Aus dem Institut für Tierzucht und Vererbungsforschung der Tierärztlichen Hochschule Hannover
Fine mapping of the genomic region of the bovine polled locus
INAUGURAL-DISSERTATION zur Erlangung des Grades einer
DOKTORIN DER VETERINÄRMEDIZIN (Dr. med. vet.)
durch die Tierärztliche Hochschule Hannover
Vorgelegt von Anne Wöhlke aus Bremen
Hannover 2004
Scientific supervision: Univ.-Prof. Dr. Dr. habil. O. Distl
Examiner: Univ.-Prof. Dr. Dr. habil. O. Distl Co-examiner: Univ.-Prof. Dr. H. Naim
Oral examination: 25.11.2004
This work has been kindly supported by the German Research Council (DFG), grant no. (DI 333/8-1) and the Dr. Dr. h. c. Karl Eibl Foundation, Neustadt/Aisch, Germany.
Dedicated to my family and Jörg
Parts of this work have been published in following journals:
Gene
Comparative and functional genomics
Contents
1 Introduction... 1
2 Literature... 3
2.1 Introduction ... 3
2.2 Phenotypic variation in development of horn size and shape in cattle ... 4
2.3 Inheritance of polledness and horn development in cattle ... 5
2.4 Mapping of the polled gene in cattle... 6
2.5 Indirect gene diagnosis for the polledness ... 9
2.6 Conclusions ... 11
3 A 4 Mb high resolution BAC contig on bovine chromosome 1q12 and comparative analysis with human chromosome 21q22... 12
3.1 Introduction ... 12
3.2 Materials and Methods ... 13
3.2.1 DNA library screening and chromosome walking... 13
3.2.2 DNA sequence analysis... 13
3.3 Results ... 14
3.4 Discussion ... 18
4 Development of 20 microsatellite and 7 single nucleotide polymorphism markers and subsequent fine mapping of the bovine polled gene region to a 1 Mb interval.... 20
4.1 Introduction ... 20
4.2 Material and methods ... 21
4.2.1 Marker development ... 21
4.2.2 Single nucleotide polymorphism development ... 21
4.2.3 Pedigree structure... 22
4.2.4 Genotyping ... 24
4.2.5 Linkage and haplotype analysis ... 24
4.3 Results and discussion... 25
4.3.1 Marker development ... 25
4.3.2 Single nucleotide polymorphism development ... 28
4.3.3 Linkage and haplotype analysis ... 28
5 Development of a reliable marker based test for polledness in different cattle breeds ... 34
5.1 Introduction ... 34
5.2 Material and methods ... 35
5.2.1 Pedigree analysis ... 35
5.2.2 Genotyping of microsatellite markers ... 36
5.2.3 Genotyping of a single nucleotide polymorphism ... 36
5.3 Results and discussion... 37
5.3.1 Properties of the microsatellite markers... 37
5.3.2 Haplotype analysis for the polled lines ... 38
5.3.3 Application of the marker based gene test in families of different cattle breeds .. 39
6 Molecular characterization of positional candidate genes for the bovine polled gene46 6.1 Introduction ... 46
6.2 Materials and methods ... 46
6.2.1 Cloning and sequencing of the bovine GART and CRYZL1 gene... 46
6.2.2 cDNA synthesis, RT-PCR... 47
6.2.3 Mutation analysis ... 48
6.3 Molecular characterization of the bovine GART gene... 49
6.3.1 Description of GART ... 49
6.3.2 Analysis of the genomic organization of the bovine GART gene ... 50
6.3.3 Analysis of the bovine GART cDNA... 54
6.3.4 Polymorphisms within the bovine GART gene ... 55
6.4 Molecular characterization of the bovine CRYZL1 gene... 57
6.4.1 Description of the CRYZL1 gene ... 57
6.4.2 Analysis of the genomic organization of the bovine CRYZL1 gene ... 58
6.4.3 Analysis of the bovine CRYZL1 cDNA ... 61
6.4.4 Polymorphisms within the bovine CRYZL1 gene ... 61
6.5 Conclusions ... 62
7 Conclusions... 63
8 Erweiterte Zusammenfassung... 64
8.1 Einleitung ... 64
8.2 Material und Methoden ... 65
8.3 Ergebnisse ... 70
8.4 Diskussion ... 72
9 References... 74
10 Appendix 1... 84
11 Appendix 2... 88
12 Appendix 3... 100
13 Appendix 4... 104
14 Appendix 5... 110
15 Acknowledgements... 119
List of abbreviations
AIRS aminoimidazole ribonucleotide synthetase APS ammoniumperoxidsulfat
AT annealing temperature
BAC bacterial artificial chromosome
BTA bovine chromosome
bp base pairs
BLASTN basic local alignment search tool nucleotide cDNA copy deoxyribonuclein acid
cM centiMorgan cR centiRay
CRYZ crystallin zeta (quinone oxidoreductase) gene CRYZL1 crystallin zeta-like 1 gene
dATP deoxy adenine triphosphate dCTP deoxy cytosine triphosphate dGTP deoxy guanine triphosphate DMSO dimethylsulfoxid dNTP deoxy nucleoside triphosphate dTTP deoxy thymine triphosphate E.coli Escherichia coli
EDTA ehtylenediamine-tetraaceticacid EST expressed sequence tag
E2F E2 transcription factor
EMBL european molecular biology laboratory FISH flourescent in situ hybridization
GART glycinamid ribonucleotide formyltransferase gene GARS glycinamid ribonucleotide synthetase
Hexp expected heterozygosity
Hobs observed heterozygosity
HSA chromosome of homo sapiens
IRD infra red dye
kb kilobase kDA kiloDalton
LG linkage group
LINE long interspersed nuclear element LOD logarithm of odds ratio
Mb megabase
MMU mouse chromosome
mRNA messenger ribonucleic acid ng nanogram nt nucleotide PCR polymerase chain reaction
PIC polymorphism information content pmol picomol
QTL quantitative trait loci
Rb retinoblastoma
RH radiation hybrid
RT-PCR reverse transcriptase polymerase chain reaction SINE short interspersed nuclear element
SNP single nucleotide polymorphism STR simple tandem repeat
STS short tag sequence
Sp1 stimulating protein 1
TBE TRIS-boric acid–EDTA
U unit
UTR untranslated region
YAC Yeast artificial chromosome µl microliter µM microMolar
1 Introduction
The hornless or polled phenotype, appeared in the early phase of domestication of cattle and this phenotype persisted until today’s cattle breeds. So this phenotype variation in horn development allows the employment of molecular genetic approaches to identify the genes involved in polledness. From the view point of phylogenesis, genes responsible for horn development should be of special interest due to the fact that pneumatic horns are characteristics for the bovine species. So the characterization of the polled gene could help to understand the development of different horn variations in bovines and the phylogenesis of the gene itself. A further interesting point to identify the gene responsible for polledness may be seen in the requirements of modern cattle housing systems as in dairy or beef cattle the risk of injuries through horn crushes increases. Hornless animals are easier to handle and safer to work with, and they are less likely to injure other cows. Normally herd owners remove the horns of their calves at an early age of live, to minimize the injury risk. Until an age of six weeks removing of horns is allowed without any anesthesia, after this age dehorning of cattle causes pain and the risk of consequential damages. So it seems expedient to breed polled cattle because for the herd owner it saves time and investments and for the animals a painful procedure, respectively.
