysis Of the 111 microsatellite markers selected (see Figure 2) for the 'total genome scan', 31 were tested using bulked segregant analysis m five different families (Table I) . The percentages of the different alleles were estimated for 'dwarf' and 'non-dwarf' pooled progeny samples and their parents and three linked microsatellite markers (MCW112 , MCW 43 , MCW18 ; all in linkage group WAU 1) showed a marked difference between 'dwarf' and 'non-dwarf' allele frequencies m the families, which is shown m Figure 3 and Table II, together with the expected percentages based on uncoupled segregation. Percentages differing more than 25% from the expected values or differing more than 25% between 'dwarfs' and 'non-dwarfs' are shown m bold. Other markers, not linked with the above mentioned ones, occasionally showed allele frequency differences between 'dwarf and 'non-dwarf animals bigger than 25%. However, these differences were not consistent in other families (Table III) . The results in Table II show differences in four of the five families and for two or three microsatellite markers. Based on this result, it was decided to test Fi and F 2 animals individually for MCW43 , MCW18 and MCW112 before continuing the 'total genome scan'. Especially the results of MCW43 m families 25/880 and 17/932 suggested linkage with the autosomal dwarfism gene . Individual analysis Individual DNA samples were tested for MCW43 , MCW18 and MCW 112. A total of 83 animals from 5 families, of which 36 were dwarfs, were analysed. Six animals, 7.2%, (two dwarfs) appeared to have a microsatellite profile that did not match with the parental profile and these animals were excluded from further analysis. Recombination fractions and LOD-scores were calculated using CRI-MAP software. Dwarfs were genotyped as homozygous recessives (adw/adw) and non-dwarfs as carriers of at least one copy of the wild-type allele (* Adw - ) . CRI-MAP" results are shown in Table IV. The significant LOD-scores (LOD-scores > 3.00) supported the initial hypothesis based on the bulked segregant data on the location of the gene. Based on these data the gene is located between MCW43 and MCW18 on linkage group WAU1 (chromosome 1) , which is the same as El (East Lansing International reference family) and C15 (Compton International reference family) . However, the big gap between the two microsatellite markers (44 cM) made it necessary to analyse more data, m order to confirm this result and to better specify the location of the Adw/adw. gene. First, the effect of adding more families was computed using CRI-MAP" . Each family was used twice m one CRI-MAP run, resulting m ten families with a progeny of 144 animals. The results of this analysis, showed m Table V, revealed that is would be useful to test more families. A quadratic increase of LOD scores was observed, which results in more significant linkage between the microsatellites and the QTL. Secondly, it was also decided to test additional microsatellite markers linked with MCW43 and MCW18 , in order to further specify the location of the Adw/adw. gene on the linkage map. Based on the results of Table V it was decided to test more families segregating for the autosomal dwarf gene. Seven additional families were incorporated in the study. All parents were used in only one of the 12 crosses 11 . Microsatellite analysis was now carried out on the 24 parents and 157 animals from the progeny of these parents (see Table VI) . Of these 157 animals, 12 (8%) animals appeared to have a genotype that did not match with the parental phenotype (an additional six animals plus the six already mentioned) . Because three animals from family 4 (24/877; 5 animals) had to be excluded from the data set, it was decided to exclude the whole family for microsatellite analysis. It is reasonable to suspect that one of the parental blood samples did not originate from the right parent. Testing of addi tional markers linked wi th MCW18 and MCW43 A test panel including 7 microsatellite markers : ADL307, ADL234 , MCW43 , MCW18 , ADL150 , MCW112 and LMU 62 was analysed . Recent research