JP2002530091A - Whole-genome radiohybrid map of the canine genome and its use for identification of genes of interest - Google Patents

Abstract

  (57) [Summary] The present invention relates to a canine genome map that includes the chromosomal location of the purified or isolated genetic markers, TOAST, and polymorphic microsatellite markers of the canine genome. Another aspect is to identify genes responsible for the phenotype and behavior of interest, identify etiological genes, analyze diseases, identify genes related to these diseases and their alleles, and study dog strains. , Is the use of this map.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

【Technical field】

The present invention relates to purified or isolated genetic markers of the canine genome, TOAST,
And a map of the canine genome including the chromosomal location of polymorphic microsatellite markers. Another aspect is the identification of genes responsible for the phenotype and behavior of interest,
Use of this map to identify etiological genes, analyze diseases, identify genes related to these diseases and their alleles, and study dog strains.

[0002]

BACKGROUND OF THE INVENTION

The only domesticated species in the family Canidae (Canidae), the Canis familiaris (Ca
nis familiaris) consists of more than 350 varieties that exhibit a very wide range of polymorphic traits, behaviors and abilities. Unfortunately, various varieties suffer from a number of genetic diseases, many of which are similar to human diseases (Ettinger and Feldman, 1995). Most varieties have been produced for quite a short period of the last 250 years. This means
It suggests that only a few important loci appear to be responsible for the peculiar properties that define varieties (Serpell, 1995; Denis, 1997). Within-breed homogeneity can be assumed to be due to a high degree of genetic homogeneity, at least in the genomic regions subjected to selection pressure.

[0003] Thus, dogs appear to be a particularly attractive and powerful model for examining the nature and association of the genes responsible for the phenotypic and behavioral variability (Ostrande
r and Giniger, 1997). The dog model should also be very useful in human genetics when identifying pathogenic genes faces two major problems. The first problem is the difficulty in pinpointing, at the molecular level, two diseases caused by two different mutant genes that exhibit characteristic signs. The second arises from the fact that there are few large genealogies that provide information. Whenever possible, these two problems are overcome by considering a pedigree from an isolated population in which a single founder effect can be assumed. Relying on dogs is another promising strategy (Galibert et al., 1998). Indeed, large, well-informed dog pedigrees are fairly easy to establish, while the unique combination of varietal variability and intra-breed uniformity makes dogs ill or otherwise complicated. It can be a model of choice for analyzing the characteristics of a particular mammal and for identifying related genes and their alleles.

[0004] Important sources for locating genes responsible for various polymorphic traits or genetic diseases are polymorphic microsatellites (type II markers) and functional genes (
1 is a genomic map containing (type I markers) at regular intervals on the genome. First, such an integrated map will allow mapping of loci by positional cloning. Second, detecting regions where synteny is conserved between different species is advantageous for identifying more genes,
One by one, the map of dogs will be enriched and a candidate gene approach will facilitate the identification of the causative gene. An integrated map containing type I and type II markers,
Radiation Hybrid panel (Cox er al., 1990; Raeymaeker
s et al., 1995; McPherson et al., 1997). More recently, integrated whole-genome maps have been developed for whole-genome radiation hybrids (Whole-
Genome Radiation Hybrid (WGRH) method (Walter et al., 1994) (Hudson et al., 1995; Gyapay et al., 1996; Schuler et al.,
1996; Stewart et al., 1997). In other mammalian species, development of the WGRH approach is at an early stage. Radiation hybrids (RH) panels were from rats (Schmitt et al., 1996
) And cattle (Womack et al., 1997). Whole genome map of mouse genome (McCarthy et al., 1997) and murine genome (Sch, ott et
al., 1996) and a chromosome-specific map of the bovine genome (Schlapfer et al., 1997;
Yang et al., 1998) was recently reported. In dogs, the first reported meiotic linkage map contains only polymorphic di- and tetranucleotide microsatellite markers (Lingaas et al., 1997; Mellersh et al., 1997).

[0005] The present invention provides that 400 markers, consisting of genetic markers, TOAST, and microsatellites, some of which have already been incorporated into meiotic maps, are mapped on a dog / hamster RH ₅₀₀₀ panel through the WGRH technique. The integrated RH map of the canine genome. Localization of these type I and type II markers yields an RH map that covers approximately 80% of the canine genome. Furthermore, comparing the gene order in this RH map with that of the human, mouse and pig maps, it is possible to characterize chromosomal segments in which synteny is conserved or conversely broken.