The polled locus causing the absence or presence of horns was mapped to the centromeric region on cattle chromosome 1 (BTA1). But all previous studies were not able to identify the responsible gene causing hornless cattle. There are several reasons that the polled locus could not be identified and characterized with molecular genetic methods: (1) the marker density of the genetic map of the proximal part of BTA1 is low. Using only the information of the available markers, many studies failed to determine the position of the polled locus on the bovine linkage map. (2) The number of recombinations in the centromeric regions are rare.
(3) No obvious candidate genes are known. The proximal part of cattle chromosome 1 shows conserved synteny with human chromosome 21 (HSA21). So the availability of the complete sequence and gene catalogue of the long arm of HSA21 can be used for a detailed comparative analysis of corresponding segments on BTA1. However, published human- bovine maps revealed several break points which were not consistent in the different studies.
In consequence of this, a conclusive map of the centromeric region of BTA1 is missing and
thus all further efforts to map the polled locus suffered from unprecise maps and low marker density.
The objectives of this work are to construct a high density marker map for the centromeric region of BTA1 based on a bacterial artificial chromosome (BAC) contig and then to develop new markers and single nucleotide polymorphisms (SNP) in order to allow gene-wise tests for linkage disequilibrium with the polled condition. The first step of the present study is to construct a genomic bovine BAC contig covering the chromosomal region with the polled locus. This bovine BAC contig should be used as a resource for developing new markers and to construct a human-bovine comparative map including all known human genes of the syntenic region. After this step we should be able to map the polled locus as precisely as possible and then to identify the responsible gene by a systematically performed linkage analysis for sets of gene-associated SNPs. DNA sequence analysis of genes significantly linked to the polled phenotype should then lead to the detection of the causative mutation responsible for polledness in cattle.
2 Literature
2.1 Introduction
Hornless cattle could be observed as early as in domesticated cattle in Egypt 3000 before Christ (LANGE, 1989). Pneumatic and round horns are characteristics of the bovine species, so it seems very interesting to know the gene responsible for the presence or absence of horns in cattle. In the modern cattle breeding, the hornless or polled phenotype is of special interest because in dairy cattle farming and beef production loose-house systems become more and more important and so the risk of injuries through horn crushes increases. Hornless animals are easier to handle and safer to work with, and they are less likely to injure other cattle or man. Furthermore, hornless cattle show a more settled character in comparison to horned cattle. There are less fights about the rank position between the animals of the herd (BRUMMER, 1976). So it seems expedient to breed polled cattle because it saves time and investments for the herd owner and a painful procedure for the animals, respectively. For a successful breeding of polled cattle it is essential to know which gene causes the polledness in cattle, because progeny testing is expensive and time-consuming and a gene test based on the causal mutation ensures the exact determination of the polled-genotype. In contrast to a direct gene test, a marker based gene test only allows to draw a probability for the polled-genotype and in addition to this, segregating family members have to be genotyped for the markers.
Furthermore, in the case of polled locus, several independent studies were not able to localize the position of the polled locus on cattle chromosome (BTA) 1 with the required precision.
In this study we give a short overview about the horn development and the inheritance of horns in cattle and discuss the molecular genetic approach to unravel the polled locus.
2.2 Phenotypic variation in development of horn size and shape in cattle
Horns are found in both sexes, but in male cattle they are developed stronger and have a broader basis as in female cattle. There is a great variation in size and shape of horns between the different cattle breeds, from very small and smooth horns up to big and strong horns or very cantilevered horns, respectively. This could be due to the fact that horns were important for the survival of the bovine species before domestication took place (SPECHT, 1999;
http://www.midohio.net/~fabouic/history.htm; ESTES, 1991), e.g. to protect themselves against enemies or food competitors. In the domesticated cattle smaller horns or polled cattle may be preferred. Selective breeding in the 19th century with the hornless phenotype has led to the development of polled breeds such as Aberdeen Angus or Galloway.
Cattle which never developed any horns or horn shapes are called polled or hornless, whereas cattle with small horns or horny scales are not classified as polled or hornless (LANGE, 1989). In addition to polled cattle, small horny growths without connection to the skull could be detected in several cattle breeds. This condition is called scurs. Scurs are referred to "wiggle horns" in German and indeed, most are moveable. Horns are usually evident at birth whereas scurs typically do not appear until about 4 months of age (AISA, 2001). “Wiggle horns” range from small and scab-like, to large and horn-like, although scurs generally do not grow as large as horns. Most of the cattle breeds are horned.
Cattle breeds with fixation of the polled allele are Aberdeen Angus, German Angus, Galloway, Polled Hereford and Red Poll. This mutation of the horn development occurs in these hornless cattle breeds, but also in breeds with mostly horned animals, like German Fleckvieh, Holstein Friesians, Charolais or Limousines. The incidence of spontaneous mutations from horned to polled phenotype is estimated at a rate of 1:20,000 (WHITE and IBSEN, 1936) and 1:50,000 to 1:100,000 (ROSENBERGER, 1981).
2.3 Inheritance of polledness and horn development in cattle
In our modern cattle breeds there were three different phenotypes distinguished: horned, hornless and scurs also named “wiggle horns”.