revealed that microsatellite marker LMY 105 is located between MCW43 and MCW18 (M .A . M . Groenen, unpublished results) , so this microsatellite marker was also included in the test panel . Figure 4 shows individual results for microsatellite LEI146, revealing strong linkage with the Adw/adw gene . All miccrosatellite data were again analysed using CRI -MAP 11 Data from Euribrid BV suggested that cock 25 was used twice (hen 880 and 887 ) , but microsatellite analysis showed that the cock used for hen 880 was not the same cock as the one used for hen 887 . Because of the Organisation of the blood sample batches , cock - hen - progeny per family it is reasonable to assume that a mistake occurred in the data . software. Table VII shows the number of informative meioses for each microsatellite marker. Since blood samples of the Fo generation were not available, there were no phase-known meioses. LOD-scores and recombination fractions are shown in Table VIII. Based on the WAU linkage map and the present data, a linkage map was build using the CRI-MAP options build and flips . The ordered loci were analysed according to their order on the WAU linkage map. All individual data were checked using the chrompic option, which determines the phase of the different alleles. Microsatellite data from individuals showing double crossovers on a short linkage distance were checked. Finally, the most likely location of the adw locus was calculated and results are shown in Table IX. Because the WAU linkage map (supplement M) is based on more informative meioses (i.e. more data; full sib families, 480 animals) than the present study, the microsatellites were put in the WAU locus order in the CRI-MAP option build, although this was not the most likely order according to the dwarf data. The recombination fraction between LEI146 and adw equals 0.03, LOD-score 23.62 (see Table VIII and Table IX). The LoglO likelihood of adw being located between MCW43 and LMU 105 is -207.67, whereas the likelihood of adw being located between LEI146 and MCW18 equals -205.34, a 10 2 - 33 difference in absolute likelihood. When CRI-MAP calculated the linkage map on itself (without knowing the WAU linkage order) the Adwladw locus was mapped on exactly the same place: closely to LEI146. Screening genomi c library wi th a mouse HMGI- c c -DNA and a chi cken LEI 146 probe The linkage between Ad/ adw and LEI146 resulted in localisation of this gene on chromosome I between MCW43 and MCW18. Interestingly, the chicken Igf-I gene is also located between these two microsatellite markers. The Igf-I gene is located on human chromosome 12 (12q22-12q24. 1 : MIM gene map , The Jackson Laboratory Mouse Genome Informatics) and on mouse chromosome 10. In both species, the gene coding for the HMGI-c protein is also located on these chromosomes: on human chromosome 12, position 12ql5 (MIM gene map) and on mouse chromosome 10, approximately 25 cM from the Igf-I gene. It was decided to screen a chicken genomic library with a mouse H gi -c cDNA probe. The library was also screened with a LEI146 probe in order to be able to use a genomic clone of known location for Fluorescent Tn Rj n Hybridisation (FISH) in combination with the putative chicken Hmgi -c genomic clone. From the screening of the genomic library, 10 positive clones for LEI146 were isolated. For Hmgi -c, 32 clones were isolated. In the second screening, further purification of the clones was carried out. Plaques from the LEI146 probe gave much better signals than the ones from Hmgi -c probe. LEI146 is a homologous chicken probe, and Hmgi -c is a heterologous mouse probe. Resul ts from sequence analysis of the isolated chicken HMGI-c clones . Southern blots derived from the DNA of 5 of the Hmgi -c positive genomic clones, digested with a number of restriction enzymes, were hybridised with the mouse H gi -c cDNA. In two of the 5 clones, specific fragments hybridised to the mouse Hmgi -c probe. From one of the genomic clones, the hybridising PstI fragments (0.9 and 1.1 Kbp) and the hybridising Hindlll fragment (1.4 Kbp) were subcloned into pBluescript , which