[0006] Accordingly, none of the above prior art documents disclose or suggest the present invention as described below.

[0007]

DISCLOSURE OF THE INVENTION

The present invention relates to the genome positions of markers selected from the group consisting of markers having the sequences of SEQ ID NOs: 1 to 804 as shown in Tables 2-1 to 2-23 (hereinafter, referred to as Table 2). The aim is to include a canine genome map.

[0008] This map shows the location on the genome of at least 50, 100, 200, 300 or preferably 400 markers selected from the group consisting of the markers of SEQ ID NOs: 1 to 804 as shown in Table 2. May be included. This map may have any combination of the markers shown in Table 2.

The expression “location” may be any relative or absolute unit of measure of two or more markers, any data indicating that a given marker is not linked to another marker, a given Means arbitrary position determination based on the relationship with the radiation group, or arbitrary data indicating that a predetermined marker belongs to a radiation group not including other markers.

[0010] Another aspect of the invention is the identification of genes responsible for the phenotype and behavior of interest, the identification of etiological genes, the analysis of disease, the relevant genes of the disease and alleles thereof. It relates to the use of the above maps for identifying genes or for studying dog strains.

[0011] A third aspect of the present invention relates to a gene related to this disease and its related genes for identifying genes responsible for the phenotypic trait and behavior of interest, for identifying pathogenic genes, for analyzing diseases, and the like. To identify alleles or to study dog strains;
The present invention relates to the use of a canine genomic marker selected from the group consisting of the markers shown in Table 2 of the sequence shown in SEQ ID NOs: 1 to 804 or a sequence complementary thereto.

Another object of the present invention is to provide, for example, a group consisting of the sequences set forth in SEQ ID NOs: 1-804, which can be used as primers for isolating the corresponding human gene sequences, in particular the genes involved in genetic diseases. This is the sequence selected from.

DETAILED DESCRIPTION OF THE INVENTION RH mapping is a powerful method due to its ability to assemble genetic and physical maps (Hudson et al., 1995; Gyapay et al., 1996; Schuler et al. .,
(1996; McCarthy et al., 1996; Schlapfer et al., 1997; Yang et al., 1998)
. The key advantages of this approach include (i) mapping of polymorphic and non-polymorphic markers,
(ii) unlimited supply of DNA, and (iii) higher resolution levels of a panel of 100-200 hybrids comparable to an equivalent number of meiosis in gene linkage mapping. For example, from theoretical considerations (Stewart et al., 1997)
The upper limit of the resolution that can be expected from the RH panel can be estimated as follows. 1
The cR corresponds to 166 kb (see below), and according to Stewart et al., 1997, the average size fragment can be estimated to be 16.6 Mb. Therefore, the theoretical limit of the mapping resolution using this panel is 16.6 Mb / [126 (cell line) × 0.21 (average retention frequency)], that is, 0.6 Mb. Furthermore, since the resolution of radiation hybrid mapping is a function of the size of the retained fragments, experimental manipulations of radiation dose can produce panels of different levels of resolution (Cox et al., 1990). ; Walter et al.,
1994; McCarthy et al., 1996).

In the human and mouse genomes, continuous maps have been generated. Skeleton (fram
ework) Genetic linkage map with microsatellite markers (Dietrich et al.,
1994; Dib et al., 1997) were first created. Then the genes STS and EST
Using the RH panel (Gyapay et al., 1996; Hayes et al., 1996; McCarthy et
al., 1997) or using YAC or BAC libraries (Hudson et al.
., 1995; Kim et al., 1996). Finally, these markers were remapped with respect to a skeletal map containing polymorphic genetic markers (Hudson et al., 1
995; McCarthy et al., 1997).

In an effort to map the canine genome with the aim of locating the gene of interest and avoiding the sequential steps involved in mapping the human or mouse genomic map, we include type I and type II markers in one step We chose to create an RH map. To this end, we mapped 400 markers (218 genes and 182 microsatellites), 121 of which were Ligaas et al., 1997; Mel
lersh et al., 1997; or previously analyzed by Werner et al., 1997. To map markers already mapped elsewhere on the RH panel,
High reliability was confirmed in the mapping results, allowing the meiotic and RH groups to be merged into an expanded and integrated map.

The extent of the map was assessed from the opportunity that any new marker would fall into one of the linkage groups. From the 180th marker, any new marker will be replaced by a rod score (
It is observed that the chance of linking with another marker at lod score 6 or higher was 80%. The range is estimated to be about 80%, assuming that the markers are most likely to be randomly distributed in the genome.

