A set of canine interrepeat sequence PCR markers for high-throughput genotyping
MANJULA DAS1,
HAKAN SAKUL2,
JULIUS KONG1,
GREGORY M. ACLAND3 and
JERRY PELLETIER1,4
1 Department of Biochemistry
4 Department of Oncology, McGill University, Montreal, Quebec, Canada H3G 1Y6
2 Department of Statistical Genetics, Parke Davis Laboratory for Molecular Genetics, Alameda, California 94502
3 James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401
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ABSTRACT
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One hundred and sixteen interspersed repetitive DNA sequence (IRS)-PCR markers have been developed and characterized from Canis familiaris for high-throughput filter-based genotyping. We present a detailed analysis of markers produced by amplification using primers directed to the conserved regions of the C. familiaris short interspersed nuclear element (Can-SINE). The majority of IRS-PCR markers developed were moderately to highly polymorphic with mean heterozygosity (HET) and polymorphism information content (PIC) values of
0.6. The HET value for 22.3% of the markers exceeded 0.7. We also demonstrate that sequence variation of Can-SINEs between breeds is significant and also represents a rich source of polymorphisms. Mapping of 73 of the markers to the existing integrated linkage-radiation hybrid map enriches the map as well as establishes the utility of the markers. The significance and utility of this new class of IRS-PCR Can-SINE-based markers for high-throughput genotyping is discussed. This method can also be extended to other species that are currently map-poor but have a sufficiently high density of SINEs to allow IRS-PCR.
IRS-PCR markers; Canis familiaris; genotyping; polymorphism; radiation hybrid mapping; polymerase chain reaction; interspersed repetitive DNA sequence
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INTRODUCTION
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ANCIENT AND MULTIPLE ORIGINS of the domestic dog, Canis familiaris, have produced over 300 diverse breeds. The extent of genetic differences between and within breeds displays a useful resource for understanding basic and applied problems in mammalian biology. The wide range of morphological and behavioral variation which is due exclusively to genetic causes (8) makes the canine an attractive model for studying congenital disorders, behavioral diversity, sporadic malignancies, and pharmacogenetics (see http://ars-genome.cornell.edu/animal.html). In addition, the relationship that canines have with humans makes them share many common environmental exposures (e.g., water source, passive cigarette smoke, insecticides). Moreover, specific types of canine malignancies are similar to their human counterparts in histopathological appearance, biological behavior, and response to therapy, giving an enormous advantage to the canine model over many existing popular animal models (14). Thus an extensive study of the genetic basis of various canine disorders can provide valuable insight into similar disorders in humans (17, 30, 13). To accomplish the former, a detailed physical and genetic map of the canine genome is necessary. As a framework to these maps, more than 500 di-, tri-, and tetranucleotide markers (28, 29, 7), dog gene markers, and traced orthologous amplified sequence tag (TOAST) (32) markers have been identified to date.
Another class of markers that has been employed in human and mouse genetics is the interspersed repetitive DNA sequence-polymerase chain reaction (IRS-PCR)-based markers. IRS-PCR allows amplification of DNA from complex sources without prior sequence knowledge. For amplification to be successful, two repeats have to be in opposite orientation with respect to each other and within a distance that allows for a conventional PCR. The unique genomic sequence derived from between the two repeats can then be assessed for polymorphism content and subsequently probed by a hybridization-based strategy to distinguish among various alleles. This approach overcomes the limitation of ordinary PCR and has supported human and mouse genetic mapping by simplifying the analysis of somatic cell hybrids, YACs, cosmids, and phages (26 16). IRS-PCR using the Alu repeats has found wide application in human genetics. For example, in isolation of chromosome region-specific polymorphic markers (2), identification of chromosome-specific YACs (3), linkage mapping (40), and contig assembly (18, 11). Utilizing B1/B2 repeats (9), IRS-PCR technology has supported the construction of integrated physical and genetic maps of the murine genome (10). Mapping of a number of mouse genes (27) and high-resolution, filter-based genotyping (21) employ IRS-PCR.
To identify genes responsible for various traits and genetic diseases, a high-density genome map including various types (types I and II) of markers is necessary. Such a map will not only allow mapping loci through positional cloning but will also help in localizing new genes by synteny mapping. A detailed whole genome comparison of human and dog by chromosome painting and fluorescent in situ hybridization (FISH) has recently been published (1). The importance and utility of whole genome radiation hybrid (WGRH) mapping has already been well established in human (35), mouse (37), rat (38), and cat (24). In dog, a significant advancement in establishing linkage and radiation hybrid (RH) has been achieved (32, 15, 25, 39). More recently, construction of an integrated linkage-RH map of the canine genome has successfully increased the density of the canine genome map with 724 markers (22).