resulted in the genomic subclones HP2 , HP3 and HH1. The inserts of these genomic subclones were partially sequenced with both the reverse and forward sequencing primers. The resulting sequence (see fig 5) clearly showed that the isolated chicken gene belonged to the HMG family of genes, and that it contained at least part of intron 4 and the complete exon 5. Moreover, since the first 100 bp of the 3 'non- coding region of the mRNA (present on exon 5) , showed 80% sequence similarity to the same region in the human and mouse gene, it is very likely that the cloned chicken gene indeed is the Hmgi -c gene . Screening of the chicken embryo cDNA library resulted in a very large number of positively hybridising clones, indicating that the gene is highly expressed in embryonic tissue. Two of the positive clones, HCC4 and HCC3 (with insert sizes of 1.1 and 2.2 Kbp respectively) were selected and partially sequenced. The resulting sequence (fig 1) showed that both clones -ere derived from the same gene as the genomic clone. Alignment of the cDNA sequence and its derived aminoacid sequence with those of the HMGI- c gene of mouse and man, clearly showed that the cloned gene indeed was the chicken Hmgi -c gene. Similar as the mammalian HMG family members, this gene coded for a putative DNA binding protein with highly conserved DNA binding domains and a highly conserved domain probably involved in protein-protein recognition. In addition, similar as in HMGI-c in mammals, the chicken protein has an extra amino acid motive (between the last DNA binding motive and the pro-prot interaction motive, see fig 6) that is only found in the HMGI-c protein, and which is absent from the other HMG family members. Particularly, this protein motive shows that we have cloned the chicken Hmgi -c gene rather than another highly conserved family member. Assignment of the chicken HMGI-c gene and marker LEI146 to a chromosomal segment on chromosome Ip To enable physical mapping of the microsatellite marker LEI146 using Fluorescent in si u hybridisation (FISH) , genomic fragments of this marker were also isolated from a chicken genomic library, and partially sequenced to identify true positive genomic clones for this marker. Both the genomic lambda clone for the chicken Hmgi -c gene and the genomic lambda clone for marker LMU146 were fluorescently labeled and hybridised to meta-phase chromosome spreads of the chicken in two seperate FISH experiments. Both clones map to a similar region on chromosome lp, close to the centromere. Subsequently, in a double labeling FISH experiment it was confirmed that both loci are located very close on the same region of chromosome lp. .Resul s from statistical analysis of chicken body weight data Animals were bred m four time periods, resulting from 11 parental crosses. Based on the original weight records, a data set containing all animals from the families used was made (see Table X) . It should be noticed that the animals actually analysed for microsatellite markers are found m Table VI . Dwarf animals not analysed are animals with missing blood samples. Only a selection of 'heavy' animals (i.e. the 'sure non-dwarf' animals) was used for microsatellite analysis. Cocks and hens were separated as being 'dwarf' (24%) or 'non-dwarf' (76%); the 'probably dwarf' animals were included m the second group. The statistical model used was : weigrh bcd = β + f mιly a + period^ + sex c + phenotype^ + errorabcd weight Body weight m grams of progeny d family Family a (1-11) in which progeny d is born period Breeding period b (1-4) in which progeny d is born sex Sex c (Male / Female) phenotype Phenotype d (Dwarf / Non-dwarf) This model proved to be the best model, since it only includes variables with significant effects (p < 0.05). All interaction terms were tested and non-signifleant (interaction) terms were stepwise deleted from the model resulting m the present model. Of the 363 animals m the data set, only 342 could be used due to missing values: 18 due to missing sex and 3 due to missing weight. In the model, weight is a variable explained by the fixed