[0017] The total size of all 57RH groups is 7995cR. RH calculated on rod 6
The data obtained from the 2PT analysis allows detection of linkage between markers located 50cR apart. This value can be added to each side of the RH group, which
Extend group reach to 13,695cR. This may be overestimated due to the fact that some markers may be located near telomeres. On the other hand, this does not take into account the assessed range corresponding to 53 unlinked markers.

RH mapping constitutes a particularly powerful tool in comparative gene mapping.
Chromosomal order can be established for expressed genes that are normally conserved among species (O'brien et al., 1993; Johansson et al., 1995; Chowdhary et al.,
1996), however, because there is no easily detectable allelic variation, there is rarely a genetic linkage mapping method. For this type of challenge, fixing the largest number of common markers is of utmost importance, as described in TOAST (Jiang et al.,
), CATS (Comparative Anchor Tagged Sequences, comparison anchor tag sequence, L
yons et al., 1997) or UMP (Universal Mammal Primer, Universal Mammal
ian Primers, Venta et al., 1996).

This RH map already reveals a high degree of conserved regions for the human, mouse and porcine genomes, even though additional assigned genes are required. These syntenic (homologous) relationships are essential for the understanding of evolution and in more methodological ways to further enhance canine maps by mapping homologous genes selected from high density map species. Can be used. In general, making a map of polymorphic markers requires nearly 10 times as many markers as needed to obtain an even distribution (one marker every 5 cM). However, if a set of regularly located genes is first described using syntenic relationships, it is now possible to select the corresponding BAC recombinant clone, and thus the cognate polymorphic microsatellite. Becomes A dense map of the canine genome with equally distributed type I and type II markers is useful for phenotypic traits and behavior, as well as localization and identification of genes involved in genetic diseases.

Radiation Hybrid Mapping A total of 400 markers consisting of 218 genetic markers and 182 microsatellites (see Table 1 below) were classified on a WGRH panel made from 126 radiation hybrid cell lines. Statistical analysis (RH2PT in the RHMAP 3.0 package, Boehnke et al., 1991
), 347 markers out of 400 were assigned to 57 radiation hybrid groups, while 53 markers were not linked as shown in Table 2 below. The RH group was created so that any marker in the group had a rod score of 6 or more with at least one other marker. Of the 57 RH groups, 11 groups contained 10 or more markers, with the largest (RH11) group being 21
17 RH groups contain 5-9 markers, and 13 groups contain 3 or 4 markers. The remaining 16 RH groups each have two markers. All RH groups with at least three markers were ordered and distances were calculated using the RHMAXLIK program of RHMAP 3.0 (Table 2).
The distance between markers is expressed in centi-rays (cR), and 1cR ₅₀₀₀ is defined as 1% of the frequency of destruction between two markers after exposure to ₅₀₀₀ rad of gamma rays (Cox et al.
al., 1990). The size of the RH group ranges from 9cR (RH06 with two markers) to 58
It is in the range of 8cR (RH14 with 18 markers). The average distance observed between two adjacent markers is 23cR.

[0021]

[Table 1]

References: (1) Jiang et al., 1998, (2) Lachaume et al., 1998; Langston et al., 1997;
Werner et al., 1997, (3) Gyapay, Genethon, France (4) Francisco et al.,
1996; Mellersh et al., 1997, (5) Ostrander et al., 1993, 1995; Lingaas et
al., 1997, (6) Holmes et al., 1993a, 1993b, 1995; Molyneux et al., 1994;
Mariat et al., 1996; Dolf et al., 1997, Printing; Thomas et al., 1997; Sch
elling et al., printing; Switonski et al., printing; Yuzbasiyan-Gurkan (http:
//www.cvm.msu.edu/text/res/c lmm.html).

[0023]

[Table 2-1]

[0024]

[Table 2-2]

[0025]

[Table 2-3]

[0026]

[Table 2-4]

[0027]

[Table 2-5]

[0028]

[Table 2-6]

[0029]

[Table 2-7]

[0030]

[Table 2-8]

[0031]

[Table 2-9]

[0032]

[Table 2-10]

[0033]

[Table 2-11]

[0034]

[Table 2-12]

[0035]

[Table 2-13]

[0036]

[Table 2-14]

[0037]

[Table 2-15]

[0038]

[Table 2-16]

[0039]

[Table 2-17]

[0040]

[Table 2-18]

[0041]

[Table 2-19]

[0042]

[Table 2-20]

[0043]

[Table 2-21]

[0044]

[Table 2-22]

[0045]

[Table 2-23]

Description of Table 2 : The marker group is sRHa (including several RHa groups) or RHa (one RHa group).
Group). The suffix a is added to determine the age of the RH map. If the chromosome assignment is known, it is designated as CFA (Canis Familiaris). Primer sequences characterized in the laboratory are shown in bold.