We have previously characterized canine-specific short interspersed nuclear elements (Can-SINE) which are
130150 bp and are present approximately every 58.3 kbp in the canine genome (6). As in other eukaryotes, canine SINEs are flanked by direct repeats and harbor (CT)n tracts followed by poly(A) tails (12). A number of Can-SINE markers were shown to be sufficiently close to allow IRS-PCR amplification and were also shown to be polymorphic (6). Two primers targeted to conserved sequences within the Can-SINE (K9S2, 5'-TGCATGGAGCCTGCTTCTCC-3'; and K9AS, 5'-GGAGAAGCAGGCTCCAT-3') when used individually allow one to amplify DNA sequences located between two interspersed repetitive elements oriented in opposite directions in the genome and separated by a distance that is within range of a conventional PCR. In this report, we have exploited the polymorphic nature of the canine IRS-PCR products and have identified and characterized 116 markers that can be used for genotyping in the canine, as well as related species. Seventy-three of these markers have also been mapped to the most recent version of the integrated linkage-RH map.
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MATERIALS AND METHODS
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Materials and general methods.
Restriction endonucleases, DNA modifying enzymes, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). [35S]deoxyadenosine 5'-(
-thio)triphosphate (1,0001,500 Ci/mmol), [
-32P]deoxyadenosine 5'-triphosphate (3,000 Ci/mmol), and [
-32P]ATP (3,000 Ci/mmol) were obtained from New England Nuclear. Preparation of high-molecular-mass genomic DNA, plasmid DNA, restriction enzyme digestions, agarose gel electrophoresis of DNA, DNA ligation, and bacterial transformations were carried out by standard methods (Ref. 33 and references therein). Subclones of PCR amplified products were sequenced by the chain termination method (34) utilizing double-stranded DNA templates.
Screening for microsatellite markers and PCR-SSCP analysis.
A small set of markers was generated from the canine SINEs, since these had been reported to be highly polymorphic (6). High-molecular-mass canine genomic DNA was prepared from blood and digested with Rsa I and Alu I. Digested DNA was fractionated on an 1.5% agarose gel, and fragments less than 300 bp were purified by glass milk (Qiagen, Mississauga, Ontario, Canada). The gel-purified DNA was then ligated into pBluescript KSII(+),which had been linearized with Sma I and treated with calf intestinal phosphatase (New England Biolabs). Following transformation into Escherichia coli DH10ß, colonies were replicated on Hybond N+ (Amersham) membranes and prepared for colony hybridization by a method described in Ref. 33. Filters were prehybridized in ExpressHyb (Clontech) for 30 min and hybridized with K9AS or K9S2 oligonucleotides (107 cpm/ml) in ExpressHyb at 37°C for 1 h. Washes were performed with 2x SSC/0.05% SDS at room temperature for 1 h with several changes and with 0.2x SSC/0.1% SDS at room temperature for 1 h with one change, after which the filters were exposed to X-OMAT film at -70°C overnight with intensifying screens. Plasmid DNA from positive clones were prepared from 1.5-ml overnight cultures and were sequenced with K9S2 and K9AS primers utilizing the dideoxy chain termination method (34).
Pairs of oligonucleotides (Table 1) were designed to flank the Can-SINEs and yielded products in the range of
200 bp. Prior to the PCR, one oligonucleotide was radiolabeled with T4 polynucleotide kinase and [
-32P]ATP (NEN). The PCR was performed using a DNA thermocycler with
50 ng of genomic DNA in a total volume of 20 µl. Thirty-five cycles of amplification were performed; each cycle consisted of 1 min at 94°C, 1 min at the optimally determined annealing temperature for each oligonucleotide pair (Table 1), and 1 min at 72°C. After PCRs, 2 µl of the reaction products were mixed with 8 µl of sample buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanole FF) and boiled for 5 min. Two microliters of this mixture was loaded on 8% polyacrylamide (50:1, acrylamide:bis-acrylamide) gels, and electrophoresis was performed in 1x TBE buffer [1x TBE is 90 mM Tris (pH 8.0), 90 mM boric acid, 2.5 mM EDTA] at 30 W in the cold room. The gel was transferred onto filter paper, dried, and exposed to X-OMAT-AR (Kodak) film at -70°C for 1224 h with an intensifying screen.
