Gilles Vergnaud

P D Thomsen

B Hùyheim

V Amarger

J Masabanda

D Ewald

J Buitkamp

K Potter

R Fries
email: fries@tz.agrar.tu-muenchen.de

V Obreque

L Coogle

P J Henney

E Bailey

R Mancilla

Garcõ Âa-Huidobro

P Hinrichsen

G Cothran

A polymorphic porcine dinucleotide repeat S0559 (BHT0107) at chromosome 6q28±q31

Keywords: The microsatellites VOLP-03, -05, -08

A polymorphic porcine dinucleotide repeat S0559

(BHT0107) at chromosome 6q28-q31.

24-mer probe [poly(dA-dC)(dG-dT), Pharmacia Biotech, Uppsala, Sweden], digoxigenin labeled using the DIG oligonucleotide 39-end labeling kit and revealed using the Genius System (Boehringer Mannheim, Mannheim, Germany). The clones giving a clear positive signal after a second hybridization were sequenced on an ABI 377A sequencer, using the M13/pUC sequencing primers (NE Biolabs, Beverly, MA, USA) and the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit. PCR primers were designed using the Oligo 5 . 0 program. PCR conditions: Reactions were made in 25 ml volumes on buffer #6 (Opti-primer PCR optimization kit, Stratagene, La Jolla, CA, USA) containing 75 mm KCl, 10 mm Tris±HCl (pH 8 . 8), 1 . 5 mm MgCl 2 , plus 0 . 2 mm each dNTP's, 0 . 2 mm each primer (one of them labeled with a fluorochrome), 0 . 5 U Taq polymerase (Boehringer Mannheim) and 50 ng template DNA. After an initial denaturation step at 95 °C for 4 min, reactions were run 30 cycles in a PTC-100 thermocycler (MJ Research, Watertown, MA, USA), each cycle including a denaturation step at 94 °C for 1 min, an annealing step at 55 °C for 1 min and an extension step at 72 °C for 1 min. Primers are described in Table 1.

Polymorphism: A group of 36 alpacas were analyzed using the ABI GeneScan software. Information regarding the locus names, repeat characteristics and allele number and size range, as well as the expected heterozygosity and PIC values, are shown in Table 2.

Chromosomal location: Not known. Source/description: Secretory carrier membrane proteins (SCAMPs) are components of secretory vesicle membranes that function as recycling carriers in the secretory pathway. SCAMPs are widely distributed and present in almost all mammalian tissues and cell types and play a fundamental role in the general cell surface recycling. We have isolated a cDNA coding for the SCAMP1 mRNA and determined the genomic structure of the porcine SCAMP1 gene by isolation and sequencing of seven recombinant phage clones. Within intron 4 (phage l7a) and intron 7 (phage l35) of the SCAMP1 gene an (AT) 10 and (GT) 23 dinucleotide repeat was identified, respectively. The DNA sequences of the SCAMP1 gene were submitted to the EMBL data library under accession numbers AJ223737, AJ223738, AJ223740, and Y15710.

Primer sequences:

Forward primer (intron 4): 4F 59-CAG AAC TGA GGC TAA AGT AC-39

Reverse primer (intron 4): 4R 59-CAG AGT TGT AGG ACT GTA GAG-39

Forward primer (intron 7): 7F 59-CAG CTC CAC TTC GAC TCC T-39

Reverse primer (intron 7): 7R 59-TTG AAG AGT GGA GTC ACT GC-39 PCR conditions: PCR primers were designed from the DNA sequences of the flanking regions. PCR reactions were optimized using the `Ready to Go' system (Pharmacia, Freiburg, Germany). A master mix was prepared containing 400 mm of each primer (the reverse primer was labelled with IRD800 fluorescence dye; MWG Biotech, Ebersberg, Germany), frozen and stored at ±20 °C. DNA (100 ng) in 1 ml TE was mixed with 12 ml master mix and one PCR bead was added (10 mm Tris±HCl pH 9 . 0, 50 mm KCl, 1 . 5 mm MgCl 2 , 200 mm of each dNTPs, and 1 . 5 U Taq polymerase). Cycling included an initial denaturation step at 94 °C for 10 min, followed by 30 cycles of 1 min at 94 °C, 30 s at 55 °C, 1 min 72 °C for and a final step of 10 min at 72 °C using a Hybaid Omnigene (Ashford, UK) cycler. PCR products were analyzed by electrophoresis on 8% denaturing polyacrylamide gels using a LI-COR Gene ReadIR 4200 automated sequencer. Analysis of the bands was performed using the RFLPscan Plus 3 . 0 (Scanalytics, Virginia, VA, USA) software.

Chromosomal localization: The porcine SCAMP1 gene has been assigned to chromosome 2q21±q22 by FISH analysis and PCR screening of a porcine-rodent somatic hybrid cell panel 1 .

Mendelian inheritance: Mendelian inheritance was followed in 48 individuals from five families of Angler Saddlebacks (Fig. 1).

Polymorphism: Polymorphisms were studied using DNA samples of 50 Angler Saddleback, five German Large White, five German Landrace, five Pie Âtrain, and six Bunte Bentheimer pigs. Among these 71 pigs, nine different alleles of the microsatellite in intron 4 were detected. The lengths of the corresponding PCR products were 193, 195, 197, 199, 201, 203, 205, 207, and Synteny mapping: The chromosomal locations of the new markers were found by PCR typing of a mouse/cattle hybrid cell panel 1 . The three microsatellites scored statistically significant syntenies between themselves and with other markers on chromosome 17.

Mendelian inheritance and linkage: Codominant segregation was observed in the International Bovine Reference Families 2 . The three microsatellites are polymorphic, showing different levels of polymorphism. Allele sizes and the number of alleles for each marker are listed in Table 1, as they were found in the typed families. Allele frequencies were calculated from the 38 parental genotypes only, assuming no close relationships between the animals. As expected, the three markers are tightly linked and map within the pericentromeric region of chromosome 17. 207, 205, 201, 199, 197, 195, 193, 191, 189, 185, 183, and 181 bp. Heterozygosity, PIC values, and allele frequencies are shown in Table 1.

Mendelian inheritance: Codominant segregation of the polymorphisms was observed in 48 individuals from five families of Angler Saddlebacks (Fig. 1).

Chromosomal localization:

The porcine V-ATPase gene was assigned to chromosome 4q15±q16 by PCR screening of a porcine rodent somatic cell hybrid panel 1 .

Acknowledgements: The authors are grateful to A. Deppe for expert technical assistance. Source/description: A genomic plasmid library was constructed in the plasmid vector pCR-Script (Stratagene, La Jolla, CA) from sizeselected (300±800 bp) rainbow trout genomic DNA digested with PvuII and HaeIII. The library was screened with a (TG) 9 probe. Positive clones were sequenced using an automatic DNA sequencer (ALF express DNA Sequencer, Pharmacia, Uppsala, Sweden). The clone Omy0002DIAS was shown to contain a (TG) 28 repeat (GenBank accession number AF039065). PCR primers were designed on the basis of the flanking sequences. The sizes of the PCR fragments were analyzed on the ALF express DNA Sequencer.

Primer sequences: 59-AGGCTAACATCGAACTATCAAT-39 59-CTGTGGTCTGGTTCTGTTCA-39 PCR conditions: Approximately 40 ng genomic DNA was amplified in a total volume of 25 ml. The reaction contained 5 pmol of each primer, 0 . 1 mm dNTP, 0 . 4 U Taq polymerase (Pharmacia), 2 . 5 mm MgCl 2 , 50 mm KCl, and 10 mm Tris±HCl. Amplification was carried out using a Touchdown Temperature Cycler (Hybaid, Teddington, Middlesex, UK). Reaction conditions were an initial denaturation step at 95 °C for 2 min followed by 30 cycles of 45 s at 94 °C, 30 s at 57 °C, and 40 s at 74 °C, and with a final extension step at 74 °C for 5 min.

Mendelian inheritance: Mendelian inheritance was confirmed by typing two halfsib families.

Polymorphism: Twenty alleles in the size range 174±254 bp were detected in samples from four hatcheries, as can be seen in Table 1. The allele sizes were determined by comparison to the PCR product from the sequenced clone and by utilization of internal size markers together with each sample. Sequence of PCR primers and PCR conditions: Primers were designed from the reported sequence (GenBank accession number U50871) of human PSEN2. ZOO±PCR was performed on bovine genomic DNA and the PCR product was sequenced to verify that the corresponding bovine locus had been amplified. PCR primers were Description of the probe and details of FISH: The same primers were used to screen bovine YAC library 1 in order to isolate PSEN2 containing YAC clones which were used for FISH experiments as described 2 .

Chromosomal location: Two YAC clones were identified as containing the PSEN2 bovine gene and one of these clones was used for FISH analysis. This YAC produced a strong signal on BTA16q16 and a secondary, faint and non-frequent signal on BTA9 (Fig. 1). To confirm the FISH result, somatic cell hybrid analysis 3 was performed by PCR and the gene was assigned to BTA16 with a correlation coefficient of 0 . 81 4 . PSEN2 has been previously mapped to human chromosome 1q31-q42. This localization agrees with the updated comparative mapping data between man and cattle that show a correspondence between bovine chromosome 16 and two different regions of human chromosome 1, p36-ter and q23±42. More information on the gene order of both chromosomes is necessary to give a precise correspondence of chromosome segments between BTA16 and HSA1.

Correspondence: C Elduque PCR conditions: Amplifications were carried out as previously described 1 . Thermocycling was performed using the following touch-down program 2 : initial denaturation for 3 min at 95 °C, two cycles each of 30 s at 95 °C, 30 s at 65 °C to 55 °C and 30 s at 72 °C, followed by 14 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C and 30 min final extension at 72 °C. Sizes of the alleles were determined on 8% denaturing polyacrylamide gels using a LI-COR DNA sequencer model 4000 L (LI-COR, Lincoln, NE).

Polymorphism: The sizes and frequencies of alleles observed in a panel of 52 dogs, representing 52 different breeds, are given in Table 1.

PIC: The calculated heterozygosity and PIC 3 were 0 . 95 and 0 . 95, respectively.

Chromosomal location: ZuBeCa6 was localized by FISH 4 to ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477 chromosome 5q12±q13 (Fig. 1) according to the partial canine standard karyotype 5 .

Mendelian inheritance: Codominant inheritance was observed in five Beagle families with seven, seven, three, 13 and nine offspring, respectively.

Other comments: ZuBeCa6 is difficult to score due to the large number and wide range of allele sizes. ZuBeCa6 amplifies in wolf and fox.

Correspondence: G Dolf Source description: A mink cosmid library was constructed from a Royal Pastel male. The cosmid clone SH248 was isolated after screening with a g 32 PdATP-labelled (GT) 9 oligonucleotide. Sequencing of the microsatellite repeat was performed after subcloning of a Sau3A digested cosmid in the BamH1 site of the pUC19 vector. The marker was named Mvi248.

Chromosomal localisation: The cosmid SH248 was localised to chromosome 10p-terminal by fluorescent in situ hybridisation.