Regarding PCR conditions: “Ta” is the annealing temperature. L at the end means a 45 second annealing step. The size of the PCR product is indicated in bp, "int" indicates the presence of an intron, and thus the size of the product is unknown. The rod score and the value of the distance between the two markers (locus 1 in the top row and locus 2 in the bottom row) are shown in the bottom row. The distance is indicated by centiRay (CR). ND: Not measured.

PCR programs: -Programs 1-4: 12 minutes at 95 ° C. 30 seconds at 94 ° C, 60 ° C (Program 1) or 55 ° C (
30 cycles of 30 seconds at program 2 and 4) or 50 ° C (program 3), 30 cycles at 72 ° C Final step at 72 ° C for 5 minutes (except program 4). -Programs 5-8: 12 minutes at 95 ° C. 30 seconds at 94 ° C, 69 ° C (program 5) or 63 ° C (
30 seconds at program 6) or 59 ° C (program 7) or 54 ° C (program 8), then lower by 0.5 ° C each cycle, and 21 cycles of 30 seconds at 72 ° C. 12 cycles of 30 seconds at 94 ° C, 30 seconds at 59 ° C (Program 5), 53 ° C (Program 6), 49 ° C (Program 7) or 44 ° C (Program 8), and 30 seconds at 72 ° C. -Program 9: 12 minutes at 95 ° C. Eight cycles of 94 ° C for 30 seconds, 62 ° C for 45 seconds, and 72 ° C for 45 seconds. 25 cycles of 30 seconds at 94 ° C, 30 seconds at 65 ° C, and 30 seconds at 72 ° C. Final step 5 minutes at 72 ° C.
-Program 10-13: 12 minutes at 95 ° C. 30 seconds at 94 ° C, 65 ° C (program 10) or 63 ° C (
30 seconds at program 11) or 59 ° C (program 12) or 61 ° C (program 13), decrease 2 ° C every 4 cycles, then 20 cycles of 30 seconds at 72 ° C. 30 seconds at 94 ° C, 55 ° C (
25 cycles of 30 seconds at program 10), 53 ° C (program 11), 49 ° C (program 12), or 55 ° C (program 13) and 30 seconds at 72 ° C. Final step 2 minutes at 72 ° C. -Programs 14-16: 12 minutes at 95 ° C. 30 seconds at 94 ° C, 45 seconds at 57 ° C (Program 14)
25 seconds at 60 ° C, 20 seconds at 58 ° C (program 15) or 45 seconds at 58 ° C (program 16), 72
8 cycles of 45 seconds at 45 ° C. 30 seconds at 94 ° C, 60 ° C (program 14) or 65 ° C (program
15) 25 cycles of 30 seconds at 61 ° C (program 16) and 30 seconds at 72 ° C. Final step 5 minutes at 72 ° C.

Marker retention The average retention frequency of the 400 markers on the RH panel is 21%, 82% of the markers is 10%
Has a retention frequency of 〜40%. The four markers with the highest retention values are more than 80%. This is due to two genes (SRP68 and Galk1) known to be located on canine chromosome 9 near the TK gene used for selection of hybrid cell lines (Werner e
t al., 1997) is not surprising. This also shows a high retention frequency,
Another marker corresponding to huESTL08069 was not linked to any other marker. It encodes a member of the DNA J family of chaperone proteins and requires careful attention before placing this marker into a linkage group. The fourth unlinked marker corresponds to the featureless microsatellite Ren01E15. This does not match any known repetitive sequence of the dog and cannot explain its high retention value.
The medium to high (40-80%) retention profile exhibited by the 28 markers may be due to a particular chromosomal location. In fact, as previously observed in the human genome, there is a general tendency for retention to increase for targets located on smaller chromosomes (Gyapay et al., 1996). This may be related to the high frequency of retention of markers near the centromere. In the canine genome, located on the canine Y chromosome, RH
An example of the SRY gene with a retention value of 40% can be illustrated on the panel. This particularly high value for genes located on the sex chromosome may be related to the fact that the Y chromosome is the smallest. Conversely, eight genetic markers known to belong to the canine haploid X chromosome (CHM, PGK1, HPRT, IL2RG, AR, DMD, F8C and F9) show a retention frequency of about half of the autosomes, This is a finding consistent with previous observations in the human genome (Gyapay et al., 1996). Finally, in all radiohybrid cell lines, the locus corresponding to the P53 oncogene (Guevara-Fujita et al., 1996) located on dog chromosome 5 is:
Although this marker was detected in the original dog cell DNA used in the RH panel, it did not retain it.