Table 1 Inheritance of polledness according to LONG and GREGORY (1978).
Sex
Horn locus
and Polled locus
Scurs locus PP Pp pp
males HH ScSc scurs scurs horned HH Scsc polled scurs horned HH scsc polled polled horned
females HH ScSc scurs scurs horned HH Scsc polled polled horned HH scsc polled polled horned Annotation:
H locus gene causes horns, fixed allele HH
P locus gene controlling development of the horned or polled phenotype, with the alleles P and p
Sc locus gene causing scurs phenotype, with the alleles Sc and sc
Based on crossing experiments with hornless Galloway and Holstein Friesian cattle WHITE and IBSEN (1936) postulated a genetic model with three independently segregating gene loci for the inheritance of polled phenotype. BREM et al. (1982) confirmed this model after the analysis of German Fleckvieh lines segregating for polledness. All taurine cattle breeds are homozygous HH for the horn gene being necessary for horn growth. Polledness is a dominantly inherited trait and not sex linked. Horned animals have the genotype pp at the polled locus, so one copy of the P allele is sufficient for the polled phenotype. BRENNEMAN et al. (1996) mapped the polled locus to cattle chromosome 1. In conclusion only the polled locus controls the absence (polled phenotype) or presence (horned phenotype) of horns in
cattle. Scurs can only develop in cattle with at least one copy of the dominant polled allele. A second independent gene locus with two dominant alleles (Sc) is required for the development of scurs. However, the phenotypic expression of the scurred condition is modified by gender dependent epistasis of the polled alleles. BREM et al. (1982) argued that females carrying one or two P alleles and homozygous for the Sc allele are scurred, whereas PP and Pp males and homozygous or heterozygous for the Sc allele express scurs. In contrast to BREM et al.
(1982), LONG and GREGORY (1978) came to the conclusion that males homozygous for the polled allele only develop scurs when they are homozygous for the Sc allele and in the case of heterozygous scurs alleles, males are polled. Further observations by KRÄUßLICH and RÖHRMOSER (1993) indicated that both above mentioned models for the expression of scurs could not explain the occurrence of scurs in three generation pedigrees of German Fleckvieh. So LAMINGER (1999) proposed maternal imprinting of the Sc allele in males.
Replacing the model with the sex dependent epistasis by maternal imprinting of the Sc allele, all inconsistencies in the phenotypic segregation of the polled and scurred animals could be resolved in the German Fleckvieh data.
2.4 Mapping of the polled gene in cattle
The first report on mapping the polled locus to bovine chromosome 1 (BTA1) was given by GEORGES et al. (1993). The polled locus was shown to be linked with the markers GMPOLL-1 (TGLA49) and GMPOLL-2 (AGLA17). With a somatic cell hybrid panel TGLA49 and AGLA17 were arranged near to the superoxid dismutase 1 gene (SOD1), that was mapped on BTA1 (O’BRIEN et al., 1991). The two-point linkage analysis between the two markers mentioned above and the polled phenotype revealed that the position of the polled locus was not between these two markers, but the relative position of the polled locus proximal or distal of the two microsatellite markers could not be determined. The precise position of the polled locus could not be resolved on the bovine genetic map of BTA1. A linkage analysis in five Charolais families including the markers TGLA49 and BM6438 confirmed the localization postulated by GEORGES et al. (1993) as no recombination was found between the two markers and the polled phenotype (SCHMUTZ et al., 1995). Due to the low density of
markers in the proximal region of BTA1, the relative position of the polled locus to the markers used could not be determined. BRENNEMAN et al. (1996) performed a linkage analysis with all available microsatellite markers from the whole BTA1. Only the marker TGLA49 exhibited a significant linkage with the polled phenotype. This was the first time a genetic map of the proximal region of BTA1 for the polled locus was released (Fig. 1). But the handicap of the previously described study was that there were no flanking markers proximal the polled locus. A further linkage study including the markers BM6438, TGLA49, IFNAR, KAP8, INRA212 and INRA117 from cattle chromosome 1 (HARLIZIUS et al., 1997) showed close linkage of the polled phenotype to all markers used, but as in previous studies the order of the polled locus relative to the markers used remained unclear. The localization of the polled locus was improved by using mapping results of 28 informative meiosis from the USDA reference families (U.S. Department of Agriculture). The polled locus was then mapped between the marker BM6438 and TGLA49 (Fig. 1; KAPPES et al., 1998) and released as horned/polled syndrome (HPS) in the bovine database BovMap (URL:http://locus.jouy.inra.fr/; EGGEN, 1998). EICHLER et al. (1999) analyzed 12 markers in 366 offspring of heterozygous males from German Fleckvieh, Holstein Friesian, Pinzgauer, Welsh Black and hybrids of these breeds. The polled locus could be mapped relative to four polymorphic markers covering a 4.3 cM interval. The polled locus was placed outside of this interval 2.0 cM proximal of BM6438 (Fig. 1). In a further work, BADER et al. (2001) genotyped 10 microsatellite markers in 7 half-sib families of heterozygous bulls German Fleckvieh. In agreement with KAPPES et al. (1998) the polled locus mapped between the markers BM6438 and TGLA49, whereby the order of markers was determined by radiation hybrid mapping and fixed in the linkage analysis (Fig. 1).
2.7 2.8 13.0
1.1 0.0
0.0 0.0
0.0
7.6 3.4
BMS1928 DVEPC141 DVEPC123 SOD1MICRO2 ARO9 ARO24 TGLA49 POLLED IFNAR PRKCBP2
BM6438 _MS
2.0 1.0 2.0 1.0
BMS1928 ARO9 POLLED
SOD1MICRO2 BM6438
EICHLER et al. 1999 BADER et al. 2001 BRENNEMAN et al. 1996
TEXAN14 BM1312
56.2 59.7
KAPPES et al. 1998
TGLA49 AGLA17
TGLA49
INRA117 BMS1928
1.5 5.0
TGLA
4.9 49
TGLA57 POLLED
52.6 0.0
POLLED BM6438
1.6 0.2 0.1
BM4307 RM95
26.3
42.1
Figure 1 Supposed localization of the polled locus on the proximal part of bovine chromosome 1.