Generation of IRS-PCR markers.
PCRs were carried out in 50 µl reaction buffer [10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, and 200 µM dNTP (Pharmacia)] containing 0.5 µg of genomic DNA, 1 µM primer, and 2.5 U of Thermus aquaticus polymerase (GIBCO BRL). Thirty-five cycles consisting of 94°C denaturation (1 min), 58°C annealing (1 min), and 72°C extension (2 min), were performed in an automated thermal cycler (Perkin-Elmer). Primers used in the PCRs were either K9S2 (5'-TGCATGGAGCCTGCTTCTCC-3') or K9AS (5'-GGAGAAGCAGGCTCCAT-3') (6).
IRS-PCR products from DNA isolated from blood of a German shepherd were fractionated on a 1.0% agarose gel, and products within 200 to 500 bp were excised and purified by the glass milk method utilizing the manufacturers recommendations (Qiagen). PCR products were treated with T4 polynucleotide kinase, followed by ligation to pBluescript KSII+ (Stratagene, Aurora, Ontario) which had been linearized with Sma I and treated with calf intestine phosphatase. Following transformation into DH10ß and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) selection for recombinant plasmids, bacterial colonies were grown in 96-well dishes at 37°C overnight in Luria broth (LB) containing 100 µg/ml ampicillin. The following morning, 2 µl of the culture was spotted onto Hybond N+ nylon membranes (Amersham), and bacteria were grown by placing the membrane onto LB agar plates containing 100 µg/ml ampicillin. Filters were processed for colony hybridization as described before (33). Cultures in 96-well plates were stored in 10% glycerol at -70°C.
Polymorphism detection.
IRS-PCR products from a number of breeds were generated and digested with Alu I. These were resolved on an 8% nondenaturing single-stranded conformational polymorphism (SSCP) gel (50:1 acrylamide/ bis-acrylamide) with the electrophoresis being carried out at 4°C at 30 W for 3 h. Samples were electroblotted to Hybond N+ in 0.6x TBE at 40 V for 2 h at 4°C. The blot was prehybridized in Churchs buffer [0.5 M sodium phosphate (pH 7.0), 1 mM EDTA, 7% SDS, and 0.5% BSA] (5) for 2 h at 65°C. Hybridizations were performed in Churchs buffer with random primed 32P-labeled IRS-PCR clone probes (106 cpm/ml) at 65°C for 1214 h. Washes were performed in 2x SSC/0.1% SDS at 65°C for 30 min with a change after 15 min and in 0.2 x SSC/0.1% SDS at 65°C for 30 min with a change after 15 min. Blots were exposed to X-OMAT-AR (Kodak) film at -70°C overnight with intensifying screen. Each blot could be reused a minimum of 50 times after deprobing them according to the manufacturers protocol.
Data analysis.
For each IRS-PCR marker DNA from 15 purebred dogs (2 German shepherd, 2 Border collie, 2 Doberman pinscher, 2 Shih Tzu, and 1 each of beagle, miniature schnauzer, golden retriever, miniature poodle, cocker spaniel, pug, and Great Dane) were analyzed. For each SSCP-PCR marker, a panel consisting of 1520 purebred dogs (and in 5 instances, a panel of 30 unrelated mixed breed dogs) were analyzed.
Heterozygosity (HET) and polymorphism information content (PIC) values were calculated according to the formulas below
where pi = frequency of the ith allele, and 1...n = n alleles of a marker.
where, pi is the population frequency of the ith allele, and pj is the frequency of the jth allele (31).
Primer generation.
Each of the marker clones was sequenced with a T3 or T7 sequencing primer. The obtained sequence was searched by BLAST to reveal regions of repetitive sequences. Oligonucleotide primer pairs were designed to give products in the range of 200 bp using PRIMER3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi).
Radiation hybrid mapping.