PCR conditions: Approximately 40 ng of DNA was amplified in a total volume of 25 ml containing 15 pmol of each primer, 0 . 2 mm dNTP, 0 . 4 U Taq polymerase (Pharmacia) and 2 . 5 ml 10´PCR buffer (100 mm Tris±HCl, pH 9 . 0, 15 mm MgCl 2 , 500 mm KCl, 0 . 1% v/w gelatine and 1% Triton X-100). Amplification was carried out in a OmniGene Temperature cycler (Hybaid). PCR cycling consisted of a denaturing step of 3 min at 93 °C, followed by 30 cycles of 52 °C for 20 s, 72 °C 10 s followed by 30 s at 93 °C, a final cycle at 72 °C for 20 min was performed.

Mendelian inheritance: Autosomal codominant segregation was demonstrated in a Danish fullsib mink pedigree.

Polymorphism: Studies for variation was performed in five populations of unrelated farm mink; two lines of Scanblack, two lines of Royal Pastel and one line of Standard mink `Wild' (Table 1). The size of the alleles were scored using a 50±500 bp ladder (Pharmacia) together with the amplified isolated clone as an external size marker.

Acknowledgements Probe description: A partial porcine orosomucoid (a 1 -acid glycoprotein) cDNA 1 (EMBL/GenBank accession number M35990) in the recombinant plasmid pAGP-3 was excised using the restriction enzyme PstI. The cDNA of 668 bp, after gel purification, was radiolabelled with [a 32 ]-dCTP and used to probe Southern blots.

Hybridization conditions: Hybridization conditions were as follows: 5´SSC, 0 . 5% SDS, 5% dextran sulphate, 5% PEG, 1% blocking reagent (Boehringer Mannheim, Milan, Italy); temperature 65 °C.

First wash: 2´SSC, 0 . 1% SDS for 15 min at room temperature.

Second wash: 0 . 2´SSC, 0 . 1% SDS for 10 min at 65 °C. The allelic fragments revealed were »2 . 6 kb (allele A) and 1 . 3 kb (allele B) (Fig. 1). Additional polymorphisms were also detected with HincII and PstI as previously reported 2,3 .

Mendelian inheritance and allele frequency: The inheritance of the PvuII alleles was confirmed in the Dutch PiGMaP reference pedigree 4 . Two-point linkage analysis was performed with CRI-MAP version 2 . 4 5 using the PvuII RFLP and the ResPig database of the PiGMaP Consortium 4 . The results showed that PvuII RFLP was linked to PstI RFLP and HincII RFLP already described at the ORM locus 2,3 (u = 0 . 00, LOD = 3 . 31 and u = 0 . 00, LOD = 3 . 91, respectively). Allele frequencies were obtained in a sample of 45 unrelated pigs belonging to six different breeds (Large White no. 18: A = 0 . 08, B = 0 . 92; Pie Âtrain no. 6: A = 0 . 17, B = 0 . 83; Duroc no. 5: A = 0 . 10, B = 0 . 90; Belgian Landrace no. 4: A = 0 . 25, B = 0 . 75; Landrace no. 4: A = 0 . 12, B = 0 . 88; Meishan no. 4: A = 1 . 00, B = 0 . 00). Source/description: The highly polymorphic mtDNA Control Region is a common molecular genetic marker. In several bird species, a nuclear pseudogene homologous to the Control Region exists 1 which makes the specific amplification of the latter difficult. The primers GluL and H401 amplify the Control Region from mtDNA enriched isolates of Eider duck (from liver 2 ; accession number AJ005266). From genomic DNA preparations of the same individual (from erythrocytes), however, the same primers amplify the nuclear pseudogene (accession number AJ005265). Novel forward primers were designed within the `hairpin' 3 of Eider duck mtDNA and pseudogene sequences: From total DNA preparations, Lmtpin selectively amplifies the mtDNA Control Region, while Lncpin amplifies the homologous nuclear pseudogene. PCR and sequencing conditions: Genomic DNA (100±1000 ng) was used as a template. PCR was performed in a total volume of 75 ml, containing 10 mm Tris±HCl, pH 9 . 0, 50 mm KCl, 1 . 5 mm MgCl 2 , 0 . 05 mm of each dNTP, 0 . 2 mm of the reverse and one of the forward primers, and 1 . 5 U Taq polymerase (Appligene, Illkirch, France).

The reaction profile was as follows: one cycle 94 °C 5 min, 88 °C 1 min (Taq was added at this step); 40 cycles 94 °C 1 min 30 s, 55 °C (with Lmtpin, Lncpin) or 48 . 5 °C (with GluL) 1 min 15 s, 72 °C 1 min 30 s; one cycle 72 °C 2 min 30 s in a GeneAmp 2400 thermocycler (Perkin Elmer, Norwalk, CT). Reaction products were purified with the PCR Purification Kit (Qiagen, Hilden, Germany). Both strands were sequenced with the Thermo-Sequenase Dye Terminator Cycle Sequencing Kit (Amersham, Buckinghamshire, UK) and analysed on an ABI 373 automatic sequencer (Applied Biosystems, Foster City, CA).

Polymorphism: The nuclear pseudogene was monomorphic in 10 Eider Ducks of different origin (accession number AJ005265); 354 bp scored sequence of the mitochondrial Control Region of nine Eider specimens from three Baltic locations contained six polymorphic positions (all transitions), defining seven different haplotypes (accession number AJ005266±AJ005272) with an average pairwise nucleotide divergence of 0 . 73%. Haplotypic diversity 4 was 0 . 94.

Comments: The amplified part of the mitochondrial Control Region is a highly polymorphic maternal marker for the Eider duck. The described primers Lmtpin and H401 were also tested for the two main domestic duck species. With an annealing temperature of 53 °C, they successfully amplify the homologous mtDNA region in Muscovy duck (Cairina moschata; cf. accession number L16769) and a shorter fragment of the Control Region in Peking duck (Anas platyrhynchos; cf. accession number L16770) 5 . The Control Regions of Muscovy duck and Peking duck differ from that of the Eider duck at 32 . 3% and 27 . 7% of 300 compared nucleotides, respectively. The difference between the Control Region and its nuclear homologous pseudogene within the Eider duck was only 19 . 7%, suggesting a relatively recent origin of the pseudogene. Therefore, the identity of PCR amplified products (mtDNA or nuclear pseudogene) should be confirmed, when applying our primers to other Anatidae. Source description: A mink cosmid library was constructed from a Royal Pastel male. The cosmid clones SH389 and SH355 were isolated after screening with a g 32 PdATP-labelled (GT) 9 oligonucleotide. Sequencing of the microsatellite repeat was performed after subcloning of Sau3A digested cosmid in the BamH1 site of pUC19 vector. The markers were named Mvi389 and Mvi355, respectively.

Chromosomal localisation: The cosmids SH389 and SH355 were localised by fluorescent in situ hybridisation to chromosome 8q1 . 2 and 12q1 . 2, respectively. PCR conditions: Approximately 40 ng of DNA was amplified in a total volume of 25 ml containing 15 pmol of each primer, 0 . 2 mm ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477 dNTP, 0 . 4 U Taq polymerase (Pharmacia) and 2 . 5 ml 10´PCR buffer (100 mm Tris±HCl, pH 9 . 0, 15 mm MgCl 2 , 500 mm KCl, 0 . 1% v/w gelatine and 1% Triton X-100). Amplification was carried out in a OmniGene Temperature cycler (Hybaid). PCR cycling consisted of a denaturing step of 3 min at 93 °C, followed by 30 cycles of 52 °C for 20 s, 72 °C 10 s followed by 30 s at 93 °C, a final cycle at 72 °C for 20 min.

Mendelian inheritance: Autosomal codominant segregation was demonstrated in a Danish fullsib mink pedigree.

Polymorphism: Studies for variation were performed in five populations of unrelated farm mink; two lines of Scanblack, two lines of Royal Pastel and one line of Standard mink `Wild' (Table 1). The size of the alleles were scored using a 50±500 bp ladder (Pharmacia) together with the amplified isolated clone as an external size marker. Source/description: Equine genomic DNA was digested with Sau3AI; DNA fragments ranging from 300 to 800 bp in length were ligated into the BamHI site of predigested Lambda Zap Express vector (Stratagene). The ligation reaction was packaged into phage particles with the Gigapack II Gold packing extract. Phage clones containing GT:AC dinucleotide repeats were identified by screening with a 32 P-labeled (GT) 10 probe. Inserts from positive phage were sequenced directly on an ABI-377 sequencer. Sequenced clones contained from 11 to 22 copies of the dinucleotide repeat. Primer pairs for PCR amplification of each microsatellite from genomic DNA were identified using the Prime program contained within the GCG Software Package (University of Wisconsin, WI).

PCR conditions: PCR reactions contained 12 . 5 ng DNA, supplied buffer (Boehringer), 1 . 5 mm MgCl 2 , 0 . 5 mm each primer, 40 mm dCTP, dGTP, dTTP, 10 mm dATP, 12 . 5 nCi [a-32 P]dATP and 0 . 5 U Taq polymerase in a final volume of 10 ml. PCR amplification was MOLECULAR GENETIC MARKERS 469 performed in 96-well plates for 30 cycles with initial denaturation at 94 °C for 30 s and the following temperature cycles were carried out: annealing at 51±56 °C for 30 s, extension at 72 °C for 30 s. The amplification was carried out using a PTC 100 thermal cycler (MJ Instruments). Electrophoresis was performed on BioRad Sequi-GenÒ 38 ´50 cm plate sequencing gel units.

Polymorphism: DNA from the 13 reference family stallions of diverse breeds from the International Equine Genome Mapping Workshop was amplified and genotypes were determined by autoradiography. In this sample population, the newly identified equine microsatellites exhibited 2±7 alleles with 1±12 of the stallions showing heterozygosity (Table 1).

Chromosomal locations: Unknown. Source/description: Equine genomic DNA was digested with Sau3AI and size selected fragments (300±600 bp) were ligated into the BamHI site of the BluescriptSK + plasmid. The library was screened with a synthetic (GT) 10 oligonucleotide end-labeled with g 32 P ATP. Positive clones were picked and rescreened and the double positives were sequenced with dye terminator chemistry on an ABI 310 sequencer. The sequences have been submitted to the GenBank database (accession numbers are listed in Table 1). Primers for the PCR were designed to amplify the regions containing the dinucleotide repeats (Table 1).

PCR conditions: The forward primers were end-labeled with g 33 P ATP. PCR amplifications were performed on a GeneAmp PCR System 9600 (Applied Biosystems, CA) thermal cycler in 10 ml reaction mixtures containing: 20±40 ng of genomic template DNA, 2 pmol of each primer, 50 mm KCl, 1 . 5 mm MgCl 2 , 10 mm Tris±HCl, 0 . 2 mm dNTP and 0 . 5 U AmpliTaq. Thermocycling parameters after denaturation at 94 °C for 5 min were 28 cycles of: 95 °C for 1 min, 60 °C for 30 s and 72 °C for 1 min followed by 10 min at 72 °C. The PCR products were run on a denaturing 6% polyacrylamide gel (7 . 5 m urea) and visualized by exposure of the gel to X-ray film.