The fact that only one of the 400 markers was not detected suggests that the RH panel covers almost the entire canine genome. Assignment of linkage groups to canine chromosomes Except for the X chromosome, all 39 pairs of canine chromosomes are terminal centromeric chromosomes, many of which are too small to be reproducibly identified by cytogenetic techniques And too similar (Langford et al., 1996; Reimann et al., 1996; Switonsky et al., 1
996). Recently, FISH analysis on canine chromosomes has resulted in an increase in the number of mapped markers. In this study, some of the latter were determined on the WGRH panel, whereby 14 RH groups were assigned to 9 dog chromosomes, as shown in Table 3 below, and one RH group was indistinguishable to dog chromosomes. (# 29 to # 35).

[0051]

[Table 3]

References to assignments: (1) Dolf et al., Printing, (2) Dutra et al., 1996, (3) Werner et al., 1997; Switonski et al., Printing, (4) Schelling et al.
(5) Dolf et al., 1997; (6) Fischer et al., 1996; (7) Deschenes et al., 1994; (8) Langston et al., 1997.

[0053] These assigned RH groups are represented as CFA (Canis familialis) followed by the chromosome number (CFA # in Table 2). However, most of these chromosomal assignments are based on the cytogenetic location of the unique marker for the RH group. Given the rather complex design of canine karyotypes, other cytogenetic assignments need to confirm these correspondences. Dog chromosome 9
Detailed physical and genetic maps have been reported for (CFA9-RH06 and RH07) (Wern
er et al., 1997), four of the assigned loci are arranged in two RH groups. As a result, the assignment of these groups to CFA9 was made with great confidence.

For the RH04 group, this means that two genes of the immunoglobulin family (I
GHE, IGHA). Conventional FISH assignment of the canine immunoglobulin heavy chain (IGH) locus to CFA 4 (Dutra et al., 1996) and the clustering mechanism of this gene family in different mammals has been described (O'Brien et al., 1993), RH04 Group
This is advantageous for assignment to CFA4. Two of the eight genes known to belong to the canine X chromosome are in the RH10 group, while six have not been found to belong to any group. This means that the size of this chromosome is large (137M
b) May be (Langford et al., 1996). Finally, the RH31 group includes the microsatellite marker AHTk336 [previously localized to a subset of chromosomes 29-35 (Fischer et al., 1996)]. Further cytogenetic assignment of markers belonging to this subset will help to characterize these small chromosomes.

Comparison of RH and Meiosis Maps Within the framework of the Human Genome Mapping Project, the power of radiohybridism lies in the ability to order type I and type II markers and obtain an integrated meiosis and physical map. (Hudson et al., 1995; Gypay et al., 1996; Schuler et al.
al., 1996). In dogs, the meiotic map has 121 markers already classified.
(Lingaas et al., 1997; Mellersh et al., 1997; Werner et al., 1997)
Positioning on the map allowed the RH and / or meiotic linkage groups to be summarized. Thus, the 26 RH groups and the three unlinked loci could then be merged into 13 larger groups, referred to as syntenic (homologous) groups, represented in Table 2 as sRH01-sRH13. As an example, sRH01 is defined by the linkage of three RH groups (RH01, 02 and 03) using seven markers belonging to a single meiotic group (L1 group reported by Mellersh et al., 1997). We were able to. The largest syntenic group, sRH06, contains 24 markers and reaches a size of 674cR. The alignment of RH and meiotic groups also
Allowed to enter some of the previously reported dog meiotic groups. Suitable examples are the syntenic sRH07 group spanning the L27 and L15 meiotic groups (Mellersh et al., 1997), and the L7 and L1 DogMap meiotic groups (Lin
Similar to the sRH12 group for gaas et al., 1997; Mellersh et al., 1997). Furthermore, the mapping of markers by two independent methods gave great confidence in the order. A detailed comparison of the most probable order of the 121 markers common to RH and the meiotic map demonstrated that both approaches were identical except for a single mismatch in the sRH06 group. Two microsatellite markers,
Cxx246 and Cxx424 were switched positions in the radiohybrid RH11 and meiotic L1 groups. However, Cxx246 is non-framework in RH maps
Marker, the location of which will undergo local optimization after capturing more markers.