Construction and characterization of the canine-rodent hybrid cell panel (RHDF5000) has been previously reported (36). PCRs were performed on 50 ng RH DNA in a final volume of 10 µl containing 25 µg of each primer, 200 µM dNTPs, 1.52 mM MgCl2, 50 mM KCl, 10 mM Tris-Cl, and 0.5 U of Taq Gold polymerase (Perkin-Elmer). Amplification was carried out in thermocyclers (DNA-Engine; MJ Research, Cambridge, MA) in 96-well format with the following cycles: initialization at 94°C for 10 min, followed by 20 cycles of denaturation at 94°C for 30 s, annealing at 5865°C for 30 s, and extension at 72°C for 30 s, followed by a final 10 cycles of 94°C for 30 s, 4855°C for 30 s, and 72°C for 30 s. PCR specificity of each primer pair was tested on a positive control consisting of canine DNA, a control of hamster DNA, a hamster-canine DNA mixture in the ratio of 75:25, and a water control to determine the appropriate amplification conditions. None of the markers used generated a product in the hamster DNA controls. In addition, all PCRs were performed in duplicate. PCR products were analyzed on 2% agarose gels in 0.5x TBE and were visualized under ultraviolet light. Images of the gels were recorded with a high-resolution CCD camera (Bioprint; Vilber Lourmat, Torcy, France). Results were scored as present, absent, or ambiguous in a semi-automated fashion on a UNIX workstation using data acquisition software developed by G. Brenterch and N. Soriano (University of Rennes, Rennes, France), and we utilized the same guidelines for RH mapping as described before (32).
The new set of RH data was merged with the latest version of the integrated map set consisting of 724 markers (22). Based on pairwise analysis, linkage groups containing new markers were identified. Each marker was linked to at least another one in the same group with a LOD score greater than 8. The best linkage value for each of the 73 novel markers was determined by 2-point analysis that had been performed with the "best-two points" function of the MultiMap package (20).
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RESULTS
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Assessment of Can-SINE informativeness.
Sequence comparison of a number of different SINEs demonstrated that canine SINEs are sufficiently conserved to allow IRS-PCR from canine DNA (6). However, it was unclear how polymorphic a SINE at a given locus would be among mixed breeds or among individuals of a given breed. To address this issue, we undertook the strategy outlined in Fig. 1A to clone and characterize markers based on SINEs. A short insert genomic DNA library was generated utilizing Rsa I or Alu I fragmented DNA. Approximately 500 colonies were picked and screened by hybridization utilizing K9S2 or K9AS oligonucleotides. Twenty positive clones were analyzed by sequencing with both the K9S2 and K9AS primers to determine the sequence of the unique region flanking the SINE repeat. Nine primer pairs were subsequently designed and used in PCRs with DNA from 1520 members of different purebreds. The products were analyzed for polymorphisms by SSCP gels. Three sets of primers based on the sequences of previously known genes, harboring Can-SINEs of the same family, were also included for analysis. These are CGMPI12 (emb-Y11309; C. familiaris gene encoding cGMP-gated channel
-subunit), COLIP (gb-M63427; dog pancreatic colipase gene), and TYRA (gb-L47165; C. familiaris tyrosine aminotransferase gene). For CGMPI, COLIP, and TYRA, 5 German shepherds, 4 Border collies, 9 Doberman pinschers, 3 golden retrievers, 1 cocker spaniel, and 30 mixed breed animals were analyzed. For other markers, 3 German shepherds, 2 Border collies, 3 Doberman pinschers, 2 Shih Tzu, 2 golden retrievers, and 1 each of miniature schnauzer, rottweiler, and miniature poodle were used for the analysis. Marker K9CH5 was also analyzed on 30 mixed breed samples. Nine markers showed detectable polymorphisms by SSCP analysis with HET and PIC values ranging from 0.380.76 and 0.340.73, respectively (Table 1). The majority of markers showed 35 alleles. In general, the mixed breed samples showed a greater number of alleles and had correspondingly higher HET/PIC values than the mixed population of purebreds. Marker CGMPI was homozygous in the five German shepherd samples (Fig. 2A, lanes 1822) and four Border collie samples tested (Fig. 2A, lanes 1417), but was polymorphic among three golden retrievers and nine Doberman pinschers (Fig. 2A, compare lanes 24 and lanes 513). Marker COLIP was homozygous in all Doberman pinscher samples tested (Fig. 2B, lanes 513) and demonstrated a two-allele system in Border collies (Fig. 2B, lanes 1417) and in German shepherds (Fig. 2B, lanes 1822). German shepherds and Border collies share a common allele (Fig. 2B, compare lanes 1422), and the second allele in Border collies is present in nine Doberman samples analyzed (Fig. 2B, compare lanes 513 and 1417). The marker TYRA was polymorphic between and within the breeds tested. Other markers were polymorphic between breeds, but sample size was too small to make any comment on their behavior within a given breed. To determine the molecular basis for the observed SSCP shifts, the PCR products obtained with CGMPI markers from a German shepherd (Fig. 2A, lane 21) and a Border collie (Fig. 2A, lane 16) were cloned. Five clones from each were sequenced to identify the allelic variation. Similarly for COLIP, PCR products from two German shepherds (Fig. 2B, lanes 20 and 21), a Border collie (Fig. 2B, lane 17), and a Doberman (Fig. 2B, lane 10) were cloned. Four clones each from the German shepherds and Border collies, as well as five clones from the Doberman PCR products, were sequenced. The sequence differences were found to be due to variations in length of the poly(A) and (CT)n tracks within the Can-SINE (M. Das, data not shown).