Polymorphism: Amplification products were tested for polymorphism in a panel of 30±34 unrelated animals representing Thoroughbred, Standardbred, Norwegian Trotter and Norwegian Fjord. Alleles were named according to their sizes as determined by comparison to an M13 sequence ladder. Estimated heterozygosity was calculated using the observed allele frequencies and assuming no null alleles (Table 2). Allele sizes for the first four horses of the 1994 ISAG/ISBC standardization test at each of the eight microsatellite loci are shown in Table 3. Correspondence: Dr Knut Rùed (E-mail: Knut.Roed@vetinst.no) ) shown previously to be homologous to a-fibrinogen sequences from humans, rhesus monkey, dog, rat, mouse and Syrian hamster 2 . The putative porcine b-fibrinogen sequence was 83 . 7% and 81 . 3% identical to sequences from exons 1 and 2 of the human b-fibrinogen gene, respectively (EMBL: J00130 and J00131). The putative porcine g-fibrinogen sequence matched g-fibrinogen sequences from humans (EMBL: X51473, 82% identity; M10014, 71% identity), European polecat (EMBL: U28494, 77% identity), cattle (EMBL: X15556, 81% identity), and rat (EMBL: J00734, 70% identity). We conclude that the cDNA clones 015±3B, 020±6FL and 019±2D represent the porcine homologues of a-fibrinogen (FGA), bfibrinogen (FGB) and g-fibrinogen (FGG), respectively.

Methods: Restriction enzyme digests, agarose gel electrophoresis, Southern blotting, hybridizations and autoradiography were performed as described previously 3 .

Polymorphisms: DNA samples from eight pigs (two of each sex from both the Chinese Meishan and British Large White breeds) were screened for polymorphisms with a panel of 10 restriction endonucleases (BamHI, EcoRI, EcoRV, HindIII, KpnI, MspI, PstI, Sac I, TaqI, and XbaI) in combination with the fibrinogen cDNA probes. These eight pigs represent the grandparents of the Scottish PiGMaP reference families 4 . Polymorphic fragments were found with the following combinations of restriction enzymes and probes ± afibrinogen (BamHI, KpnI, PstI, SacI, TaqI, XbaI); b-fibrinogen (EcoRI, MspI, TaqI, PstI); and g-fibrinogen (BamHI, HindIII, KpnI, MspI, PstI, SacI and TaqI). The a-fibrinogen probe revealed three allelic fragments with XbaI: 7 . 2 kb, 3 . 15 kb and 2 . 95 kb (Fig. 1). The 8 . 8 kb, 6 . 5 kb and 5 . 6 kb fragments revealed with the b-fibrinogen probe in EcoRI digested DNA (Fig. 2) could be attributed to three allelic combinations: 8 . 8 kb + 6 . 5 kb (B1); 6 . 5 kb + 5 . 6 kb (B2) and 6 . 5 kb (B3). Two allelic fragments of 3 . 0 kb and 2 . 65 kb were found in TaqI digests hybridized with the g-fibrinogen probe as well as three constant fragments of 1 . 75 kb, 1 . 28 kb and 1 . 05 kb (Fig. 3). The a-fibrinogen (XbaI), b-fibrinogen (EcoRI) and g-fibrinogen (TaqI) RFLPs were shown to under simple autosomal monogenic control by segregation analysis of up to seven three-generation families with a total of 73 F 2 offspring. Interpretation of the inheritance patterns for the b-fibrinogen (EcoRI) RFLP was difficult as the 6 . 5 kb fragment behaved not only as an allele on its own but also as a component of the other two alleles.

Linkage analysis and mapping: No recombinant haplotypes were observed amongst 40 F 2 offspring from intercross matings between F 1 individuals heterozygous at all three RFLP loci described here. Thus, the porcine FGA, FGB and FGG are tightly linked. These fibrinogen loci have been mapped previously to chromosome 8 by linkage analysis 4 . The tight linkage observed amongst the porcine fibrinogen loci had been expected as the human FGA, FGB and FGG loci are found together in a 50-kb region on chromosome 4q23±q32 5 . The synteny of FGA, FGB, FGG, ALB, IL2, PDGFRA and SPP1 is conserved in humans and pigs 4 and provides further support that these RFLPs map to the porcine homologues of FGA, FGB and FGG.

1 Buchman T.G. et al. (1990) Surgery 108, 559±66. A perfect (AC)17 repeat microsatellite, IDVGA75 (EMBL accession number: X89117; D number D15S24) was isolated by screening a partial cosmid library of Sau3AI digested bovine DNA in the vector SuperCos 1 with a labeled (TG)8 probe. The microsatellite repeat region was sequenced directly from the cosmid 1 . Primers were designed from the sequence flanking the microsatellite repeat using the OLIGO program (National Biosciences, Plymouth, MN). Oligonucleotides primers without self-complementary and with a Tm between 60 and 65 °C were selected.

PCR conditions: PCR was carried out in a volume of 10 ml, containing 50 ng genomic DNA, 1 mm of each primer (pAG5 U2 (TG strand): 59GGGGTCTCAAAGAGTCAGATAC39 and pAG5 (AC strand): 59TTTCTGGAGAACTTTGGCTATG39), 200 mm dNTP, 1 mm MgCl2 and 1 U Taq DNA polymerase (Life Technologies, Paisley, UK). The pAG5 (AC strand) primer was labeled with [g-33 P]ATP (Amersham International, Little Chalfont, Buckinghamshire, UK) by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). DNA amplification was performed on a PTC-100 thermal cycler (MJ, Watertown, MA) using the following conditions: five initial cycles of denaturation at 94 °C (30 s), annealing at 60 °C (30 s) and elongation at 72 °C (45 s) followed by 30 additional cycles with the annealing temperature reduced to 55 °C. Finally an elongation step at 72 °C (3 min) was performed.

Allele size and frequency: Alleles were scored in a panel of 25 unrelated animals of five Italian and one Chinese breeds (Chianina, Frisona, Maremmana, Piemontese Vladostana and Nanyan). PCR products were separated on denaturing polyacrylamide-urea (7%-7 m) gels with a sequencing ladder and the microsatellite allele amplified from the parent cosmid clone (188 bp) as size markers. Seven alleles were observed with the corresponding allele frequency indicated in parentheses: 180 bp (0 . 28), 182 bp (0 . 20), 184 bp (0 . 16), 186 bp (0 . 08), 188 bp (0 . 16), 190 bp (0 . 10) and 192 bp (0 . 02).

Mendelian inheritance: A halfsib family composed of a Piemontese sire, five Nanyan dams and six progeny were typed for this microsatellite marker in order to confirm the Mendelian inheritance of the alleles; six alleles are shown to segregate in this family material (Fig. 1).

Chromosome location: The cosmid clone from which the IDVGA75 microsatellite was derived was localized to Q-banded metaphase chromosomes by fluorescence in situ hybridization (FISH) as described elsewhere 2 . A specific signal was identified on BTA 15q25. four DNA bands (306, 160, 146 and 66 bp), respectively (Fig. 1a). The D allele is detected since primer D2 is not an exact match for the DNA template with respect to a C replacing a G at position 23. This substitution creates a new MaeIII site (position 210), while leaving a common MaeIII site (position 157) unchanged in amplified DNA fragments from nonD individuals. Fragments yielded after digestion with MaeIII of the 237-bp amplified DNA allowed us to distinguish the D allele (Fig. 1b). Homozygous individuals of D/D genotype give two fragments (157 and 80 bp). The heterozygous nonD/D show restriction pattern with four fragments (157, 80, 53 and 27 bp). Likewise, nonD/nonD present a three-band pattern (157, 53 and 27 bp). The DNA analysis agreed with the phenotype determined by IEF starting from milk of 50 individuals.

Comments: Genetic variants of milk protein can be related to the quantity and quality of milk production, observed for the ovine a-s1 casein D allele 3 . The aim of the present study was to develop a method to discriminate between the A, C and D alleles at DNA level. This method could be employed to eliminate from the breeding scheme males and non-lactating females carrying the unfavourable D allele as well as, facilitate the detection of polymorphisms occurring in the population for basic studies. Mendelian inheritance: Co-dominant Mendelian inheritance was seen in a seven-member German shepherd dog pedigree (Fig. 1).

Chromosomal location: The DLA-DQA1 gene, located within the major histocompatibility complex, is found on chromosome 12qter 3 .

The human gene is found on chromosome 6p21 . 3.

Acknowledgements: This work was supported by the American Shetland Sheepdog Association. Polymorphism and Mendelian inheritance: Two alleles were observed in the 177 bp fragment. Segregation analyses were performed in the East Lansing 4 chicken reference back-cross mapping population using the Map Manager software 5 .

Linkage analysis and chromosomal location: The segregation pattern of ME1 alleles in the reference population showed that this locus is located on chromosome 3, between ADL024 (5 . 8 cm, LOD 10 . 7) and ADL015 (9 . 6 cm, LOD 8 . 5). The homologous Mod1 gene has been localized on mouse chromosome 9 and the human ME1 one on chromosome 6q12 6 . This result extends the synteny conservation between a fragment of chicken chromosome 3 and HSA 6 to the proximal part of the q arm of this chromosome. Some MOLECULAR GENETIC MARKERS 475 rearrangements have occurred, since ME1 is localized on chicken chromosome 3 between TCP1 and PLN, located on human 6q26±q27 and 6q22, respectively. Concerning the conservation of this genomic segment in the mouse genome, only one other gene of this region is localized on mouse chromosome 9, i.e. GSTA2. ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

Fig. 1 .

 1 Fig. 1. Partial bovine metaphase spread before (a; QFQ-banded) and after (b; DAPI-counterstained) FISH with a YAC positive for bovine FBN1. The arrow indicates chromosome 10.

Acknowledgements:

  Fig. 1. Multiplex PCR analysis of the dinucleotide repeats in intron 4 and intron 7 of the SCAMP1 gene showing Mendelian inheritance. Allele sizes are indicated.

  Chromosomal location: The LIPE gene has been assigned by FISH to porcine chromosome 6q122 . Three new microsatellite markers on bovine chromosome 17 B A Konfortov, J R Miller Laboratory of Genome Mapping, Department of Immunology, The Babraham Institute, Cambridge CB2 4AQ, UK Accepted 27 March 1998 Source/description: A YAC clone, mapping to bovine chromosome 17 was digested with MboI and subcloned into pUC18. The resulting plasmid sublibrary was screened with oligolabeled d(CA)-d(GT) repeat probe and the positive transformants isolated. Three strongly hybridizing microsatellite containing clones were sequenced and PCR primers chosen from the repeat flanking regions. One primer of each pair was labeled with a fluorescent tag.PCR conditions: Amplifications were carried out in microtitre plates in 10 ml final volumes. Each reaction contained 10±20 ng genomic DNA, 200 mm each dNTP, 0 . 25 mm corresponding primers, 2 mm MgCl 2 , 0 . 125 U Tth polymerase (Epicentre Technologies, Madison, WI, USA) and 1´reaction buffer. Thermocycling consisted of initial denaturation at 95 °C for 5 min, followed by 40 cycles of 10 s at 95 °C denaturation, 40 s at the specific annealing temperature and 20 s extension at 72 °C. Each reaction was quantitated by running 4 ml product on 3% agarose gel and the remainder of the samples diluted, aliquoted and pooled. The pooled DNA fragments were separated on an ABI 373 automatic sequencer. Allele sizes and genotypes were estimated by Genescan 2 . 1 analysis software.

Fig. 1 .

 1 Fig. 1. Mendelian inheritance of the Hsp92I polymorphism at the porcine LIPE gene (allele A ± two predominant bands, 308 and 190 bp; allele G ± two predominant bands, 241 and 190 bp; the 67 bp band is barely visible). The weak zones, which do not obscure the genotypes, are apparently due to the incomplete digestion that could not be avoided. The 100 bp ladder has bands 1000±100 bp, and pUC19/MspI marker possesses bands 501/489, 404, 331, 242, 190, 147, 111/110, 67, 34, and 26 bp. S, sire; D, dam; 1±9, offspring.