The comparison of RH map units and RH and meiotic maps in relation to genetic and physical distances, in addition to increasing the informativeness of the mapping, shows that cR ₅₀₀₀ for the same set of markers in different regions of the canine genome. It is now possible to associate genetic units with map units. In this study, distances at Centilay and Kosambi centimorgan (cM) were recorded for 26 pairs of markers as reported in Table 4 below. Since these markers are from different groups and likely from different chromosomes, the calculated cR / cM ratios ranging from 1 to 33 are:
It can provide a good assessment of mutations that are spread throughout the canine genome. Such mutations reported in previous human chromosome mapping analyzes reflect the fact that genetic distances are not uniform in the genome. This also gives γ
Of “hot spots” for destruction during X-ray irradiation (Rayemakers et al., 1
995). Similarly, a 25-fold mutation was found in the RH mapping of human chromosome 18 (Francke et al., 1994). For the purpose of this map, the values shown in Table 4 resulted in 1 cM corresponding to 6 cR ₅₀₀₀ . When recombination rate of dogs and humans in the genome is assumed to be similar, observed in common human genome, the correspondence between 1cM respect 1Mb is applied to the RH panel, 1Cr _50
00 can be evaluated to 166 kb. This assessment is found on different human chromosomes (Cox et
al., 1990; Francke et al., 1994; Gypay et al., 1996; McPherson et al.,
1997), or other relationships found in the mouse genome for comparable radiation doses (3000-8000 Rad) (Schmitt et al., 1996; McCarthy et al., 1997).

Similarly, the Genebridge 4 panel (Gyapay et al., 1996), which served as a reference in the establishment of our dog RH panel (Vignaux et al., In press), also had a 110 kb / cR
Shown are ratios in the range of 305 kb / cR.

[0058]

[Table 4]

A: distance in centi-rays (cR ₅₀₀₀ ) obtained in the RH-a group reported in this study b: according to Lingaas et al., 1997; Mellersh et al., 1997; Werner et al., 1997 The previously reported distance in Kosambi centiMorgan (cM) obtained from the meiotic group c: From these two data series, the comparative gene mapping polymorphism microsatellite that gives a 6CR correspondence to 1 cM (2737 / 449.6) is Although not generally conserved between species, it is a useful reagent in linkage studies, but of little value in comparative mapping studies. In contrast, mapping type I markers makes it possible to take advantage of data collected by cytogenetic studies and / or physical mapping in different mammalian species.

Of the 218 genetic markers mapped in this study, 174 are present in humans (HSA
), 93 were on the mouse (MMU) and 23 were on the pig chromosome (SSC). Given only those genes located in humans, 147 are distributed in all RH groups and 27 genes are unlinked. And, all human chromosomes, except HSA18, have counterparts in at least one dog linkage group. The smallest canine chromosome, the Y chromosome, has only one unlinked marker, the SRY gene.

The counterpart of a human chromosome in dogs is most commonly 2-5 RH or sR.
Divided into H groups. For example, HSA12 occurs in dog RH groups 27, 33, 34, 35 and 39. This is not surprising when comparing the size of the HSA and the size of the dog RH group. Nevertheless, the majority of the RH groups correspond to 2-5 different human chromosomal fragments. Furthermore, when some dog RH groups were assigned to some dog chromosomes (see Table 3), a relationship between CFA and HSA chromosomal fragments could be pointed out. For example, HSA4 corresponds to CFA3 (sRH01) and CFA13 (RH29),
CFA3 has homology to HSA 4p, 5q and 15q (Table 2). Taken together, these data show the degree of chromosome segment conservation previously observed in comparisons of other maps (O'Brien et al., 1993, 1997a, 1997b; Johansson et al., 1995; W
omack et al., 1997).