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Fig. 1. Schematic representation of strategies undertaken to assess the polymorphisms of Canis familiaris short interspersed nuclear element (Can-SINEs; A) and to generate interspersed repetitive DNA sequence (IRS)-PCR markers (B). SSCP, single-stranded conformational polymorphism.
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Fig. 2. SSCP analysis using forward and reverse CGMPI (A) and COLIP (B) primers. The breed of origin of each product is shown above blocks of lanes. Following PCR amplification, the products were analyzed on an SSCP gel as described in the MATERIALS AND METHODS. Arrows to the left indicate the position of migration of different alleles observed in the indicated breeds.
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Development of IRS-PCR product markers.
The results described above indicate that a given SINE at a particular locus can be a rich source of sequence variation. To extend these results, we undertook to produce a series of markers based on IRS-PCR, utilizing the strategy outlined in Fig. 1B. We produced two libraries of IRS-PCR markers, with each one generated utilizing a different SINE primer. Both libraries were produced from DNA isolated from a German shepherd. One primer (K9AS) includes most of the Can-SINE sequence in the final PCR product, whereas the other (K9S2) resides toward the end of the Can-SINE and extends out into the unique genomic region. A total of 1,056 colonies from the K9AS library and 5,568 colonies from the K9S2 library were picked and grown in 96-well plates. Colonies were also spotted onto filters for colony hybridization to eliminate duplicate clones. The usefulness of individual IRS-PCR clones for polymorphism detection was assessed by hybridizing the radiolabeled marker to Hybond N+ filters containing SSCP fractionated IRS-PCR products from individuals of different breeds (called "D-blots") (Fig. 1B).
Hybridization to the D-blots allowed for the direct determination of polymorphic content, whereas hybridization to the colony blots allowed us to eliminate duplicate clones from future screens. To further confirm the uniqueness of the clones, and that indeed they had arisen by IRS-PCR, a number of clones were end-sequenced. In this fashion, 193 and 204 unique markers from the K9S2 and K9AS library, respectively, were analyzed.
The hybridization pattern obtained with one marker, S1H7, on a D-blot is presented (Fig. 3). On this panel, there are two alleles for marker S1H7 (Fig. 3). Clearly, the IRS-PCR products are informative for polymorphism detection. We therefore determined the HET/PIC values of sixty-one K9S2 and fifty-five K9AS markers (Table 2). Thirty-four of the markers were also used to probe a blot of 1530 unrelated mixed breed dogs (Table 2), and 21 of the markers were tested on a panel of 8 purebred German shepherds, Border collies, and Doberman pinschers (Table 3). HET and PIC values are summarized in Table 2. Although a few of the markers, such as S7B9 and S1B3, revealed the existence of a larger number of alleles when tested on the mixed breed samples, in general we did not observe a higher number of alleles and/or HET/PIC values for the mixed breed samples tested compared with the values obtained from the mixed population of purebreds. Information for markers S3F1 and S7B5 was obtained only from the mixed breed panel. The PIC values of most of the K9AS markers are in the range of 0.30.7, and these have HET values between 0.4 and 0.7 (Fig. 4A). Similarly, the PIC and HET values of the K9S2 group of markers clustered around 0.30.6 and 0.40.7, respectively (Fig. 4B). The average values (both PIC and HET) are 0.6 for K9S2 and 0.55 for K9AS markers. Most of the markers that have lower HET/PIC values are biallelic systems (Table 2) though the average value for such system is between 0.3 and 0.4. In general, a direct relation between the number of alleles and the HET/PIC values has been observed in all the markers (Fig. 5). When tested on a small panel of purebred dogs, nine of the markers did not show any polymorphisms (single allele only) in the three breeds tested, and three markers were informative in all three breeds tested (sample size of eight dogs from each breed) (Table 3).