Reference 1

 1 Yerle M. et al. (1996) Cytogenet Cell Genet 73, 194±202. Correspondence: Dr Bertram Brenig (E-mail: bbrenig@gwdg.de) Omy0002DIAS: a highly polymorphic dinucleotide microsatellite in rainbow trout (Oncorhynchus mykiss) L-E Holm Danish Institute of Agricultural Sciences, Department of Animal Breeding and Genetics, Research Centre Foulum, PO Box 50, 8830 Tjele, Denmark Accepted 28 March 1998

1

  Chromosomal location: Unknown.Correspondence: Lars-Erik Holm (E-mail: leh@genetics.sh.dk) Assignment of the presenilin 2 gene (Laboratoire de Ge Âne Âtique biochimique et de Cytoge Âne Âtique, INRA-CRJ 78350 Jouy-en-Josas, France; 2 ABS Global Inc., 6908 River Road, DeForest, WI 53532 USA Accepted 29 March 1998

Fig. 1 .

 1 Fig. 1. QFQ-banded partial metaphase (A) prior to FISH (B) with the cosmid ZuBeCa6. Chromosome 5 (A) and hybridization signals (B) are indicated by arrows. Inset is a schematic representation of chromosome 5 adapted to the GTG banding system.

Fig. 1 .

 1 Fig. 1. R-banded metaphase spread after in situ hybridization with a YAC clone containing the bovine PSEN2 gene. Arrows indicate signal on BTA16q16.

  and Genetics, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark; 2 Department of Animal Science and Animal Health, The Royal Veterinary and Agricultural University, 1870 Frederiksberg, Denmark; 3 Department of Product Quality, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark; 4 Institute of Human Genetics, Aarhus University, DK-8000 A Ê rhus, Denmark Accepted 29 March 1998

Fig. 1 .

 1 Fig. 1. Autoradiogram showing the PvuII RFLP genotypes at the orosomucoid (ORM) locus in five pigs.

  Chromosomal localization: Chromosome 1 4,6 . Correspondence: L Fontanesi Novel primers for the mitochondrial Control Region and its homologous nuclear pseudogene in the Eider duck Somateria mollissima R Tiedemann, K G von Kistowski Institut fu È r Haustierkunde, Christian-Albrechts-Universita Èt zu Kiel, Olshausenstrasse 40, D-24118 Kiel, Germany Accepted 5 May 1998

Correspondence 2 1

 2 Department of Breeding and Genetics; 2 Department of Product Quality, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark Accepted 8 May 1998

Fourteen 1 1

 1 Acknowledgements: We thank Mr S. Svendsen for skilful technical assistance. This work was supported by the Danish Project AVL 93-SH-2. Correspondence: Klaus Brusgaard, Institute of Clinical Research, Department of Clinical Biochemistry and Genetics, Odense University Hospital, DK-5000 Odense C, Denmark (E-mail: klb@imbmed.ou.dk) Department of Veterinary Pathobiology, 2 Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, MN 55108, USA Accepted 10 May 1998

  Acknowledgements: We are grateful to the International Equine Genome Mapping Workshop for providing the reference family stallion DNA. This work was supported in part by a Grant in Aid from the University of Minnesota Graduate School and a grant from the North American Equine Ranching Information Council. Correspondence: Dr J R Mickelson (E-mail: micke001@tc.umn.edu) Eight new equine dinucleotide repeat microsatellites at the NVHEQ26, NVHEQ29, NVHEQ31, NVHEQ40, NVHEQ43, NVHEQ90, NVHEQ98 and NVHEQ100 loci K H Rùed, L Midthjell, G Bjùrnstad Department of Morphology, Genetics and Aquatic Biology, Norwegian College of Veterinary Medicine, PO Box 8146 Dep., N-0033, Oslo, Norway Accepted 10 May 1998

  Chromosomal locations: Unknown. Acknowledgements: This work was partly supported by the Norwegian Research Council and by the Norwegian Trotter Association.

  RFLP and linkage analysis of the porcine fibrinogen loci ± FGA, FGB and FGG Roslin, Midlothian, EH25 9PS, UK; 2 Washington School of Medicine, Washington University, St Louis, MO, USA Accepted 14 May 1998 Probes: Porcine cDNAs encoding a-fibrinogen (015±3B; EMBL accession number: M29514), b-fibrinogen (020±6FL; EMBL accession number: M29513) and g-fibrinogen (019±2D; EMBL accession number: M29510) were isolated previously as pBluescript clones from a liver cDNA library established in lZAPII 1 . The EMBL database was searched (19th April 1998) with partial DNA sequences of these cDNA clones. The putative a-fibrinogen sequence was 97 . 2% identical to a pig DNA sequence (EMBL: D43760

Fig. 2 .

 2 Fig. 2. The EcoRI RFLP at the FGB locus. Alleles: B1 = 8 . 8 kb + 6 . 5 kb; and B2 = 6 . 5 kb + 5 . 6 kb and B3 = 6 . 5 kb. Genotypes from left to right are B2/B2, B1/B1, B1/B2, B1/B1, B1/B1, B2/B3, B1/B3, B1/B2, B2/B3, B2/B3, B1/B3 B2/B3, B2/B3, B1/B1, B1/B1, B1/B1, B2/B3, B1/B3, B1/B3. The relative intensities of the fragments were used to determine whether the B3 allele was present or not.

Fig. 1 .

 1 Fig. 1. The XbaI RFLP at the FGA locus. from left to right are: A1/ A1, A2/A3, A1/A2, A1/A3, A2/A3. Alleles: A1 = 7 . 2 kb, A2 = 3 . 15 kb and A3 = 2 . 95 kb. Fig. 3. The TaqI RFLP at the FGG locus. Alleles: C1 = 3 . 0 kb and C2 = 2 . 65 kb.

1 2 1Fig. 1 .

 21 Fig. 1. Amplification products of microsatellite IDVGA75 in a halfsib family of a Piemontese sire crossed to five Nanyan dams. C, Cloned allele from cosmid; S, Piemontese sire; D1, D2, D4±D6, Nanyan dams; P1±P6, progeny. The size of alleles are shown to the left, with the cloned allele underlined.

References 1

 1 Ferranti P. et al. (1995) J Dairy Res 62, 281±96. 2 Lien S. et al. (1992) Anim Genet 23, 333±8. 3 Piredda G. et al. (1993) Scienza e tecnica lattiero-casearia 44, 135±43.Correspondence: Fabio Pilla (E-mail: pilla@hpsrv.unimol.it)PCR-RFLP test for direct determination of b-

Fig. 1 .Fig. 1 .

 11 Fig. 1. (a) Genotyping at the ovine a-s1 casein locus, using PCR followed by MboII digestion. Agarose gel (2% Metaphore + 2% agarose) electrophoresis analysis of DNA fragments amplified with the primer pair A1 and A2 from homozygous A/A (lanes 2 and 3), heterozygous nonA/A (lanes 4 and 5) and `homozygous' nonA/nonA (lanes 6 and 7). Lanes 2, 4 and 6 show undigested PCR products, whereas lanes 3, 5 and 7 correspond to the same fragments digested with MboII. Lane 1, molecular weight marker (pBR322, HaeIII digested). Sizes (in bp) of DNA fragments are given on the right. (b)Genotyping at the ovine a-s1 casein locus, using PCR followed by MaeIII digestion. Agarose gel (4:2% Metaphore agarose + 2% agarose) electrophoresis analysis of DNA fragments amplified with the primer pair D1 and D2 from homozygous D/D (lanes 2 and 3), heterozygous nonD/D (lanes 4 and 5) and `homozygous' nonD/nonD (lanes 6 and 7). Lanes 2, 4 and 6 show undigested PCR products, whereas lanes 3, 5 and 7 correspond to the same fragments digested with MaeIII. Lane 1, molecular weight marker (pBR3222, HaeIII digested). Sizes (in bp) of DNA fragments are given on the right.

  -F141L-D: GTTCCTGAGGTGACTGTGTT DLA-DQA1-F141L-U: GAAGGAATGATCCCCCTTGG The altered nucleotide that creates the BslI site with the L allele is in italics. PCR/RSP conditions: Reaction components were: 10 mm Tris±HCl (pH 8 . 3 at 20 °C), 50 mm KCl, 2 mm MgCl 2 , 80 mm dNTPs, 0 . 13 pmol of each primer, and 0 . 5 U Taq DNA polymerase. Target DNA was derived from blood or cheek swab DNA (50±200 ng). PCRs were performed in a final volume of 25 ml with the following cycle times: 94 °C,

References 1 1 1

 11 Sarmiento U.M. et al. (1992) Immunogenetics 35, 416±20. 2 Lingaas F. et al. (1997) Mamm Genome 8, 218±21. 3 Dutra A.S. et al. (1996) Cytogenet Cell Genet 74, 113±7. Laboratoire de Ge Âne Âtique Cellulaire, INRA-CRT, Castanet-Tolosan 31326, France; 2 Laboratoire de Ge Âne Âtique, INRA-ENSAR, Rennes 35000, France Accepted 1 July 1998 Source/description: The chicken ME1 gene (Malic Enzyme 1) has been partially cloned and sequenced 1 . From the published sequence (EMBL accession number U49693), we designed two PCR primers to amplify a promoter fragment of this gene. The expected length of the fragment was 177 bp. Primer sequences: MAL1U: 59-CCG ATC CCC TCC AGA GAA GG-39 MAL1L: 59-TGC TGC GTG CGG TAC TGC CG-39 PCR conditions: PCR amplifications were performed in 25 ml on a Techne PHC-3 thermocycler. The reaction mixes comprised 20 ng genomic DNA, 200 mm each dNTP, 1 mm each primer, 1 . 5 mm MgCl 2 , 20 mm Tris±HCl (pH 8 . 4), 50 mm KCl and 1 U Taq polymerase (Gibco BRL, Rockfield, MD). The profile for thermal cycling was, for 30 cycles: denaturation 94 °C, 30 s; annealing 65 °C, 30 s; elongation 72 °C, 30 s. The amplified fragment was denatured by heating at 95 °C and loaded onto a non-denaturing acrylamide/bisacrylamide (49:1) gel containing 5% glycerol, to visualize a Single Strand Conformation Polymorphism 2 through silver staining 3 .

Fig. 1 .

 1 Fig. 1. Co-dominant Mendelian inheritance in a seven-member German shepherd dog pedigree. M indicates 100 bp ladder lanes and W indicates a water blank lane. ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

Fig. 1 .

 1 Fig. 1. Fluorogramy showing Mendelian inheritance in a family of Angler Saddlebacks. Allele sizes are indicated.

Table 1 .

 1 Primer sequences

	Locus	Primer sequence (59±39)
	VOLP-03	AGACGGTTGGGAAGGTGGTA
		CGACAGCAAGGCACAGGA
	VOLP-05	ACTTAATCACCTGGATGTAT
		ATATGGTTCACTGTGTTTACT
	VOLP-08	CCATTCACCCCATCTCTC
		TCGCCAGTGACCTTATTTAGA
	VOLP-10	CTTTCTCCTTTCCTCCCTACT
		CGTCCACTTCCTTCATTTC
	VOLP-12	TTGTTCTCAACAGGGACTGC
		TCTGGCCCACCCACTAA
	VOLP-17	GACTTCCAGCCACAAC
		CCATCAGTTTTAAGCAACATAC
	VOLP-32	GTGATCGGAATGGCTTGAAA
		CAGCGAGCACCTGAAAGAA
	VOLP-33	TAATCCTTAAGAACCGACCCT
		GGACCCGCTTCATCTATTG
	VOLP-59	CCTTCCTCAGAATCCGCCACC
		CCCGCGCACCAAGCAG
	VOLP-67	TTAGAGGGTCTATCCAGTTTC
		TGGACCTAAAAGAGTGGAG

Table 2 .