family, period, sex and phenotype effects and an error term. Results are m Table XI. The SAS General Linear Model estimates an overall mean (μ= 994.5 g) and a Lsmean for eacn variable. Of course, the goal of this procedure was to determine the 'phenotype' effect: to show the significant difference between 'dwarf and 'non-dwarf' chicken. This is possible by correcting the observation on weight for estimated Lsmean for family, period and sex. Weigh a bcd - Lsmean - Lsmean Sex c μ + Lsmean Phenotype^ + error a bcd (2) The corrected weights were calculated and plotted m histograms (Figure 7) These corrected weights can be combined with phenotypic data. Figure 7 shows the differences in body weight between 'dwarfs' and 'non- dwarfs' after correction for sex, family and period effects. The mean of the corrected 'dwarf body weights, 989.9 g, differs significantly from the 'non-dwarf body weights, 1297.4 g (p < 0.001, one-sided Student's t-test with unequal variance, equal variance of the two groups was tested with a Fisher-test, p < 0.01) . The right-end of the distribution in Figure 7 does suggests a difference between heterozygous and homozygous dominant animals: there seems to be a heavy subgroup of 'non-dwarfs', which might result from the double dominant animals. The dominant allele in the heterozygous animals might not have a full penetrance. This can only be confirmed by analysing all animals and subsequent statistical analysis. Since LEI146 is strongly linked to Adw/adw and because the 276 bp allele is linked to the adw allele in all families, it is useful to compare weights of the three possible LEI146 genotypes. The heterozygous animals, animals carrying one copy of the 276 bp allele, might have lower body weights than the animals not carrying the 276 bp allele. Indeed, Figure 8 shows a difference between these animals. However, the corrected body weights of these animals do not differ significantly in a Student's t-test (p = 0.45 in a one-sided test with equal variance, again confirmed with a Fisher-test) . It should be noted that LMU105 has a recombination fraction of 0.03, which means that 3 out of 100 animals are recombinants . Individual Linkage analysis In order to verify the results from the bulked segregant analysis experiment and in order to obtain a more accurate estimation of the location of the adw locus on the genetic linkage map, DNA samples from 145 dwarf and non-dwarf chickens were subjected to individual linkage analysis. Five microsatellite markers (MCW43 , MCW18 , ADL307 , LEI71 , LEI146) , all being located in the region of the adw locus, were analyzed. One of these markers (LEI146) was located in the region between MCW43 and MCW18 . Using two point analysis, positive linkage with the adw locus was found for all markers except ADL307 and LEI71 , the recombination fraction between LEI146 and the adw locus being as low as 0.03 (LOD lιnkage score= 31.98). A multipoint analysis revealed the most likely position of the adw locus with respect to the markers present on linkage group WAUl (Figure 9) . O 98/30689 PCT7NL98/00021 Table I Families used for bulked segregant analysis. 1 Parents Progeny Progeny Dwarf Non-dwarf Family Cock Hen Cocks Hens Unknown 1 Cocks Hens Unknown 1 Total 1 , 20 858 4 2 0 8 0 0 14 2 25 880 2 5 0 8 2 0 17 3 15 923 3 4 0 6 3 2 18 4 17 932 3 5 0 4 6 0 18 5 6 969 3 5 0 5 2 1 16 Total 15 21 0 31 13 3 83 unknown sex Table II Percentages of different alleles for MCW43, MCW18 and MCW772 determined using bulked segregant analysis Family 15/923 25/880 6/969 Number of Animals 18 Total, 7 Dwarf 17 Total, 7 Dwarf 16 Total, 8 Dwarf Microsatellite Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 MCW43 Cock 130 bp 134 bp 130 bp 134 bp 130 bp 134 bp Hen 134 bp 134 bp 130 bp 134 bp 130 bp 134 bp 130 bp 134 bp 130 bp 134 bp 130 bp 134 bp Non-dwarf 50 0 o 50% 45% 55% 59% 41% Dwarf 31% 69% 0% 100% 42% 58% Expected 25% 75% 50% 50% 50% 50% MCW18 Cock 225 bp 233 bp 219 bp 233 bp 231 bp 233 bp Hen 225 bp 233 bp 225 bp 233 bp 225 bp 233 bp 225 bp 233 bp 219 bp 225 bp 233 bp 225 bp 231 bp 233 bp Non -dwarf 58% 42% 51% 26% 22% 34% 38% 28% Dwarf 20% 80% 13% 2% 85% 15% 40% 46% Expected 50% 50% 25% 25% 50% 25% 25% 50% MCW112 Cock 274 bp 276 bp 272 bp 274 bp 261 bp 274 bp Hen 274 bp 276 bp 261 bp 276 bp 261 bp 276 bp 274 bp 276bp 261 bp 272 bp 274 bp 276 bp 261 bp 274 bp 276 bp Non-dwarf 44% 56% 29% 32% 12% 28% 35% 24% 41 % Dwarf 68% 32% 39% 14% 32% 15% 50% 29% 21% Expected 50% 50% 25% 25% 25% 25% 50% 25% 25% Table II (continued) Family 20/858 17/932 Number of Animals 14 Total. 