Synteny has been analyzed whenever it is easy to compare positions on the dog RH group, human, mouse and pig chromosomes. As expected, the canine X chromosome (sR
The part represented by the H05 group) is its human and murine counterpart (O'Brien
et al., 1993). The other seven RH groups were conserved together among dog, human and mouse species. The largest block is HSA1
It is part of the sRH03 group (CFA9), homologous to 7q and MMU11, reaching 300cR (slightly less than 50Mb). If synteny is preserved between the four species, 10
Proven in the sRH06 group over a distance of 0cR (16.6Mb). The RH28 group corresponding to CFA12 contains 14 markers aligned over 195cR. Six of these are MHC genes and two are genes commonly associated with MHC loci, namely tumor necrosis factor (TNF) and colipase (CLPS). Such clustering is due to the conservation of the entire MHC machinery in the human (HSA6p12-p21), mouse (MMU17), pig (SSC7) and rat (chromosome 20) genomes (O'Brien et al.,
1993; Yamada et al., 1994; Archibald et al., 1995) and reinforce the concept of high conservation during mammalian evolution.

However, one unexpected synteny disruption is associated with one canine MHC class I gene, DLA-79. Its human counterpart is located at 6p21 (Burnett et
al., 1995), three different markers corresponding to this gene were tested independently.
(Data not shown), but this gene was always incorporated into a different group, RH30. Thus, differences in location may be due to unexpected translocations made by the canine DNA used to establish the RH cell line, or to the destruction of synteny in chromosomal regions previously found only in dogs.

The majority of other syntenic disruptions were unique to the canine genome compared to the human and mouse genomes and were observed in nine RH groups. For example, group sRH04 is 2
Shows homology to two human chromosomes, HSA 3p and 19p, and two mouse chromosomes, MMU6 and 9. In addition, for the two dog RH groups for which pig data is available, the destruction seen in the dog-human and dog-mouse comparisons is also seen in the dog-pig comparison (RH36 and RH33 groups). Conversely, cases in which synteny is conserved between dogs and humans, but is disrupted between mice and dogs, are observed less frequently and illustrate only two RH groups (RH30 and RH38). You. The genetic organization observed in this RH map sheds light on a number of syntenic conservation between dog, human and mouse genomes. The observed syntenic destruction reflects ancestral rearrangements that occurred between carnivore, human and murine species.

[0065]

【Example】

Example 1 Generation of Radiation Hybrid Cell Lines A panel of radiation hybrid cell lines was generated as described by Vignaux et al., (In press). Hybrid dog fibroblasts were exposed and exposed to 5000 rads of gamma radiation. The cells were then fused with thymidine kinase deficient hamster cells (HTK3-1) in the presence of polyethylene glycol according to Benham et al., 1989. After selection in HAT medium and cell culture in roller bottles, DNA was extracted from individual clones (H
oglund et al., 1995). After confirmation of the presence of dog DNA and analysis of the retention profile, a total of 126 radiation hybrid cells were grown and a minimum of 2.5 mg genomic DNA was prepared. This WGRH panel was called RHDF ₅₀₀₀ and was used to map the canine genome.

Example 2: Development of Markers Using the canine gene- PRIMER program (GCG, 94), a pair of oligonucleotide primers was designed from the canine gene sequences present in the Genbank database (
Table 1).

TOAST (Traced Orthologous Amplified Sequence Tags) (orthologous amplified sequence tags) (a gene whose sequence has been determined in two or more mammalian species, including at least at the genomic level) Gene-specific universal primers were selected to develop. All fragments amplified by PCR with TOAST primers were sequenced before use to confirm by BLAST search that they corresponded to the orthologous gene (Table 1; Jiang et al.
., Printing).

HuEST (Expressed Sequencing Tag) —optimizes the PCR conditions on canine genomic DNA for some of a number of EST primers (provided by G. Gyapay, Genethon, France) designed for mapping the human genome. Decided (Table 1).

[0069] Microsatellites - classifying the smaller insertion canine genome library and additional birds and a set of dinucleotide microsatellite characterized from canine microsatellites from the literature (Table 1).

Example 3: Radiation Hybrid Map Screening of Canine-Specific Markers on Hamsters: Each marker was tested with dog, hamster and dog / hamster mixed (1: 2) DNA to mimic RH DNA content. Tested. PCR was optimized to obtain dog-specific PCR products.