In conclusion the most likely position of the polled locus on BTA1 is between the markers BM6438 and TGLA49. Although the map published by KAPPES et al. (1997) and the regularly updated MARC/USDA in the internet map (http://www.marc.usda.gov/) comprised 1250 markers with an average distance of 2,5 centiMorgan (cM) between the markers, and incorporating 2990 cM, especially the density of markers at the proximal region of BTA1 was very low. For the last ten years no new genetic markers have been published for this genomic region. So a further refinement of the position of the polled locus was not possible before the development of new markers for the polled region. The sequence data of the long arm of human chromosome 21 (HATTORI et al., 2000) opened a new perspective to increase the marker density for BTA1 through comparative gene mapping in cattle. The first two comparative maps between the proximal part of BTA1 and HSA21q22 were published by REXROAD and WOMACK (1999) and BAND et al. (2000) (Fig. 2), and a third comparative map between BTA1 and HSA21q22 followed by DRÖGEMÜLLER et al. (2002). The human bovine comparative maps of BTA1 were not consistent with regard to the existence of breakpoints and the arrangement of the markers. The reason for this may be due to the low resolution of the maps constructed. Although, construction of a genome-wide BAC contig for cattle is in progress (LARKIN et al., 2003; SCHIBLER et al., 2004), especially for the proximal part of BTA1 no new genes were identified. In a second generation comparative map constructed by EVERTS-VAN DER WIND et al. (2004) five new positions of genes were mapped in the proximal region of BTA1. The most important step towards a very high
resolution map for the proximal part of BTA1 was the construction of a bovine bacterial artificial chromosome (BAC) contig for this genomic region (Chapter 3). In total, 31 genes were assigned in this bovine BAC contig, of which 16 genes had been newly mapped. This contig provides a valuable tool towards the determination of the position of the polled locus ing and analysing the genes mapped in the contig. This bovine BAC contig built was facilitated by the fact that in the last years more and ore expressed sequence tags (EST) were released in the bovine databases (TAKASUGA et
e mentioned markers. As all ulls and their progeny exhibited the same haplotype for the markers BM6438 and SOD1MICRO2 segregating with the polled alleles, the conclusion was drawn that only the better two markers would be necessary for an i direct gene test for polledness. The reliability of this indirect gene test was not tested in larger samples or breeds other than German on cattle chromosome 1 and the basis for sequenc
m
al., 2001; STONE et al., 2002) and the sequence data of the syntenic region of HSA21q22.
2.5 Indirect gene diagnosis for the polledness
Based on the results of their study EICHLER et al. (1999) developed a marker based gene test (indirect gene test) to diagnose the polled phenotype in cattle. This indirect gene test requires genotyping of families segregating in polled and horned animals to draw conclusions on the polled phenotype. Using the genotyping results of the pedigree the most likely genotype for the polled locus can be calculated. For the indirect gene diagnosis at the polled locus four microsatellite markers had to be genotyped: BM6438, SOD1MICRO2, ARO9, and BMS1928.
In cattle families with only polled members the test was not applicable. So this indirect gene test only worked when the breeders started to intensify interbreeding polled with horned cattle. Furthermore, when both parents were polled the markerset was not able to differentiate between heterozygous and homozygous polled progeny. In contrast to these results, BROCKMANN et al. (2000) suggested an indirect gene test based on the following four markers on BTA1: BM6438, SOD1MICRO2, BMS4015, and BMS4017. This recommendation was based on a linkage study with three half sib families descending from three heterozygous German Fleckvieh bulls for the polled allele and the four abov
b
n
Fleckvieh. So it remains unknown if the marker haplotype for BM6438 and SOD1MICRO2 is fixed for the polled allele in all cattle breeds and in all animals.
BTA1 and HSA21q22 according to REXROAD and WOMACK (1999), BAND et al. (2000), and DRÖGEMÜLLER et al. (2002).
Figure 2 Human-bovine comparative radiation hybrid map between the proximal part of
APP GRIK1 SOD1 PRED33 EST1413 SYNJ1 KCNE2 BSMIT IFNAR1 IL10RB PRKCBP2 EST0601 AGLA17
BM6438
TGLA49 ARO9 KAP8 ARO24
S1M2
BMS1928 DVEPC141 INRA117
DVEPC123 Proximal BTA1 RH Map (cR3000)
329 (Mb)
HSA21
APP
ETS2
AGLA17 KAP8 IFNAR BM6438 TGLA49 BSMIT BMS1928 APP
SOD1
BMS2321
TGLA57 CSSM4 BMS2725
POUF1
ETS2 STCH
1,41
12,83 SOD1
18,61 IFNAR
20,27 ATP5O
20,85 SLC5A3 (SMIT)
21,02
25,75
ATP5 BTA1
(CR5000 )
O
Rexroad and Womack, 1999 Band et al., 2000
Drögemüller et al., 2002
2.6 Conclusions
In the past few years the decipherment of the bovine genome has made large progress. The bovine comparative BAC contig can be used as a powerful and effective tool for the exact mapping and molecular genetic characterization of the polled locus. Using this bovine BAC contig, single nucleotide polymorphisms (SNP) markers for all genes in this genomic region can be developed to increase the marker density in this region and to identify the responsible gene for polledness. Then the responsible gene can be sequenced and mutation analysis performed. If the causal mutation or mutations for bovine polledness are identified, a gene test can be developed to be used efficiently in breeding programs for polled cattle.
3 A 4 Mb high resolution BAC contig on bovine chromosome 1q12 and comparative analysis with human chromosome 21q22
3.1 Introduction
A bovine physical map consisting of a contiguous assembly of overlapping BAC clones (contig) is considered a necessary prerequisite for the accurate assembly of whole genome shotgun sequences in the current efforts to obtain the bovine genome sequence (GIBBS et al., 2002). Although construction of preliminary genome-wide BAC contigs for cattle (Bos taurus) is in progress (LARKIN et al., 2003; SCHIBLER et al., 2004), there is a need to construct highly accurate physical maps of targeted regions to facilitate targeted sequencing and the discovery of species specific genes or QTL affecting economically important traits.