6 Dwarf 18 Total, 8 Dwarf Microsatellite Allele 1 Allele 2 Allele 1 Allele 2 MCW43 Cock 130 bp 134 bp 130 bp 134 bp Hen 130 bp 134 bp 130 bp 134 bp 130 bp 134 bp 130 bp 134 bp Non-dwarf 61% 39% 45% 55% Dwarf 22% 78% 0% 100 % Expected 50% 50% 50% 50% MCW18 Cock 227 bp 233 bp 231 bp 233 bp Hen 225 bp 233 bp 219 bp 233 bp 225 bp 227 bp 233 bp 219 bp 231 bp 233 op Non-dwarf 37% 37% 26% 33% 34% 33% Dwarf 0% 9% 91% 10% 30% 60% Expected 25% 25% 50% 25% 25% 50% MCW112 Cock 261 bp 274 bp 261 bp 274 bp Hen 261 bp 276 bp 261 bp 274 bp 261 bp 274 bp 276 bp 261 bp 274 bp Non-dwarf 43% 28% 30% 52% 48% Dwarf 48% 52% 0% 25% 75% Expected 50% 25% 25% 50% 50% Table III Percentages of different alleles for MCW68, MCW109 and MCW200 determined using bulked segregant analysis. Family 15/923 25/880 6/969 Number of Animals 18 Total. 7 Dwarf 17 Total, 7 Dwarf 16 Total, 8 Dwarf Microsatellite Allele 1 Allele 2 Allθlel Allele 2 Aileiei Allele 2 MCW68 Cock 171 bp 196 bp 174 bp 181 bp 174 bp 177 bp Hen 181 bp 181 bp 171 bp 196 bp 171 bp 181 bp 171 bp 181 bp 196 bp 171 bp 174bp 181 bp 196 bp 171bp 174 bp 177 bp 181 bp Non-dwarf 21% 59% 20% 30% 38% 11% 20% 32% 15% 37% 17% Dwarf 49% 51% 0% 35% 31% 20% 15 % 22 % 36% 15% 27% Expected 25% 50% 25% 25% 25% 25% 25% 25% 25% 25% 25% MCW109 Cock 132 bp 132 bp 132 bp 155 bp 153 bp 155 bp Hen 132 bp 155 bp 132 bp 155 bp 134 bp 157 bp 132 bp 155 bp 132 bp 155 bp 134 bp 153 bp 155 bp 157 bp l Non-dwarf 69% 31% 61% 39% 21% 22% 30% 26% Dwarf 66% 34% 49% 51% 13% 17% 38% 32% Expected 75% 25% 50% 50% 25% 25% 25% 25% MCW200 Cock 240 bp 240 bp 240 bp 248 bp 238 bp 258 bp Hen 249 bp 258 bp 240 bp 240 bp 238 bp 248 bp 240 bp 249 bp 258 bp 240 bp 248 bp 238 bp 248 bp 258 bp Non-dwarf 66% 20% 14% 78% 22% 39% 36% 25% Dwarf 65% 20% 15% 58% 42% 55% 34% 11 % Expected 50% 25% 75% 5% 25% 50% 25% 25 % Table III (continued) Family 20/858 17/932 Number of Animals 14 Total. 6 Dwarf 18 Total, 8 Dwarf Microsatellite Allele 1 Allele 2 Allele 1 Allele 2 MCW68 Cock 177 bp 196bp 181 bp 181 bp Hen 172 bp I896bp 181 bp 196bp 172 bp 177 bp 196 bp 181 bp 196 bp Non-dwarf 29% 44% 27% 81% 19% Dwarf 39% 7% 54% 92% 8% Expected 25% 25% 50% 75% 25% MCW109 Cock 132 bp 141 bp 151 bp 153 bp Hen 134 bp 155 bp 132 bp 155 bp 132 bp 134 bp 141 bp 155 bp 132 bp 151 bp 153 bp 155 bp Non-dwarf 26% 12% 39% 23% 30% 15% 39% 22% Dwarf 67% 5% 7% 21% 35% 26% 23% 22% Expected 25% 25% 25% 25% 25% 25% 25 % 25% MCW200 Coc 246 bp 246 bp 238 bp 248 bp Hen 238 bp 238 bp 238 bp 238 bp 238 bp 246 bp 238 bp 248 bp Non-dwarf 62% 38% 83% 17% Dwarf 70% 30% 91% 10% Expected 50% 50% 75% 25% Table IV CRI-MAP results of individual analysis of family 1-5. Table V CRI-MAP results of individual analysis of family 1-5, each family incorporated tvace in the data set. Coupling between: j recombination fraction LOD-score MCW43 - Adw/adw 1 0.10 13.10 MCW18 - Adw/adw 0.19 6.78 mcw18 - MCW43 0.28 4.61 MCW112 - Adw/adw 0.21 5.60 MCWH2 - MCW18 0.09 39.81 Table VI Families used for individual analysis unknown sex Table VII Number of informative meioses for individuals all families. Table VIII CRI-MAP results of individual analysis of all families . Table IX Linkage maps as calculated from CRI-MAP. Table X Families used for statistical analysis (SAS) Parents Progeny Progeny Dwarf Non-dwarf Family Cock Hen Cocks Hens Unknown 1 Cocks Hens Unknown 1 Total 1 19 853 4 2 0 16 14 0 36 2 20 858 5 6 0 13 11 1 36 3 23 873 2 5 0 12 12 1 32 4 25 880 2 6 1 14 8 2 33 5 225 887 1 4 0 11 15 4 35 6 10 889 2 5 0 10 15 3 35 7 15 923 4 5 0 10 17 1 37 8 17 932 5 6 0 8 14 1 34 9 2 941 3 6 0 ' 9 0 25 10 1 946 1 5 0 7 11 2 26 11 6 969 3 6 0 12 11 2 34 Total 32 56 1 120 137 17 363 Due to missing values, only 342 