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Fig. 3. D-blot analysis of clone S1H7 against total IRS-PCR product fractionated on an SSCP gel and transferred to Hybond N+. The breed of origin of each product is shown above each lane, and numbers indicates different animals from each breed. The various alleles detected by each marker are indicated on the left of the photograph.
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Fig. 4. Distribution of markers with respect to their heterozygosity (HET, y-axis) and polymorphism information content (PIC) values (x-axis) from the K9AS (A) and K9S2 (B) libraries.
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Radiation hybrid mapping.
To map some of the markers, 116 of these were sequenced and PCR primers were generated to the unique genomic region contained within the SINE pair. Of these, 73 markers gave specific PCR products when used in typing experiments on a WGRH panel consisting of 126 RH cell lines. The sequences of these primer pairs are given in Table 4, and the markers they generate were mapped relative to the existing comprehensive canine RH map (22). Fifty-nine (80%) markers were linked to at least one other marker of the RH map with a significant statistical support (LOD score > 8). The best linkage value for each of the 73 new markers was then determined against the markers of the RH map (Table 5). These values indicate the relative position within the group for each of the 59 linked markers. The RH mapping demonstrates that the markers described in this work are spread throughout the genome. The 59 linked markers map on most of the canine chromosomes (CFA1, 2, 3, 5, 6, 9, 12, 16, 19, 20, 22, 26, 2935, and 30), as well as to different RH and synteny groups for which chromosome numbers have not yet been assigned. Finally, two of them form an individual new RH group not yet linked to any of the existing linkage groups. Fourteen (20%) of the markers remain unlinked (Table 5).
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DISCUSSION
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The usefulness of IRS-PCR in the physical and genetic mapping of the canine genome depends on the ability of PCR primers to amplify the region between two repeat elements. IRS-PCR based on canine SINE repeats has the potential to generate a new class of markers amenable to filter-based genotyping in the dog (6). Allelic variation between inter-Alu PCR products arises as a consequence of 1) length variability, 2) sequence variability in the intervening sequence, or 3) the presence or absence of a specific IRS product (for example, among inbred mouse strains).
The abundant distribution and polymorphisms of the Can-SINEs indicate that they present an efficient, rapid, and useful way of developing markers for genome mapping (6). We have developed, characterized, and mapped to the existing RH panel a set of canine SINE-based markers that are amenable to high-throughput filter-based genotyping. We also evaluated a set of markers to be used in SSCP analysis for conventional PCR-based genotyping. PCR amplification utilizing oligonucleotides flanking the repeat sequence, followed by SSCP gel analysis demonstrated that 70% of the markers tested were polymorphic, with an average HET and PIC value of 0.58 and 0.52, respectively (Table 1). The results obtained with the markers derived from the CGMPI and COLIP genes indicate that some breeds may have skewed allelic distribution of some variants, suggesting that this type of marker could be developed to yield good breed-specific markers (Fig. 2). The highly polymorphic nature of these markers indicates that they could be used within the same breed, one immediate application of which is paternity testing. Allele detection, based at present on SSCP, is a rate-limiting step for some applications but should not be a permanent barrier.
Among the previously developed canine markers, the majority are microsatellite markers (28, 7, 32). The average PIC values of canine dinucleotide (CA)n repeat markers are 0.53 (28), whereas many of the tetranucleotide (GAAA)n markers have PIC values greater than 0.75 (7). The informativeness of the IRS-PCR markers indicates that these should also be useful for mapping studies since the average PIC value of these markers is
0.6, with a significant number of markers (34%) showing values greater than 0.7 (Table 2). We have analyzed our markers on more than one representative from each group of dogs (hounds, nonsporting, sporting, herding, working, and toy) to widen the window of different alleles detected. It is likely that the PIC/HET values of our markers are underestimated given that a number of parameters influence the ability to detect a mobility shift by SSCP analysis. These include the surrounding sequence context of the polymorphism, the electrophoresis conditions, the presence of glycerol in the gel, and the size of the DNA fragment under study (19). Thus most of the markers presented herein should be useful in further genetic studies.