 2 Summary data for loci and primers Lund et al.(1996) Nucl Acids Res 24, 800±1.

			Alleles	Size	Hetero-
	Locus	Repeat	(n)	range (bp)	zygosity PIC
	VOLP-03 (TG) 13	8	129±169 0.799	0.74
	VOLP-05 (TG) 13	6	134±150 0.79	0.753
	VOLP-08 (TG) 10 TCCG(TG) 2 TCCG(TG) 5 3	148±152 0.226	0.214
	VOLP-10 (TG) 2 TA(TG) 7 TA(TG) 7	3	231±235 0.596	0.521
	VOLP-12 (TG) 3 CG(TG) 4 CG(TG) 7	2	136±148 0.08	0.077
		CT(TG) 2 CA(TG) 2
	VOLP-17 (GA) 5 CAGATACAGA(TA) 2	12	184±228 0.784	0.733
		GATTT(TG) 14 (TTG) 4 TG
	VOLP-32 (TG) 20	12	192±247 0.797	0.802
	VOLP-33 (A) 4 G(A) 5 (TG) 12 T(A) 5	4	178±184 0.676	0.591
	VOLP-59 (TG) 7 [(CG) 2 (TG) 5 ] 2 (CG) 2	5	112±136 0.683	0.614
		(TG) 4 CT(TG) 4 AGCT(TG) 3
	VOLP-67 (TG) 5 (G) 4 (TG) 9 CG(TG) 7	5	158±170 0.671	0.584

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477 connection with a project of the University of Kentucky Agricultural Experiment Station as paper number 98-14-15.

Reference 1

Table 1 .

 1 Polymorphic information content, heterozygosity, allele sizes and frequencies of the two SCAMP1 gene microsatellites

	Intron 4	Intron 7

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477 PCR conditions/cloning/sequencing: PCR was performed in 50 ml reactions using 100 ng porcine genomic DNA, standard PCR buffer, 2 mm MgCl 2, 200 mm each dNTP, 0 . 4 mm each primer and 1 . 3 U Taq polymerase. After an initial 95 °C denaturation step (2 min) the PCR was carried out at 95 °C (45 s), 60 °C (1 min) and 72 °C (1 min; the final extension 5 min) for 30 cycles. Amplification products were electrophoresed on a 4% NuSieve agarose gel. A single fragment of the expected size was observed. The PCR products generated from two crossbred pigs (Meishan ´Pie Âtrain and Wild Boar ´Pie

Âtrain)

were ligated into pUC18, and four recombinant clones were sequenced using an ALFexpress Sequencing System (Pharmacia Biotech, Uppsala, Sweden). The sequence (EMBL accession number AJ224692) was almost identical to the recently available data on porcine LIPE (EMBL accession number AJ000482).

Polymorphism: Comparison of the sequences of the four clones of porcine LIPE revealed a 433 A®G polymorphism (numbering from the 59 end of the PCR fragment), which was found to be present within a recognition sequence for enzyme Hsp92I. Digestion of the PCR product with the enzyme revealed two alleles: A (fragments 190 and 308 bp) and G (fragments 190, 241 and 67 bp). The three genotypes observed are shown in Fig.

1

. The substitution of 433 A®G also results in an amino acid substitution (ATC ± Ile; GTC ± Val) in the corresponding protein.

Mendelian inheritance/allele frequencies: Codominant inheritance of the LIPE alleles was demonstrated in the Hohenheim PiGMaP Meishan ´Pie Âtrain three-generation pedigree 1 . Both alleles were present in Meishan; in Pie Âtrain allele G was fixed. Unrelated animals of Landrace (n = 8) and Large White (n = 7) breeds were monomorphic for allele G, while Duroc (n = 11) was polymorphic (frequencies: A ± 0 . 23; G ± 0 . 77).

  Mendelian inheritance of the Hsp92I polymorphism at the porcine LIPE gene (allele A ± two predominant bands, 308 and 190 bp; allele G ± two predominant bands, 241 and 190 bp; the 67 bp band is barely visible). The weak zones, which do not obscure the genotypes, are apparently due to the incomplete digestion that could not be avoided. The 100 bp ladder has bands 1000±100 bp, and pUC19/MspI marker possesses bands 501/489, 404, 331, 242, 190, 147, 111/110, 67, 34, and 26 bp. S, sire; D, dam; 1±9, offspring.

		A highly polymorphic microsatellite within
		intron 5 of the porcine 54/56 kDa vacuolar H(+)-
		ATPase subunit gene (V-ATPase)
		I Pfeiffer, B Brenig
		Institute of Veterinary Medicine, Groner Landstrasse 2, 37073
	1 Konfortov B.A. et al. (1998) Anim Genet 29, 302±6.	Goettingen, Germany
	2 Barendse W. et al. (1997) Mamm Genome 8, 21±8.	Accepted 1 July 1998
	Correspondence: B A Konfortov (E-mail: bernard.konfortov@bbsr-c.ac.uk)	Source/description: The vacuolar proton-translocation ATPases are multienzyme complexes located in the membranes of eukaryotic
		cells regulating the cytoplasmic pH. We have isolated and
	A polymorphic horse microsatellite locus:	characterized a cDNA coding for the porcine 54 and 56 kDa subunit
	VHL150 W A van Haeringen 1 J A Lenstra 2 , H van Haeringen , L H P van de Goor 1 , N van den Hout 1 1 Dr Van Haeringen Laboratorium B.V., PO Box 408, 6700 AK Wageningen, The Netherlands; 2 Faculty of Veterinary Medicine, PO 1 ,	of the V-ATPase. The genomic structure was determined by sequencing of recombinant phage clones and assigned it to chromosome 4q15±q16 by PCR screening of a porcine-rodent hybrid cell panel. The V-ATPase gene consists of 14 exons with sizes ranging from 90 to 336 bp with a non-coding first exon and an alternatively spliced seventh exon. Within the fifth intron of the V-
	Box 80165, 3508 TD Utrecht, The Netherlands Accepted 28 March 1998	ATPase gene a ((CA) 13 *(CT) 17 ) dinucleotide repeat was identified. The DNA sequence of the V-ATPase gene was submitted to the EMBL data library under accession number AJ223748.
	Source/description: A plasmid clone was isolated from a library of size-selected horse fragments following screening with a (CA) 25 oligonucleotide. The sequence of this clone which contains a (AC) 14 microsatellite was determined. The sequence of this locus has been submitted to EMBL data library (accession number X86450).	Primer sequences: Forward primer (EXO 6f): 59-CCA CAC ACT TGC TAA CAG AAA-39 Reverse primer (EXO 6r): 59-CAC TGC AAT AAA TCT GGA AGA G-39
	Primer sequences:	PCR conditions and analysis: PCR reactions were optimized using
	59-CTCCTATGGGTGTCAGAAAG-39 (forward) 59-TGTAATCTGTGGGGGAGACT-39 (reverse) PCR conditions: As a template for PCR amplification 75±125 ng DNA was used in a total reaction volume of 15 ml, containing 10 mm Tris±HCl (pH 8 . 3), 50 mm KCl, 1 . 5 mm MgCl 2 , 5 pmol of each primer (Perkin Elmer, Foster City, CA, USA) and 1 . 2 U Amplitaq (Perkin Elmer). PCR amplification was carried out on a Perkin Elmer GeneAmp PCR system. Thirty cycles of 10 s 94 °C, 10 s 55 °C, and 10 s 72 °C, were performed. Allelic frequencies: The sizes of allelic PCR products were determined with the ABI 373 DNA sequencer and Genescan software (Perkin Elmer-ABI, Foster City, CA, USA). The frequencies of the microsatellite alleles are based on samples submitted for parentage control. Frequencies are presented for Dutch Warmblood,	the `Ready to Go' system (Pharmacia, Freiburg, Germany). A master mix was prepared containing 400 mm of each primer (the forward primer was labelled with IRD 800 fluorescence dye; MWG Biotech, Ebersberg, Germany), frozen and stored at ±20 °C. DNA (100 ng) in 1 ml TE (10:1) was mixed with 12 ml master mix and one PCR bead was added (10 mm Tris±HCl pH 9 . 0, 50 mm KCl, 1 . 5 mm MgCl 2 , 200 mm of each dNTP and 1 . 5 U Taq polymerase). Cycling included an initial denaturation step at 94 °C for 10 min, 30 cycles of 1 min at 94 °C, 30 s at 55 °C, 72 °C for 1 min and a final step of 10 min at 72 °C using a Hybaid Omnigene cycler. PCR products were analyzed by electrophoresis on 8% denaturing polyacrylamide gels using a LI-COR Gene ReadIR 4200 automated sequencer. Analysis of the bands was performed using the RFLPscan Plus 3 . 0 (Scanalytics, Virginia, VA) software. Polymorphism: Polymorphism was studied using DNA samples of
	Fjord Horse, Friesian Horse, Shetland Pony, and Icelandic Horse
	(Table 1).
	Table 1. Allelic frequencies, PIC, heterozygosity and PE values for VHL150	Repeat sequence	(CA) 13 *(CT) 17 )
	Table 1. Primer information, allele sizes and levels of polymorphism Marker accession no. Primer sequences T°a (°C) Allele sizes (bp) and frequencies AF6 GAATGGCCAAGAGAACACATACAGAACATC 61 230 240 242 244 Y15071 CCCCTGGAGGAGGGCATGGC (0 . 013) (0 . 316) (0 . 276) (0 . 395) AF7 GGACAGAGGAGCCTGGCAGGG 62 177 187 189 191 Y15072 AATGAATGGCCAAGAGAACACATACAGAAC (0 . 013) (0 . 316) (0 . 276) (0 . 395) AF8 GATCCACAGGGTCGCCAAGAGTTG 62 164 166 168 170 Y15073 TTGTCTTACAGGAGGAGATGAGCACATGTC (0 . 066) (0 . 026) (0 . 290) (0 . 211) (0 . 092) (0 . 065) (0 . 105) (0 . 013) (0 . 132) 172 174 176 178 180 Allele frequencies are shown in parentheses under the corresponding alleles. Mendelian inheritance: Transmission of the microsatellite alleles was consistent with codominant, autosomal inheritance. Correspondence: W A van Haeringen Breeds DW FjH FrH SP IH Alleles (bp) n = 1350 n = 650 n = 25 n = 10 n = 12 96 0 . 158 0 . 424 0 . 059 0 0 . 095 98 0 0 . 064 0 0 0 100 (M*) 0 . 654 0 . 415 0 . 137 0 . 214 0 . 476 102 0 . 163 0 . 093 0 . 471 0 . 714 0 . 429 104 0 . 008 0 . 005 0 0 0 106 0 . 016 0 0 . 333 0 . 071 0 PIC 0 . 475 0 . 565 0 . 581 0 . 387 0 . 490 Heterozygosity 0 . 520 0 . 636 0 . 439 0 . 439 0 . 586 PE 0 . 291 0 . 363 0 . 218 0 . 218 0 . 286 DW, Dutch Warmblood; FjH, Fjord Horse; FrH, Friesian Horse; SP, Shetland Pony; IH, Icelandic Horse. PIC 0 . 91 Heterozygosity 0 . 92 Allele size (bp) and frequency 227 (0 . 028) 225 (0 . 011) 223 (0 . 011) 221 (0 . 017) 219 (0 . 011) 217 (0 . 011) 215 (0 . 086) 213 (0 . 022) 211 (0 . 011) 209 (0 . 006) 207 (0 . 051) 205 (0 . 028) 201 (0 . 022) 199 (0 . 086) 197 (0 . 115) 195 (0 . 033) 193 (0 . 057) 191 (0 . 068) 189 (0 . 057) 185 (0 . 167) 183 (0 . 051) 181 (0 . 051)
	ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

*In the international nomenclature for parentage control with horse microsatellites, base pair values of alleles are renamed to and presented in an alphabetical order (e.g. 98 bp = `L', 100 bp = `M', 102 bp = `N', etc.). Table 1. Polymorphic information content, heterozgosity, allele sizes and frequencies of the V-ATPase gene microsatellite ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477 77 Angler Saddleback, five German Large White, five German Landrace, five Pietrain, and six Bunte Bentheimer pigs. Among these 98 pigs 22 different alleles of the microsatellite were detected. The lengths of the corresponding PCR products were 227, 225, 223, 221, 219, 217, 215, 213, 211, 209,

Table 1 .