animals could be used for statistical analysis 1 unknown sex Table XI Results from SAS ' GLM procedure . Dependent Variable: Weight Sum of Mean Source DF Squares Square F Value Pr>F Model 15 10307877.049 687191.803 39.20 0.0001 Error 326 5715510.448 17532.241 Corrected Total 341 16023387.497 R-Square C.V. Root MSE Weight Mean 0.64 13.3 132.4 994.5 Source DF Type III SS Mean Square F Value Pr>F Family 10 564679.9319 56467.9932 3.22 0.0006 Period 3 3186031.5131 1062010.5044 60.57 0.0001 Phenotype 1 5838521.6702 5838521.6702 333.02 0.0001 Sex 1 828696.2176 828696.2176 47.27 0.0001 Family μ + Lsmean 853 1022.6 g 858 903.2 g 873 888.1 g 880 876.5 g 887 891.8 g 898 954.3 g 923 939.2 g 932 903.1 g 941 924.5 g 946 908.4 g 969 907.7 g Period μ + Lsmean 1. 982.8 g 2. 748.4 g 3. 982.4 g 4. 966.2 g Phenotype μ+Lsmean Dwarf 766.2 g Non-dwarf 1073.7 g Sex μ + Lsmean Hens 869.1 g Cocks 970.8 g

Claims: CLAIMS 1. A recombinant nucleic acid associated with autosomal dwarfism in poultry, the chicken homolog of which recombinant nucleic acid being located within 6 Mega base pairs, preferably 2 Mega base pairs of microsatellite marker LEI 146. 2 . A recombinant nucleic acid associated with autosomal dwarfism in poultry, the chicken homolog of which recombinant nucleic acid being located within 5 centi Morgan, preferably 2 centi Morgan of microsatellite marker LEI146. 3. A recombinant nucleic acid associated with autosomal dwarfism in poultry, the chicken homolog of which recombinant nucleic acid having a LOD-score of at least 10, preferably at least 20, more preferably at least 25 with microsatellite marker LEI146. 4. A recombinant nucleic acid according to any one of claims 1-3 having a sequence comprising at least a functional part of the Hmgi -c gene or a functional derivative thereof of a bird. 5. A nucleic acid according to claim 4 whereby the Hmgi -c gene is from the species chicken, turkey, duck or goose. 6. A recombinant nucleic acid according to anyone of claims 1-5 which leads to the phenotype dwarf. 7. A nucleic acid probe capable of distinguishing the 'dwarf' allele of the Hmgi-c gene from the non-dwarf allele . 8. A method for detecting alleles of a gene responsible for autosomal dwarfism having a sequence located within a recombinant nucleic acid according to anyone of claims 1-7, comprising hybridisation of at least one nucleic acid probe derived from said recombinant nucleic acid to a sample comprising at least a functional part of such a gene or a sequence located in close vicinity to said gene. 9. A method according to claim 8 comprising an amplification step. 10. A testkit for distinguishing between alleles of a Hmgi-c gene comprised within a recombinant nucleic acid according to anyone of claims 1-7 comprising at least one nucleic acid probe and a hybridization buffer. 11. A testkit according to claim 10 further comprising at least one nucleic acid polymerase . 12. A method for selecting birds which are homozygous or heterozygous for the 'autosomal dwarf ' -allele comprising carrying out a method according to anyone of claims 8-9, or comprising the use of a testkit according to claim 10 or 11, or comprising the use of a nucleic acid probe according to claim 7. 13. A method for making a breeding line consisting of 1 dwarf ' -birds , comprising selecting birds homozygous for the ' dwarf ' allele using a method according to claim 12. 14. A breeding line obtainable by the method of claim 13. 15. A method for breeding birds comprising crossing a bird from a breeding line according to claim 14 with non-dwarf birds . 16. A method for producing broiler birds comprising crossing a bird from a breeding line according to claim 14 with non-dwarf birds.

Inventors: ALBERS GERARDUS ANTONIUS ARNOL, GROENEN MARTINUS ANTONIUS MATH, ALBERS, GERARDUS, ANTONIUS, ARNOLDUS, GROENEN, MARTINUS, ANTONIUS, MATHILDA
Applicants: ALBERS GERARDUS ANTONIUS ARNOL, EURIBRID B V, GROENEN MARTINUS ANTONIUS MATH, ALBERS, GERARDUS, ANTONIUS, ARNOLDUS, EURIBRID B.V., GROENEN, MARTINUS, ANTONIUS, MATHILDA
Application year: 1998
Main classification: C12N15
IPC codes: A01K67, C07K14, C12N15