Presently, successful positional cloning studies using detailed contig maps of specific cattle genome regions have been rare, e.g. the identification of the bovine LIMBIN gene causing dwarfism in Japanese brown cattle (TAKEDA et al., 2002; TAKEDA and SUGIMOTO, 2003) or the analysis of the bovine DGAT1 gene as functional candidate for milk yield and composition (GRISAT et al., 2002, 2004; WINTER et al., 2002, 2004).
In cattle, the hornless or polled phenotype is of special interest due to its economical importance in beef production. Hornless individuals are much safer to work with and they are less likely to injure themselves or other animals. The bovine polled phenotype shows a monogenic autosomal dominant inheritance and the still unknown gene has been genetically mapped to the centromeric region of bovine chromosome (BTA) 1 (GEORGES et al., 1993;
SCHMUTZ et al., 1995; HARLIZIUS et al., 1997). The first cattle-human comparative maps have been determined at low resolution by chromosome painting experiments and revealed that the proximal part of BTA1 shows conserved synteny with human chromosome (HSA) 21 (THREADGILL et al., 1991; CHOWDHARY et al., 1996). The recent expansion in the available number of bovine ESTs (SMITH et al., 2001) in combination with sequence information of the nearly finished human genome project provided the resources for detailed comparative maps. Subsequently, a medium-resolution bovine-human whole genome
comparative map was generated by RH-mapping (BAND et al., 2000). Additionally, different comparative RH maps of the centromeric BTA1 region were constructed but revealed inconsistencies concerning the existence of chromosomal rearrangements between BTA1q12 and HSA21q22 (REXROAD et al., 1999, 2000; DRÖGEMÜLLER et al., 2002). Considering the difficulties with high-resolution RH mapping, a successful comparative positional cloning strategy of the polled gene should be complemented by a precise clone-based physical map of this region.
Herein we describe the construction of a BAC contig covering a ~4 Mb segment on BTA1q12 and its comparative analysis with the syntenic region on HSA21q22, which has previously been shown to contain the polled mutation. This genomic contig integrates a large number of genes and markers of physical, genetic, cytogenetic, and RH maps of BTA1q12. As a first step towards positional cloning of the polled gene in cattle, this high-resolution BAC contig map represents a valuable resource for future fine mapping and sequencing efforts.
3.2 Materials and Methods
3.2.1 DNA library screening and chromosome walking
Library screenings with cDNA clones were done as described (DRÖGEMÜLLER et al., 2002). PCR-amplified DNA fragments were labeled with 32P and hybridized as probes on the high-density clone filters of the bovine genomic BAC library RPCI-42 (WARREN et al., 2000) according to the RPCI protocol (http://www.chori.org/bacpac/). BAC DNA was prepared from 100 ml overnight cultures using the Qiagen Midi plasmid kit according to the modified protocol for BACs (Qiagen, Hilden, Germany). Insert sizes were determined as described (MARTINS-WESS and LEEB, 2003).
3.2.2 DNA sequence analysis
Isolated BAC DNA was sequenced with the ThermoSequenase kit (Amersham Biosciences, Freiburg, Germany) and a LICOR 4200L automated sequencer. BAC DNA was sequenced with IRD-labelled T7 and Sp6 sequencing primers. Sequence data were analyzed with
Sequencher 4.2 (GeneCodes, Ann Arbor, MI, USA). BLAST database searches were performed at NCBI (http://www.ncbi.nlm.nih.gov/) for human mRNA alignments against bovine EST entries and for the bovine-human comparison against the whole human genome sequence (build 34.3). Repetitive elements were identified with the RepeatMasker searching tool (http://www.repeatmasker.org/). Single copy sequences were used to design primer pairs for the chromosome walking using the program GeneFisher (http://bibiserv.techfak.uni- bielefeld.de/genefisher/).
3.3 Results
To construct a BAC contig of the bovine polled gene region we started to screen a bovine BAC library by hybridization of 12 different heterologous human IMAGE cDNA clones (Table 1).
Table 1 Human cDNA hybridization probes within the bovine BAC contig.
human gene
symbol Genname IMAGE-ID RZPD clone ID TIAM1 T-cell lymphoma invasion and
metastasis 1
3197030 IMAGp 998 O157814 SOD1 superoxid dismutase 1 436140 IMAGp 998 B131026 HUNK Hormonally upregulated Neu-
associated kinase 768063 IMAGp 998 H161890 C21orf108 Chromosome 21 open reading
frame 108
25729 IMAGp 998 G19138 C21orf59 Chromosome 21 open reading
frame 59
124398 IMAGp 998 E07121 SYNJ1 Synaptojonin-1 2038462 IMAGp 998 M235017 OLIG2 Oligodendrocyte lineage
transcription factor 2170611 IMAGp 998 P045361 IL10RB Interleukin 10 receptor, beta 842859 IMAGp 998 E042085 GART Glycinamide ribonucleotide
formyltransferase 2901218 IMAGp 998 J037162 SON SON DNA-binding protein 1696332 IMAGp 998 N134307 KCNE2 potassium voltage-gated
channel, Isk-related family, member 2
2308895 IMAGp 998 A245722
DSCR1 Down syndrome critical region gene 1
324006 IMAGp 998 B07734
The physical localizations of six representative gene associated BAC clones were established by RH mapping and FISH on BTA1q12 (DRÖGEMÜLLER et al., 2002) prior to the beginning of a chromosome walking strategy. Further sequence tagged site (STS) probes that allowed the gradual joining of the individual emerging contigs into one large contig were generated from the obtained BAC end sequences of appropriate clones. Overlaps between clones were determined by STS content analysis. In total, 109 new STS markers were generated (Appendix 1, Table 2). The complete BAC contig consisted of 92 clones (Figure 1).
The physical mapping information derived from the contig assembly was refined by taking into account estimated BAC insert sizes from fingerprint analyses and pulsed field gels. The average insert size of the 92 BAC clones was 162 kb (range from 30 to 200 kb). The entire contig spans approximately 4 Mb and can be covered with a minimal tiling path of 32 clones (Figure 1).
The clone-based physical map was anchored to the linkage and RH map of BTA1 by STS content mapping of five previously described bovine microsatellites (ARO9, ARO24, TGLA49, SOD1MICRO2, BM6438) and two EST markers (EST0601, EST1413) (Figure 1).
During construction of the bovine contig primers were designed for 25 HSA21q22 genes from corresponding bovine EST sequences (Appendix 1, Table 3). PCR analysis of all 92 BAC clones with the gene-specific EST primer pairs revealed positive clones and the localization of these genes on the contig (Figure 1).