PCR analysis on radiation hybrid cell lines: 15 ng of each primer, 200 μM dNTPs, 1.5-3 mM MgCl ₂ , 50 mM KCl, 10 mM Tris-
50 in a final volume of 10 μl containing HCl and 0.5 U Taq Gold Polymerer (Perkin Elmer)
PCR was performed on ngRH DNA. Amplification is Techne (Techne, Cambridge, MA) or MJ
(MJ Research, Cambridge, MA) I went with a thermocycler. PCR products
Analyzed by migration in a 1.8% agarose gel (Hybaid electrophoresis system) for 30 minutes at 120 V in 5 × TBE buffer, visualized under ultraviolet light after ethidium bromide staining. Gel images were recorded using a high-resolution CCD camera (BioPrint, Vilber Rourmat, Torcy, France). Results are semi-automatically present, absent or unknown on a UNIX workstation using data acquisition software (Nicolas, Soriano, @ unov-rennes 1.fr) developed by G. Brenterch and N. Soriano. Scored as clear. PCR analysis of each of the 400 markers was performed in a single assay, and scoring of the results was done with great care. In addition, 40 markers were performed twice on the panel with the same result. Slight discrepancies in the presence or absence of PCR products were only found in at most 1-4 (of 126) hybrid cell lines. None of these had much effect on the mapping results.

Statistical Analysis of Radiation Hybrid Data: Two-Point and Multipoint Likelihood Methods for Statistical Analysis (Boehnke
et al., 1991) were used to determine the order and distance between markers. This method
It is assumed that gamma-ray destruction in the genome is random and fragments are retained independently. The statistical package RHMAP 3.0 consists of three programs: RH2PT, RHMIN
BRK and RHMAXLIK. Using a two-point analysis program (RH2PT), pairs of loci with a rod score of 6 or more were identified and from these data the RH group was obtained. RH
MINBRK ordered multiple loci by minimizing the inevitable disruptions needed to explain the data. For the largest linkage group, RHMINBRK order, RHMA to order multiple loci by maximizing the prospect of hybrid data,
Used as a candidate for XLIK analysis. Multipoint analysis is 1000: 1 and 100: 1
Was performed by determining the position of a marker as a skeleton. Next, the non-skeletal markers were located in the most favorable order. Stepwise locus ordering was used to reduce computation time. Finally, the selected locus model 1 was used to confirm the locus order of the linkage group containing the TK locus, which allows one retention chance for the fragment containing the selectable locus.

Example 4: Comparative Mapping Cytogenetic data for human, mouse and pig genes were extracted from the Genome database: http://bisance.citi2.fr/GENATLAS; http: //www.ncbi.nlm. nih.gov/UniGene/index.html. We applied the chromosome name of ISCN (1978): the dog chromosome was CFA (Canis familiaris), the human chromosome was HSA (Homo sapiens, Homo sapie).
ns), the mouse chromosome is designated as MMU (Mus musculus) and the pig chromosome is designated as SSC (Sus scrofa).

[0074]

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[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor André, Catherine France, 35630 Saint-Brug-de-Ifs, La Thiere (No address) F-term (reference) 4B024 AA11 BA80 CA01 HA11 4B063 QA01 QA13 QA18 QQ42 QR08 QR55 QR62 QS25 QS34

Claims (10)

[Claims] 1. A canine genome map containing the positions on the genome of markers selected from the group consisting of the markers of SEQ ID NOs: 1 to 804 shown in Table 2. 2. Includes at least 50, 100, 200, 300 or preferably 400 markers in the genome of at least 50, 100, 200, 300 or preferably 400 markers selected from the group consisting of the markers set forth in SEQ ID NOs: 1-804 as shown in Table 2. The map according to claim 1. 3. Use of a map according to any of claims 1 and 2 for identifying genes responsible for the phenotypic trait and behavior of interest. 4. Use of the map according to any one of claims 1 and 2 for identifying a pathogenic gene. 5. Use of the map according to any one of claims 1 and 2 for analyzing a disease or identifying a gene related to the disease and an allele thereof. 6. Use of a map according to any of claims 1 and 2 for studying dog strains. 7. The sequence of SEQ ID NO: 1 to 804 or a sequence complementary thereto selected from the group consisting of the markers shown in Table 2 for identifying a gene responsible for a phenotype and behavior of interest. Use of canine genomic markers. 8. Use of a canine genomic marker selected from the group consisting of the markers shown in Table 2 of the sequence set forth in SEQ ID NOs: 1 to 804 or a sequence complementary thereto for identification of a pathogenic gene. 9. A group consisting of the markers shown in Table 2 of the sequences of SEQ ID NOs: 1 to 804 or the sequences complementary thereto for analyzing a disease or identifying a gene related to the disease and an allele thereof. Use of a canine genomic marker selected from: 10. Use of a canine genomic marker selected from the group consisting of the markers set forth in Table 2 of the sequence set forth in SEQ ID NOs: 1-804 or a sequence complementary thereto for the study of canine strains.