 1 Allele frequencies, observed heterozygosity (H o ), and polymorphism information content (PIC) for Omy0002DIAS in rainbow trout from four Danish hatcheries

		Allele frequency
	Allele size 174 176 178 180 182 186 190 202 206 208 212 222 224 236 238 240 242 246 248 254	Hatchery E 0 . 20 0 . 11 ± 0 . 05 ± ± 0 . 13 ± ± 0 . 20 ± 0 . 05 0 . 09 0 . 05 0 . 07 0 . 02 0 . 04 ± ± ±	Hatchery H ± 0 . 26 ± 0 . 01 0 . 01 ± 0 . 12 ± ± 0 . 12 ± 0 . 06 0 . 24 0 . 03 ± ± ± ± 0 . 12 0 . 03	Hatchery C 0 . 02 0 . 13 0 . 02 0 . 11 0 . 02 0 . 14 ± 0 . 05 0 . 07 0 . 07 ± ± 0 . 30 ± ± 0 . 05 ± ± 0 . 02 ±	Hatchery D ± 0 . 16 0 . 06 ± 0 . 18 ± ± ± 0 . 02 0 . 04 0 . 02 0 . 10 0 . 25 ± 0 . 02 0 . 07 ± 0 . 02 0 . 07 ±
	H o PIC	0 . 71 0 . 85	0 . 74 0 . 80	0 . 86 0 . 83	0 . 82 0 . 84
	n	28	39	28	34

Fig. 1. PCR analysis of the dinucleotide repeat in intron 5 of the V-ATPAse subunit gene demonstrating Mendelian inheritance. Allele sizes are indicated. ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477 A.1: 59 GATGGGAGGCCAGGGTGTC 39 and A.2: 59 CCTCCTGCT-GCTTGCTGTGTT 39. A 128-bp fragment was amplified in 10 ml PCR containing 50 ng of genomic DNA and 1 . 5 mm MgCl 2 . Samples were preheated for 5 min at 94 °C and subjected to 30 cycles at 94 °C for 30 s, 62 °C for 30 s, 72 °C for 30 s, and to a final extension step of 5 min at 72 °C in a Cetus 9600 thermocycler, using a Promega PCR kit (Promega, Madison, WI).

Table 1 .

 1 Sizes and frequencies of alleles observed

	Allele (bp) Frequency	290 0 . 038	294 0 . 019	300 0 . 019	302 0 . 105	306 0 . 086	310 0 . 019	314 0 . 067	318 0 . 010	322 0 . 029
	Allele (bp) Frequency	325 0 . 029	327 0 . 029	329 0 . 019	330 0 . 048	333 0 . 019	334 0 . 019	336 0 . 067	340 0 . 038	341 0 . 010
	Allele (bp) Frequency	343 0 . 038	346 0 . 048	348 0 . 010	350 0 . 056	352 0 . 010	353 0 . 010	356 0 . 048	359 0 . 010	363 0 . 029
	Allele (bp) Frequency	365 0 . 010	367 0 . 019	369 0 . 010	376 0 . 010	383 0 . 010	387 0 . 010

  : We thank Mr S. Svendsen for skilful technical assistance. This work was supported by the Danish Project AVL 93-SH-2.Correspondence: Klaus Brusgaard, Institute of Clinical Research, Department of Clinical Biochemistry and Genetics, Odense University Hospital, DK-5000 Odense C, Denmark (E-mail: klb@imbmed.ou.dk)

	A PvuII RFLP at the porcine orosomucoid
	(ORM) locus
	L Fontanesi, R Davoli, V Russo
	DIPROVAL, Sezione di Allevamenti Zootecnici, University of
	Bologna, Via F.lli Rosselli 107, 42100 Reggio Emilia, Italy
	Accepted 29 March 1998

Table 1 .

 1

	Mvi248 GAATGGAGCACTTATTCAGAGG 7	145	0 . 79	0 . 76 52	U87249
	AAGAGACCCAATTAACACAGG

Microsatellite loci from American mink Marker Primer sequence (59-39) No. of Alleles Product size (bp) Heterozygosity PIC Annealing temperature GenBank accession no.

Table 1 .

 1 Genotyping primers and conditions

			Annealing	[MgCl 2 ]	Size	Alleles	No. of heterozygous
	Locus	Primer sequence (59®39)	temperature (°C)	(mm)	range (bp)		stallions
	UM026	CCCAAAATCAATTAGGTCTC	56	1 . 5	208±220	4	12
	UM027	ATCAGTTGCTCTCTACTTTTC TGCAAGAATTGTGAGGGAC	56	1 . 5	235±239	3	3
	UM028	GTGCTCAGTTAGTGGTATTC GCTCTGCCTCATTCTTATC	56	1 . 5	127±129	2	1
	UM029	TTGAGGACGGAGTGTTG CTTTCATCTTTTGGCCCC	56	1 . 5	177±185	3	7
	UM030	CTGTCTTGCTTTCCCACC CCGTGAAGTCACAGACTTAG	51	1 . 5	127±143	6	9
	UM031	ACAGTTTCTACAACAAACTGA AGCTCAAACCAACCTTTC	56	1 . 5	136±148	5	9
	UM033	CCTTAAAACAGACACACTAC CATTGTCCTGAGCAAGTC	56	1 . 5	126±140	4	10
	UM034	CTATCCGTCAAGTGTTTC TTGAAACCCTTCAGACCC	56	1 . 5	102±117	4	7
	UM035	CAGAGAGCAAGTGTGAG GTGATGGATGACATGAGG	52	1 . 5	203±207	2	1
	UM037	GCATTTAAAACACTAGAACAC TCATTTTATCCTCCACCTC	52	1 . 5	107±118	5	6
	UM038	AAAAGGGCGTAATATGG CAAGACAGAACAGAAGAAGAC	56	1 . 5	113±137	7	8
	UM040	ATATGGCTCGCTCCTAC CTCTTGTACATGTCTCCTTGTC	51	1 . 5	250±270	6	9
	UM042	TACTTTCTCTCTTCCAAACC GGCATCCCACATACAAAG	56	1 . 5	218±226	4	8
	UM043	GAAGCAACAGTCAATTCAG CCTCAATCTTTTCTTCTCC	56	1 . 5	145±150	3	10
		TCAAGAGAGACGCTACAC

Table 1 .

 1 Microsatellite loci from American mink

			No. of	Product			Annealing	GenBank
	Marker ID	Primer sequence (59-39)	alleles	size (bp)	Heterozygosity	PIC	temperature	accession no.
	Mvi248	GCTCGTGTTTCTGGCTCT	2	210	0 . 50	0 . 38	55	U87248
		CCTGGGCTTTCCTGTTTAT
	Mvi389	GAAGGCTAGGTAGGCATTAACC	3	112	0 . 42	0 . 33	55	U87251
		TTGTCCCATCCTTCACAGC

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

Table 1 .

 1 Sequence and primer information for eight equine microsatellite loci

	Locus (accession no.)	Repeat sequence	Primer sequences
	NVHEQ26	(GT) 11	F: GGAAGTGGAACCAACTATGACA
	(AF056392)		R: TGGAGGTTTAAGGGACGCTAT
	NVHEQ29	(TG) 17	F: GAGATTTTGCCCCAAAGGTTA
	(AF056393)		R: CTCTTCTTTCTTCCCCAGGTCT
	NVHEQ31	(CA) 12	F: AACTCAAAAGCACAAGCCCACAC
	(AF056394)		R: CAACACAGTGATGACCCAGGTAGG
	NVHEQ40	(CA) 3 TG(CA) 13 AG(CA) 2	F: TGGCATCTGAATGGAGAATG
	(AF056395)		R: GATTATGATGCTACAGGGGAAAG
	NVHEQ43	(CCA) 5 (CA) 22	F: TGACACAAGATAAAAGCCCCAGG
	(AF056396)		R: GATTGGGAAAAGAGCACAGCC
	NVHEQ90	(GT) 13	F: TCTATGTGTCAGAATGAGGAGGT
	(AF056397)		R: TTTTGCAAGGTAGATAGATGCC
	NVHEQ98	(CA) 10	F: TGCAATGACCATCAGAACTTC
	(AF056398)		R: CGGTGGTTTTGGAAATGAA
	NVHEQ100	(TG) 22 T(TG) 2	F: CCAAAGCAGAACATGTGAAGTT
	(AF056399)		R: TGGCATAGATGTTAGCTCAGTGA
	F, forward; R, reverse.

Table 2 .

 2 Polymorphism of equine microsatellite loci

		Alleles	Size range	Size of
	Locus	(n)	of products (bp)	M-allele a	Heterozygosity
	NVHEQ26 NVHEQ29 NVHEQ31 NVHEQ40 NVHEQ43 NVHEQ90 NVHEQ98 NVHEQ100	2 6 2 6 7 3 2 8	140±142 91±113 128±130 144±158 135±159 99±105 167±169 185±207	140 101 128 152 151 101 167 197	0 . 36 0 . 77 0 . 33 0 . 68 0 . 78 0 . 35 0 . 33 0 . 74

a For alphabetic nomenclature.

Table 3 .