In total 165 BAC end sequences with an average read length of 726 bp totalling approximately 120 kb of genomic survey sequences were generated. Thus, the BAC end sequences cover approximately 3% of the studied genomic region. The sequence information of 165 BAC ends has been deposited in the EMBL nucleotide database under accession nos.
AJ698510 - AJ698674. Sequence alignments revealed 8 pairs of identical BAC ends. The end sequences contain an average GC content of 44.3% marginally exceeding the value of 41%
that is generally accepted as the average GC content in mammalian genomes. The GC content analysis further suggests that BTA1q12 is indeed closely related to HSA21q22, which has a GC content of 43.2% in the corresponding 4 Mb region. An analysis of repetitive sequences revealed that 39.1% of the BAC end sequences consisted of bovine repetitive DNA, mainly LINE (18.9%) and SINE (14.9%) elements, only 3.4% were of retroviral origin (LTRs), and 1.3% represented DNA transposons. In 56 cases, all or the majority of the BAC end
sequences represented repetitive sequences and were therefore discarded for STS design. The repeat masked BAC end sequences were subjected to BLAST comparisons against the sequence of the human genome (build 34.3). The obtained matches confirmed the homology between the cloned chromosomal region in cattle with HSA21q22. Significant and unique matches (e-value < 10-5) against human genomic sequences were observed for 38 (23%) bovine BAC end sequences. All but one of the 38 matches mapped to the expected location on HSA21q22 (Appendix 1, Table 4). All these BLAST matches corresponded well with the overall clone order in the bovine BAC contig and confirmed the correct assembly. In some cases the BLAST searches revealed the presence of genes within BAC end sequences and confirmed the previously obtained mapping results (Appendix 1, Table 4). The C21orf62 and SFRS15 genes could be localized in silico by this approach on the contig for the fist time (Figure 1). Only one single sequence (380C19-SP6) matched to a different human chromosome during the BLAST search. This unexpected BLAST result probably indicates a chimeric clone, as this BAC has been anchored in the contig by 4 STS markers and a gene specific bovine EST primer pair (Figure 1).
In total, the construction of this contig confirms the mapping of 15 previously mapped BTA1 genes and provides 16 new chromosomal assignments of bovine orthologs to the human genes SFRS15, C21orf45, C21orf108, C21orf63, C21orf59, C21orf66, C21orf62, IFNGR2, C21orf4, SON, MRPS6, C21orf82, C21orf51, KCNE1, DSCR1, and CLIC6. The gene order of the 31 assigned genes in the bovine BAC clone contig (Figure 1) corresponds exactly to the gene order of the NCBI HSA21q22 map (http://www.ncbi.nlm.nih.gov/mapview/; build 34.3), which lists 50 gene loci in the interval between KRTAP8P1 and CLIC6. Of these 50 loci 7 represent computer predicted hypothetical genes and 5 pseudogenes while 38 genes have at least some experimental evidence. The physical size of the investigated region and the distances between the mapped genes seems to be conserved between human and cattle. A high degree of gene order conservation can also be observed with respect to annotated murine genes. Some of the mapped bovine genes are assigned to the linkage map of mouse chromosome (MMU) 16. The current NCBI sequence map of MMU 16 (http://www.ncbi.nlm.nih.gov/mapview/; build 32.1) lists 19 of the 31 analyzed genes in a similar order as in cattle or human.
383 K23 63 O12 496 H4 394 A5 386 F4 352 O20 44 B5 292 J15 234 N12 506 K17 23 E5 506 K15 311 D23 320 O18 26 I16 180 G7
323 G5
301 M9 447 G24
316 N2
196 M18
292 J17
420 A17
199 N3
266 O23
46 I17
213 N17
553 A8
314 I19
493 P3 320 F13 293 I14 68 K7 249 E18 271 E18 470 N12 217 G23 518 G6
569 F23 161 B10
420 E6 76 J4 351 B8 219 G21 21 K598 P9
554 P19
52 K19
487A22
69 E7
552B21 241 F8
332 I5
564 N14
200 A7
543 J10
79 M3 346 B6 509P 22
534 N15 376 M15 538 E7
368 A9 242 D1 520 B16
370 F8 79 N19 204 M10
372 L18 221 H19 380 B9 218 J7
374 D19
T7 end Sp6 end 51 I7 420 O24
382 D7
244 B6
167 I16
182 B8
400 B6 400 D6
206 B24
328 M7
566 F20
80 B9
132 D12
37 H23
494 B13 540 F4
31 K20
543 J23
380 C19
BTA 1 *************** + _HSA 21 NCBI build 34.3
* *
*
* *
*
* *
*
* *
* *
* *
* * *
* 100 kb 100 kb Figure 1 Physical map of the isolated bovine BAC contig on BTA1q12. All mapped loci are indicated vertically on the top. Previously published BTA1 mapping results are marked by one (genes), two (ESTs), or three (microsatellites) asterisks. Underlined gene markers were initially assigned by human cDNA hybridization probes. Two framed genes were localized on the contigin silico. RPCI-42 BAC clones are given horizontally below the markers by continuous lines with their corresponding abbreviated clone names. A single chimeric BAC is shown by a dashed horizontal line. A minimal tiling path of 32 clones is indicated by thick lines. Bovine microsatellite, EST, and STS markers are represented by vertical solid lines. Bovine markers that are associated to corresponding human genes are plotted by dotted vertical lines and linked to 31 genes on the 4 Mb sequence segment of HSA21q22 (NCBI build 34.3) at the bottom. Comparative mapping of 31 gene- associated markers revealed a complete conservation of the gene order across the entire 4 Mb interval between Bos taurus and Homo sapiens.