The general utility of these IRS-PCR markers is based on the following: 1) that it is relatively rapid and easy to develop a large number of markers and 2) canine chromosome-specific markers can be developed easily from mixed DNA sources (e.g., BAC clone DNA, somatic cell hybrid DNA) or from very small source quantities like single chromosomes or microdissected chromosomes. The Can-SINE is very specific for the canid family and closely related species (6) and therefore can be used as anchor points when amplifying from somatic cell hybrids harboring canine chromosomal fragments. 3) There is also the potential to utilize these markers in a non-gel-based readout assay. We are currently evaluating the nucleotide basis for the variation observed within our IRS-PCR markers. Clearly, a contribution to variation among the K9AS marker set will be length variation within the (CT)n and poly(A) tract of the SINE, since this primer directs DNA synthesis across this region of the repeat element (6). Of greater interest to us are sequence-variations within the unique genomic region flanked by the two SINEs, since the advantage of using IRS-PCR-based markers is that once the underlying basis for the polymorphism has been identified, one can spot the total IRS-PCR product and use hybridization of allele-specific oligonucleotides (ASO) to determine HET of the subject at the particular locus being sampled. For these purposes, even markers demonstrating a two-allele system are useful. 4) Moreover, this method can also be extended to other species that are currently map-poor but have a sufficiently high density of SINEs to enable IRS-PCR.
Our markers should also be useful in the area of conservation biology. Our previous results (6), and those of others (5, 23) indicate that members of the Can-SINE family are also present in wolf, jackal, and the more distantly related fox. Thus some of the markers we have identified in the domestic dog should effectively amplify sequences from related species, such as wolves, jackals, coyotes, and wild dogs. It may also be possible to develop IRS-PCR markers unique to a given species. The presence or absence of a specific IRS product among inbred mouse strains is a powerful mapping tool in mouse genetics (10). It is relatively easy to determine whether a given marker is conserved (or not) among species, since this requires a simple hybridization to be performed on total IRS-PCR products from the species of interest.
In this report our markers have been linked to a recent integrated linkage-RH map of the canine genome (22). The positioning of the IRS-PCR markers on this framework will help to improve the resolution of the map and usefulness of the markers. Second, one needs a much denser physical map, which ideally will bridge the gap between the linkage map and the genomic sequence. As well, a canine BAC library has been developed (see http://www.chori.org/bacpac/), and the integration of this resource with the canine RH and linkage maps will be another step which could be facilitated by the analysis of markers such as the IRS-PCR canine SINE-based markers.
The IRS-PCR markers could not be mapped onto the RH panel by an hybridization approach because the IRS-PCR products are from sources of different complexity. The markers were developed by IRS-PCR amplification of total genomic DNA, whereas the products from the individual RH panels were from a hamster-canine background containing a small fraction of canine genomic DNA. Hence, the mapping was done by the classic PCR method. Although we have reported the development of only a modest number of markers, the resource for rapidly developing and mapping more such informative markers is readily available to any small laboratory. As indicated above, the present set of markers was generated from a size fraction cut of the IRS-PCR lying between 200500 bp. We believe that the IRS-PCR library from the 500- to 1,000-bp size range would also yield a good number of markers. Development of this series of higher size range markers would also add another dimension of multiplex analysis to this already high-throughput system. It is also fairly easy and valuable to incorporate the IRS-PCR markers into the already existing maps to increase the density further and to make them available to the other researchers. The markers we characterized also appear to be well distributed (Table 5). Since the D-blots can be used multiple times (at least 50), simultaneously hybridizing with a number of markers having different sizes would accelerate the process severalfold. It should also be mentioned that multiplexing and reusability of the primary blot significantly reduces the amount of valuable primary sample. One PCR (50 µl) from one sample is required and sufficient for the generation of 10 blots. Thus these IRS-PCR markers can be used easily in laboratories not equipped for rapid high-throughput conventional genotyping.
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ACKNOWLEDGMENTS
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We thank Dr. Francis Galibert, Christophe Hitte, Catherine Andre, and Corinne Rennier from the University of Rennes (France) for the RH panel, for constant help and encouragement throughout this work, and for comments on the manuscript.
J. Pelletier is an Medical Research Council of Canada scientist. This work has been supported by a grant from the Medical Research Council of Canada (to J. Pelletier) and by National Eye Institute Grant EY-06855 (to G. M. Acland).
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: J. Pelletier, McIntyre Medical Science Bldg., McGill Univ., Rm 902, 3655 Drummond, Montreal, Quebec, Canada H3G 1Y6 (E-mail: jerry{at}med.mcgill.ca).
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REFERENCES
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