 3 Allele sizes for the first four horses of the 1994 ISAG/ISBC standardization test

	Horse	1	2	3	4
	NVHEQ26	140/142	140/142	140	142
	NVHEQ29	101	107	101	101
	NVHEQ31	128/130	128/130	130	130
	NVHEQ40	154/156	154	154	154
	NVHEQ43	141/145	141/145	153	151/153
	NVHEQ90	101	101	101	101
	NVHEQ98	167	167/169	167	167
	NVHEQ100	207	207	207	191/207

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

  After a denaturation of 10 min at 95 °C the temperature cycling was as follows: 35 cycles of 15 s at 93 °C, 15 s at 60 °C, 30 s at 72 °C and a final extension of 10 min at 72 °C. Ten

	Microbiology and Small Animal Clinical Sciences, College of
	Veterinary Medicine, Michigan State University, East Lansing, MI
	48824±1314, USA
	Accepted 15 June 1998
	Source/description: A sequence variation that distinguishes several
	DLA (dog lymphocyte antigen)-DQA1 alleles occurs as a phenyla-
	microlitres of the reaction was phenol/chloroform purified, pre-cipitated and digested with 1 U of RsaI (Boehringer Mannheim, lanine-leucine interchange at amino acid position 141 (F141L; e.g. GenBank accession numbers: M74908, M74910) 1 . A PCR-based test was developed to distinguish the underlying nucleotide difference Milano, Italy) in a final volume of 15 ml for 1 h at 37 °C. The result by the artificial introduction of a restriction site (AIRS). The two was directly analysed by electrophoresis in 3% agarose gel stained alleles can be distinguished by digestion of the PCR product with with ethidium bromide, followed by ultraviolet exposition. BslI. The PCR product is 177 bp and the F allele is not cut. The L
	allele is cut into 157 and 20 bp fragments.

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

PCR and digestion conditions: The 50 ml reaction mix comprised of 100 ng genomic DNA, 150 nm of each primer, 1 . 25 U Taq Gold DNA polymerase (Perkin Elmer, Monza, Italy), 50 mm KCl, 10 mm Tris± HCl (pH 8 . 3), 2 . 5 mm MgCl 2 , dNTPs each at 200 mm and 0 . 01% gelatin. Amplification was carried out using a 9600 DNA Thermal Cycler (Perkin Elmer).

  4 min (1 cycle); 94 °C, 40 s, 63 °C, 1 min, 72 °C, 1 min (50 cycles). After PCR, 3 ml of 50 mm MgCl 2 and 10 U of BslI were added directly to the PCR mix and the reaction was incubated at 55 °C for at least 1 h. Eight microlitres of digested product were run on a 2% agarose (1´TBE) gel.Allele frequencies: The frequency of each allele was examined in 10 Shetland sheepdogs, 10 Doberman pinschers, nine Cairn terriers, 14 American Cocker spaniels, 16 Beagles, and seven German shepherd dogs. The animals for the last two breeds are the parents of the DogMap panel 2 . The respective F and L allele frequencies (and heterozygosities) for the six breeds were: 0 . 65, 0 . 35 (0 . 4); 0 . 3, 0 . 7 (0 . 4); 0 . 72, 0 . 28 (0 . 11); 0 . 64, 0 . 36 (0 . 29); 0 . 78, 0 . 22 (0 . 44); and 0 . 79, 0 . 21 (0 . 14). Values for the combined data are: 0 . 65, 0 . 35 (0 . 32).

Table 1 .

 1 Polymorphic information content, heterozgosity, allele sizes and frequencies of the triadin gene microsatellite

	Repeat sequence	((CT) 21 *(CA) 14 )
	PIC	0 . 88
	Heterozygosity	0 . 89
	Allele size (bp) and frequency	234 (0 . 01)
		232 (0 . 07)
		230 (0 . 05)
		228 (0 . 16)
		226 (0 . 09)
		224 (0 . 17)
		222 (0 . 07)
		220 (0 . 03)
		218 (0 . 01)
		214 (0 . 01)
		210 (0 . 02)
		208 (0 . 15)
		206 (0 . 08)
		204 (0 . 02)
		202 (0 . 03)
		190 (0 . 02)
		186 (0 . 01)

Table 1 .

 1 Characterization of nine canine microsatellites

	Microsatellite				Number of		PCR product
	number	Primers	Clone	H a	alleles	Conditions b	length (bp)
	AHT116	59ATTCAGTCAATATGCAGGCT39	(GT) 16	0 . 11	2	2 mM/54	229±231
	AHT128	59CTATGCTGTGAGGCTTTCCA39 59AGTTTGAAGCAGCATGGTAGTG39	(GT) 18	0 . 76	6	1 mM/TD59	80±90
	AHT140	59CGCAGTCGCATGTGTGTCTA39 59GTCGTGCATGGGAAAATTCT39	(GT) 17	0 . 81	8	2 mM/TD59	101±117
	AHT142	59TATGGTCCAGGCTGTGAACA39 59GGTGAGGCACTTAGCTTTTCC39	(TG) 14	0 . 69	5	2 mM/TD59	188±198
	AHTf12	59AGAAATTACCCAAACCTAGCCC39 59CAAGCTTTCTGAAAATGGTGC39	(CA) 15	0 . 77	5	2 mM/TD62	132±146
	AHTf16	59CTCTGCCTGTCTCTGCCC39 59GAGGTGGGGGTATAGGAAGC39	(CA) 24	0 . 79	6	2 mM/TD59	155±165
	AHTf44	59CCTGCTTCTCCCTCTGCC39 59CCCGAGATACAGGAAATGGA39	(GT) 17	0 . 74	4	2 mM/TD59	153±159
	AHTf64	59CCTGCTGCATGGTGTTACAC39 59CACTCCCCAGGACTGCAG39	(TG) 16	0 . 71	4	1 mM/TD61	118±124
	AHTf66	59GGGTTACTGAGCAGAGCCTG39 59CAAAGGCTTCCCATCTGTC39	(CA) 24	0 . 74	5	1 mM/TD58	171±179
		59TCGGTTACTCATATCTGGGG39
	a Heterozygosity (unbiased) 3 .

b TD are Touchdown programs 1 .

ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

Acknowledgements: This work was supported by a grant from the Fundacio Ân FIA (Chile) project #016-94. This study is published in Acknowledgements: We thank Professor H. Geldermann for providing DNA samples from the Hohenheim PiGMaP pedigrees. This study was supported by Grant Agency of the Czech Republic (Grant no. 523/97/1305) and EC (Contract no. ERBIC15CT960902).

Acknowledgements: This work was supported by the UK Meat and Livestock Commission. We thank Dr W. Barendse for carrying out the linkage analysis and mapping, and The Resource Center/Primary Database of the German Human Genome Project for providing the YAC clone. References Acknowledgements: The author wishes to thank Ms H. Jensen and Ms. B. Flu È gel for their excellent technical assistance. This work was supported by a grant from the Danish Agricultural and Veterinary Research Council and in part by the European Community: Project FAIR CT96-1591 SALMAP. Acknowledgements: The authors thank C. Giraud-Delville and P. Laurent for technical assistance. Acknowledgements: We thank E. Garbely, U. Sattler and B. Colomb for their technical assistance and P. Schawalder for providing dog blood samples. This work was supported by the Albert-Heim Stiftung, Switzerland, and by the Union Bank of Switzerland acting on behalf of a client. Acknowledgements: We wish to thank Dr R. T. Stone (USDA, ARS, Roman L. Hruska US Meat Animal Research Center, Clay Center, NE, USA) who provided the cDNA probe. This study was funded by the Italian MURST 60% and was associated with the PiGMaP Consortium. Acknowledgements: Malte Bussiek and Insa Cassens provided technical assistance. Heinrich Luttmann, Henning Noer and Joachim Oesert provided duck samples. This work was supported by Deutsche Forschungsgemeinschaft (TI 267/1±1). Acknowledgements: This study was conducted in the context of the European Pig Gene Mapping Project (PiGMaP) which is supported at the Roslin Institute with funds from the European Commission, the Biotechnology and Biological Sciences Council and the Ministry of Agriculture, Fisheries and Food. References Acknowledgements: This work was possible thanks to a fellowship granted to H. Cao under the co-operation treaty between the National Research Council of Italy and the Chinese Academy of Agricultural Sciences, 1995±1997.

References Acknowledgements: This study was supported by MURST 40% (G. Enne). We thank Dr A. Nudda for milk samples.

Acknowledgements: We thank P. Vellema, M.F. de Jong, J. Table 1. Polymorphic markers Bos taurus Ovis aries Acknowledgements: We would like to thank L.B. Crittenden and Hans Cheng for providing DNA samples of the East Lansing chicken reference family. Acknowledgements: The authors are grateful to A. Deppe for expert technical assistance. Acknowledgements: We are grateful to Dr K.C. Barnett, Mrs P.J. Rothoff-Rook and Ms D.J. Flack for help in obtaining blood samples. Work on the canine genome at the Animal Health Trust and at the University of Leicester is supported by the Guide Dogs for the Blind Association. PRIMERS and PIC were made available through the Human Genome Mapping Project Resource Centre (Wellcome Genome Campus, Hinxton, Cambridge, UK).

 1Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3508 TD Utrecht, The Netherlands; 2 Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, UK; 3 Norwegian College of Veterinary Medicine, Department of Morphology, Genetics and Aquatic Biology, Oslo, Norway; 4 School of Veterinary Medicine, Department for Animal Breeding and Genetics, Hannover, Germany Accepted 15 June 1998 Source/description: We analysed 29 previously published bovine microsatellites on 25 sheep of the Dutch Texel breed. Animals were tested in pools of four or five; it was checked that this allowed the detection of all individual alleles. Markers were assigned a rank to represent the ease of interpretation as previously described 1 . (Rank 1, clear bands for each allele; rank 2, stutter bands which did not interfere with the scoring; and rank 3, severe stutter but alleles could still be identified.) PCR conditions: Genomic DNA (50 ng) was amplified in 10 ml containing 10 pmol of 32 P-labelled left primer, 10 pmol of right primer, 0 . 2 mm dNTPs, 1 . 5 mm MgCl 2 , 50 mm KCl, 10 mm Tris±HCl pH 8 . 3, 0 . 001% gelatin and 0 . 2 U Taq polymerase (Promega, Madison, WI). After 2 min at 93 °C, 25 cycles of 15 s at 92 °C, 45 s at the indicated annealing temperature (Table 1) and 45 s at 72 °C were performed. The final step was 2 min at 72 °C.

Analysis of PCR generated fragments: PCR products were separated on a denaturing polyacrylamide gel (7%) and amplified fragments were visualised by autoradiography. Apparent allele sizes were determined by comparison with a M13 sequencing ladder.

Chromosomal location: The homologous karyotype of cattle and sheep chromosomes and extensive genome conservation between these species allows tentative assignments of the markers to sheep chromosomes 3,16 (Table 1).

Polymorphism: Seventeen bovine microsatellite primers amplified specifically in sheep and 13 were polymorphic with allele numbers ranging from 2 to 11 (Table 1). One of these markers (CSSM22) was previously reported to be monomorphic in sheep 16 . Four markers were monomorphic (IOBT730, IOBT918, IDVGA80, and HEL9) and 12 either did not amplify or gave multiple products (IOBT930, IOBT959, IOBT313, IOBT528, INRA209, CSSM42, CSSM14, HAUT24, HAUT37, HAUT29, HAUT27, and RM28). A similar succesrate of bovine primers in sheep has been described previously 2,16 ã 1998 International Society for Animal Genetics, Animal Genetics 29, 460±477

Hagendijk, G. Huisman, and S.J. Henstra for collecting blood samples. J.L. Williams acknowledges the Ministry of Agriculture, Fisheries and Food (LK0602) for supporting this work.

Accepted 1 July 1998 Source/description: Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum.