3.4 Discussion
Here we describe a ~4 Mb single BAC contig that is predicted to contain the putative bovine polled gene. It establishes the physical order of the genetic microsatellite markers from different linkage maps that define the linked region and enables an exact determination of the candidate interval size. The physical map of this work has a higher resolution and accuracy than other currently available maps, which often have conflicting data with respect to marker order (REXROAD et al., 1999, 2000; DRÖGEMÜLLER et al., 2002). The recombination frequency could not be reliably estimated in the investigated region as there were inconsistencies between the different genetic maps of the BTA1 centromere (TAYLOR et al., 1998). The markers TGLA49 and BM6438 that are separated by 0.3 cM on the current MARC cattle linkage map (http://www.marc.usda.gov) are separated by roughly 1.4 Mb and the recombination frequency would be approximately 0.2 cM/Mb. This low value for the recombination frequency seems reasonable considering that the investigated region is located close to the centromere, where low recombination frequencies have to be expected. The precise physical assignment of the linked microsatellites will benefit future efforts towards the positional cloning of the bovine polled gene, as the precise marker position with respect to coding genes is now available. The generated BAC contig represents also a resource for the isolation of additional polymorphic markers for fine mapping efforts.
In this study three techniques were used to localize bovine genes on the contig. During the first phase of contig construction we applied a comparative approach. The recent availability of the complete sequence and gene catalogue of the long arm of HSA21 (HATTORI et al., 2000) has facilitated the procedure using appropriate human heterologous screening probes to isolate bovine BAC clones. In the second phase of contig construction we increased the marker density by exploiting the available bovine EST resources that allowed the generation of bovine gene-specific primers for bovine orthologs of human genes. To develop these primers we used the rapidly growing bovine EST sequence information in combination with data on exon/intron boundaries from the human genome. Finally, in some cases genes could be localized on the contig in silico according to the BLAST search results of BAC end sequences. Using these three approaches 31 genes could be assigned to the BAC contig, of which the following 15 gene loci had previously been mapped to cattle chromosome 1 with
low precision: KRTAP8P1 (HARLIZIUS et al., 1997); SOD1, IFNAR1, IFNAR2 (THREADGILL et al., 1991); GART (CHOWDHARY et al., 1996); ATP50 (SMITH et al., 2001); SLC5A3 (REXROAD et al., 1999); TIAM1, HUNK, SYNJ1, OLIG2, IL10RB, KCNE2 (DRÖGEMÜLLER et al., 2002); ITSN1 (LAURENT et al., 2000);. CRYZL1 (STONE et al., 2002), respectively.
The presented bovine-human comparative map provides the highest resolution comparative map of HSA21q22 with the centromeric region of BTA1 reported to date. The analysis of gene content of the investigated genomic region on BTA1q12 revealed a perfect synteny conservation between cattle and human. In contrast to the current bovine RH maps (REXROAD et al., 1999, 2000; DRÖGEMÜLLER et al., 2002), we found no evidence for the existence of chromosomal rearrangements in cattle, which is in part due to recent changes in the human genome assembly. High overall gene order conservation can also be observed with respect to the mouse. In other studies different gene orders within conserved synteny groups were observed across mammalian species (SCHIBLER et al., 1998). One possible explanation for the strong conservation observed here could be that the high gene content of BTA1q12 interfered with major chromosome rearrangements during mammalian evolution.
In conclusion, the constructed BAC contig is an essential preliminary step toward the targeted positional cloning of the bovine polled gene. The presented mapping information will facilitate the accurate assembly of whole genome shotgun DNA sequences of this region during the upcoming cattle genome project.
4 Development of 20 microsatellite and 7 single nucleotide polymorphism markers and subsequent fine mapping of the bovine polled gene region to a 1 Mb interval
4.1 Introduction
In Bos taurus the monogenic autosomal dominant inherited hornless or polled phenotype shows tight linkage with genetic markers of the centromeric region of bovine chromosome (BTA) 1q12, but until now several independent studies have not been able to order the polled locus relative to the markers used (GEORGES et al., 1993; SCHMUTZ et al., 1995;
BRENNEMAN et al., 1996; HARLIZIUS et al., 1997). Without knowledge of polled gene flanking markers, the selection of positional candidate genes using comparative mapping data may lead to failure. Therefore, a fine mapping of this chromosomal region has been initiated with the construction of a high-resolution comparative RH map of the proximal part of BTA1 (DRÖGEMÜLLER et al., 2002). Recently, a sequence-ready ~4 Mb single BAC contig on BTA1q12 could be constructed by integration of the generated contig map with existing genetic and physical maps of this region (Chapter 3). For the first time the precise physical assignment of the polled linked microsatellite markers TGLA49 and BM6438 that are separated by roughly 1.4 Mb has been determined. The aim of this study was a targeted development of additional microsatellites and SNPs spread over this BAC contig to generate the resource for the subsequent fine mapping of the polled gene using paternal half sib families of three different German cattle breeds.
4.2 Material and methods
4.2.1 Marker development
BAC DNA was isolated using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). This DNA was restricted independently with two enzymes (SacI and XbaI) and separated on 0.8 % agarose gels. The resulting fragments were cloned into the polylinker of pGEM-4Z (Promega, Mannheim, Germany). Recombinant plasmid DNA was end sequenced with the ThermoSequenase kit (Amersham Biosciences, Freiburg, Germany) and a LICOR 4200 automated sequencer. Sequence data were analyzed with the RepeatMasker 2 searching tool for repetitive elements and microsatellites (Smit, A.F.A. and Green, P., http://repeatmasker.genome.washington.edu/). Single copy sequences were used to design flanking primers for the identified microsatellites using the web program Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The precise localization of the new microsatellites on the contig map was determined by subsequent STS content mapping (Chapter 3).
4.2.2 Single nucleotide polymorphism development
DNA from BAC clones were isolated using the Qiagen large construct kit (Qiagen). BAC DNA was mechanically sheared to obtain fragments of approximately 2 kb. To determine new single nucleotide polymorphisms (SNP) shotgun plasmid libraries of four bacterial artificial chromosome (BAC) clones were constructed. Plasmid subclones were sequenced with the ThermoSequenase kit (Amersham Biosciences) and a LICOR 4200 automated sequencer (MWG Biotech, Ebersberg, Germany). Sequence data were analyzed with the RepeatMasker 2 searching tool for repetitive elements (Smit, A.F.A. and Green, P.
http://repeatmasker.genome.washington.edu/). In the BLASTN (Basic Local Alignment Search Tool Nucleotide) program (http://www.ncbi.nlm.nih.gov/BLAST/) the subclone sequences was used as queries against the human database to detect homologies between the sequences and HSA21. The homologous sequences were used to design primers for the identification of SNPs using the web program Primer3 (http://www-genome.wi.mit.edu/cgi- bin/primer/primer3_www.cgi).
4.2.3 Pedigree structure
The families comprised 809 animals in 30 two generation half sib families representing the six cattle breeds German Fleckvieh, Pinzgauer, German Holstein, Limousin, Shorthorn and Charolais (Table 1 and Appendix 2, Figure 3). In total, due to the decease of dams, DNA samples of 736 family members (91 %) could be collected. All 30 founder sires were heterozygous polled animals P/p mated to horned p/p (305 / 377: 81 %) or polled P/p dams (72 / 377: 19 %). A total of 248 (62 %) polled versus 154 (38 %) horned offspring was observed. Segregation of the paternal polled (P or p) allele was clear in 345 of 402 calves (86 %). In 43.5 % of the family members the polled phenotype could be observed.
Table 1 Family structure.
Breed1
Family
name Generations Total pedigree member
Available DNA samples
pp1 dam
P.1 dam
Pp1 offspring
pp1 offspring
Total offspring
Informative P/p meioses FV 03 2 26 26 12 0 8 5 13 13 FV 04 2 43 42 20 1 14 7 21 21 FV 07 2 31 30 14 1 10 5 15 14 FV 10 2 28 28 13 0 7 7 14 14 FV 12 2 41 41 20 0 9 11 20 20 FV 14 2 45 45 21 1 10 12 22 22
FV 17 2 11 11 1 4 3 2 5 3
FV 19 2 13 13 5 1 3 3 6 5
FV 76 2 75 65 35 2 19 18 37 35
FV 78 2 9 9 2 2 3 1 4 2
PI 31 2 23 14 11 0 7 4 11 11
PI 67 2 44 39 8 12 14 9 23 13 PI 68 2 36 28 13 4 15 3 18 14 PI 69 2 47 39 14 8 15 9 24 19
PI 72 2 7 6 0 3 2 1 3 1
PI 73 2 25 22 6 6 5 7 12 9
PI 74 2 26 26 3 9 12 1 13 4
DH 26 2 19 14 7 2 7 2 9 7
DH 260 2 7 6 3 0 2 1 3 3
DH 51 2 10 7 3 0 3 3 6 6
DH 52 2 12 9 3 1 4 3 7 6
DH 53 2 15 10 6 0 6 2 8 8
DH 54 2 12 8 4 0 4 3 7 7
DH 55 2 19 12 7 0 7 4 11 11 DH 57 2 69 69 34 0 21 13 34 34 LIM 81 2 11 11 3 2 4 1 5 3
LIM 82 2 9 9 4 0 2 2 4 4
SH 83 2 36 35 8 9 11 7 18 9 CHA 40 2 13 10 6 0 4 2 6 6 CHA 61 2 47 24 19 4 16 7 23 19
Total 809 736 305 72 248 154 402 345
1Annotation to Table 1
pp dam/offspring horned
P. dam polled phenotype, genotype unknown Pp offspring polled phenotype, heterozygous genotype
FV German Fleckvieh
PI Pinzgauer breed
DH Holstein Friesian
LIM Limousin
SH Shorthorn breed
CHA Charolais
4.2.4 Genotyping
A PCR amplification (12 µl final volume) was performed using 20 ng of genomic bovine DNA, 100 µM dNTPs, 10 pmol of each primer, and 0.5 U Taq polymerase in the reaction buffer supplied by the manufacturer (QBiogene, Heidelberg, Germany). The thermocycler profile was 94°C for 4 min; 32 cycles of 94°C for 30 s, primer pair specific annealing temperature (AT) (see Table 2; AT for all SNPs: 58°C) for 30 s, and 72°C for 30 s; followed by a final cooling step at 4°C for 10 min. Infra red dye (IRD) labelled PCR products were separated on 0.2 mm 6 % denaturing polyacrylamide gels using a LI-COR Gene ReadIR 4200 automated sequencer (MWG Biotech). The sequences for the SNP detection were sequenced with internal IRD labelled primer on 0.2 mm 8 % denaturing polyacrylamide gels using a LI- COR Gene ReadIR 4200 automated sequencer (MWG Biotech). Scoring and size determination of alleles was carried out independently by two investigators by comparison to a simple tandem repeat (STR) size standard marker (LI-COR, Bad Homburg, Germany).
4.2.5 Linkage and haplotype analysis
The evaluation of the data using the Merlin software was based on a multipoint analysis, including identical by descent calculations, kinship calculations and nonparametric linkage analysis. Furthermore, we estimated haplotypes by finding the most likely path of gene flow (ABECASIS et al., 2002). The generated genotype information of the families were tested for two-point linkage using the pairs and for multipoint analyses using the npl option. To reconstruct likely haplotypes for individuals and identify recombinations we applied the best option.
4.3 Results and discussion
4.3.1 Marker development
From 13 different BAC clones we generated approximately 390 kb genomic sequence data. In relation to the BAC clone insert size, we cloned on average 30 subclones per BAC and sequenced them from both ends with an average read length of 500 bp. A search for microsatellites within these sequences resulted in the development of 20 microsatellite markers, one to four on each of the processed BAC clones (Table 2). Most of the new microsatellites are (CA)n or (GT)n dinucleotide repeats (Table 2) and the number of observed alleles ranged between 2 and 11 (Table 3). Marker characteristics were determined by genotyping animals of six different cattle breeds and the average PIC value (45 %) for the 20 new markers (Table 3) was comparable to the PIC value of the 6 genotyped markers from the literature (52 %) (Table 4). The mendelian nature of these markers was confirmed by observing their inheritance through two or three generations of the genotyped cattle families.
There are several methods of developing microsatellite markers, but however, most of these methods have time-consuming laborious steps such as isolation of targeted clones by hybridization. Our results demonstrated that the method of random sequencing of subclones from before mapped BAC clones was effective for developing microsatellite markers in specific regions of interest. Additionally, we obtained about 30 kb raw sequences per BAC, which provide valuable resources for further marker development (SNP) or sequencing efforts. The selected 13 BAC clones are distributed over 3 Mb of the recently reported BTA1 BAC contig and finally, all new markers were exactly mapped by STS content mapping on the BAC contig (Chapter 3) in relation to already mapped markers (Figure 1).