It is considered to play an important role in excitation contraction coupling. However, structural analysis indicates that the triadin is co-localised with the ryanodine receptor/Ca 2+ release channel 1 . It contains a single transmembrane domain that separates the protein into a cytoplasmic and a luminal segment 2 . Wei and Campbell 3 suggest that triadin mediates the functional coupling between the ryanodine receptor/Ca 2+ release channel and calsequestrin in the lumen of the sarcoplasmic reticulum. We have isolated a phage, designated l5, from a porcine genomic DNA library cloned in lamba FIXII (Stratagene, Heidelberg, Germany) coding for triadin. The partial genomic structure of the triadin gene was determined by sequencing of phage l5. Within the last intron of the triadin gene a ((CT) 21 *(CA) 14 ) dinucleotide repeat was identified. The DNA sequence of the triadin gene was submitted to the EMBL data library under accession number AJ224992.

Primer sequences:

Forward primer (TRIMS1 f): 59-GCT TAC AGG AAT CTA CTG G-39

Reverse primer (TRIMS2 r): 59-GAC TTT AGG AGA CAC AGG-39

PCR conditions and analysis: PCR reactions were optimised using the `Ready to Go' system (Pharmacia, Freiburg, Germany). A master mix was prepared containing 400 mm of each primer (the forward primer was labelled with IRD 800 fluorescence dye; MWG Biotech, Ebersberg, Germany), frozen and stored at ±20 °C. DNA (100 ng) in 1 ml TE (10:1) was mixed with 12 ml master mix and one PCR bead was added (10 mm Tris±HCl pH 9 . 0, 50 mm KCl, 1 . 5 mm MgCl 2 , 200 mm of each dNTP and 1 . 5 U Taq polymerase). Cycling included an initial denaturation step at 94 °C for 10 min, 30 cycles of 1 min at 94 °C, 30 s at 55 °C, 72 °C for 1 min and a final step of 10 min at 72 °C using a Hybaid Omnigene cycler. PCR products were analysed by electrophoresis on 8% denaturing polyacrylamide gels using a LI-COR Gene ReadIR 4200 automated sequencer. Analysis of the bands was performed using the RFLPscan Plus 3 . 0 (Scanalytics, Virginia, VA) software. 1.

Mendelian inheritance: Co-dominant segregation of the polymorphisms was observed in 48 individuals from five families of Angler Saddlebacks (Fig. 1).

Chromosomal location: The porcine triadin gene was assigned to chromosome 1 by PCR screening of a porcine rodent somatic cell hybrid panel 4 .

Nine canine microsatellites N G Holmes Source/description: Partial genomic libraries were constructed in SmaI-cleaved M13mp10 using HaeIII-cut DNA from several dogs and screened with a poly (dA-dC).poly(dG-dT) probe (Pharmacia, Uppsala, Sweden) labelled using deoxyadenosine 59-[a-32 P]triphosphate (Amersham, Buckinghamshire, UK). Positive clones were sequenced using a Sequenase kit (Amersham, Buckinghamshire, UK). Primers flanking the dinucleotide (CA/GT) repeats were selected using the program PRIMERS (see Table 1).

PCR conditions: PCR conditions are shown in Table 1 with PCR carried out as described previously 2 .

Heterozygosity: Heterozygosity was calculated using the program PIC (see Table 1). A minimum of nine unrelated dogs of mixed breeds were typed for each microsatellite.

Allele sizes: Allele lengths were estimated by comparison with an M13 sequence ladder on 5% denaturing acrylamide-urea gels. The product amplified from the appropriate M13 clone was run at the same time.

Chromosomal location: Unknown.


Hang Zou

Youba Nait-Belaid

Michel Kieffer
email: michel.kieffer@l2s.centralesupelec.fr

Optimal Opponent Selection for Distributed Multi-Agent Self-Classification

Keywords: Distributed algorithms, Estimation, Multi-agent systems

This paper considers a mobile multi-agent system (e.g., a crowdsensing or crowdsourcing application) in which agents are characterized by their discrete-valued Level of Ability (LoA) at doing some sensing or data processing task. Agents are not aware of their LoA and are willing to estimate it without the help of a central classification authority, in order to determine whether their contribution will improve or degrade the performance of the global network. Using their estimated LoA, agents may then voluntarily restrain themselves to participate to the activity of the multi-agent system, without being banned by some central control authority.

For that purpose, agents, when they meet, perform pairwise comparison tests (PCT) able to determine which is the best agent of a pair of competing agents. Two maximum a posteriori (MAP) estimators of the LoA of each agent are proposed using the results of several PCTs. These MAP estimators are then employed to determine, for a given agent, the LoA of the next opponent that minimizes its probability of LoA estimation error. Simulations results show that the proposed optimal opponent selection approach provides better results than simply choosing opponents at random, or always choosing the opponents with the best LoA.

I. INTRODUCTION

In crowdsensing applications [START_REF] Borcea | Mobile Crowdsensing[END_REF], [START_REF] Zamora | A survey on smartphone-based crowdsensing solutions[END_REF], a large group of agents equipped with sensing and computing capabilities (smartphones, tablets, cars...) collaborate to collect data and extract information to measure, map, estimate [START_REF] Fagnani | A distributed classification/estimation algorithm for sensor networks[END_REF], [START_REF] Nihar | A permutation-based model for crowd labeling: Optimal estimation and robustness[END_REF], classify [START_REF] Luo | Distributed classification in peer-to-peer networks[END_REF], [START_REF] Hock | Communication-efficient classification in P2P networks[END_REF], or analyze any process of common interest [START_REF] Guo | Mobile crowd sensing and computing: the review of an emerginghumanpowered sensing paradigm[END_REF], [START_REF] Zamora | A survey on smartphone-based crowdsensing solutions[END_REF].

An important problem in such applications is to evaluate the Level of Ability (LoA), i.e., the performance, of the participating agents at doing sensing or data processing tasks, in order to keep only information coming from agents with the best LoA. For example, in a sensing application, agents may only have a very partial knowledge of the performance of their sensing device (biased or unbiased, very noisy or accurate) [START_REF] Chiuso | Gossip algorithms for simultaneous distributed estimation and classification in sensor networks[END_REF]. In crowdsourcing applications, agents may not be totally aware of their LoA at doing some task.

Most of the prior work assume that some byzantine agents are voluntarily injecting erroneous data in data collection and processing networks [START_REF] Kailkhura | Data falsification attacks on consensus-based detection systems[END_REF], [START_REF] Gentz | Data injection attacks in randomized gossiping[END_REF], or fake news in social networks to disturb the network [START_REF] Waiy | Identifying susceptible agents in time varying opinion dynamics through compressive measurements[END_REF]. Here, we assume that agents which may degrade the performance of the network are not aware of this behavior. Such situation may occur due to degraded sensors, or to a virus perturbing the sensors [START_REF] Suarez-Tangil | Evolution, detection and analysis of malware for smart devices[END_REF]. Nevertheless, agents are willing to detect whether the information they produce and transmit may degrade the performance of the network, before being banned by some central authority.

The aim of this paper is to help each agent in estimating in a distributed way its own LoA at doing the basic tasks required by some crowdsensing or crowdsourcing application. Agents with LoA not high enough may then, by themselves, restrain participating in the application. Most of the approaches addressing this problem consider centralized reputation-based mechanisms [START_REF] Yu | Reputation-based incentive protocols in crowdsourcing applications[END_REF], [START_REF] Kantarci | Reputation-based sensing-as-a-service for crowd management over the cloud[END_REF], [START_REF] Ren | SACRM: Social Aware Crowdsourcing with Reputation Management in mobile sensing[END_REF] to reject poorly performing agents based on previous results. Distributed approaches have also been proposed, see, e.g., [START_REF] Fagnani | A distributed classification/estimation algorithm for sensor networks[END_REF], [START_REF] Li | Distributed faulty node detection in delay tolerant networks: Design and analysis[END_REF]. The main advantage of the proposed approach is to help agents protecting their reputation in a decentralized way, since the detection is performed with the help of peers, and not by the network management authority.

For that purpose, following the idea of [START_REF] Li | Peerassisted individual assessment in a multi-agent system[END_REF], one assumes that two agents, when they meet, are able to perform a pairwise comparison test (PCT). PCTs provide some information about the agent with the best LoA in the pair of competing agents. Such PCTs are found, e.g., in weakly supervised learning and preference learning [START_REF] Furnkranz | Preference Learning[END_REF], [START_REF] Busa-Fekete | A Survey of Preference-Based Online Learning with Bandit Algorithms[END_REF], [START_REF]Pairwise Ranking Aggregation in a Crowdsourced Setting[END_REF], [START_REF] Graepel | Score-based bayesian skill learning[END_REF]. In [START_REF] Li | Peerassisted individual assessment in a multi-agent system[END_REF], each agent, using the proportion of PCTs for which it was deemed as the best agent, estimates its own LoA.

In this paper, one assumes that each agent can decide whether it will perform a PCT when meeting an other agent. This decision may depend on the estimate of their respective LoAs exchanged at the beginning of the meeting. Two MAP estimators of the LoA of an agent are then proposed using the results of a succession of PCTs. Then, this paper introduces a technique to determine, for a given agent, the LoA of the opponent with which the PCT will be the most informative, in the sense that the PCT outcome will minimize its probability of LoA estimation error.

The LoA estimation problem is presented in Section II. Section III describes two MAP estimators of the LoA from PCT outcomes. Section IV introduces a criterion to determine the LoA of the most informative opponent against which an agent has to perform its next PCT to minimize its LoA estimation error. Some simulation results are described in Section V before drawing conclusions in Section VI.

II. PROBLEM FORMULATION

The notations introduced in [START_REF] Li | Peerassisted individual assessment in a multi-agent system[END_REF] are briefly recalled here before describing the specific problem addressed in this work.

Consider a multi-agent system consisting of N a mobile agents. Each agent i has a given LoA θ i at doing some task. One assumes that the θ i s are realizations of independently and identically distributed (iid) discrete-valued random variables Θ i , i = 1, . . . , N a . The set of possible LoAs is L = {1, 2, ..., K}, with θ i = 1 representing the best LoA and θ i = K representing the worst one. The vector p = (p 1 , . . . , p K )

T gathers the a priori probabilities associated to each LoA.

One assumes that the agents are not aware of their LoA, and are willing to estimate it. For that purpose, two agents i and j, when they come in vicinity, can perform a PCT. The outcome y i,j of such PCT is modeled as a realization of a binary random variable Y i,j which distribution is conditioned by the LoAs of the competing agents, i.e.,

Pr (Y i,j = 1 | θ i , θ j ) = q (θ i , θ j ) . ( 1
)
Two types of PCTs have been considered in [START_REF] Li | Peerassisted individual assessment in a multi-agent system[END_REF]. One focuses here on the win-lose PCT, also considered in preference-based learning [START_REF] Furnkranz | Preference Learning[END_REF], such that

q (θ i , θ j ) = 1 -q (θ j , θ i ) . ( 2
)
Such PCT typically represents the outcome of a game between two players. The various q (θ i , θ j ) are gathered in a preference matrix [START_REF] Thurstone | A law of comparative judgement[END_REF], [START_REF] Bradley | Rank analysis of incomplete block designs: I. The method of paired comparisons[END_REF], [START_REF] Luce | Individual choice behaviour: A theoretical analysis[END_REF]

Q = ⎛ ⎜ ⎝ q (1, 1) . . . q (1, K) . . . . . . . . . q (K, 1) . . . q (K, K) ⎞ ⎟ ⎠ . ( 3
)
We assume that the time interval between two successive meetings follows an exponential distribution with an intercontact rate λ [START_REF] Zhu | Recognizing exponential inter-contact time in vanets[END_REF], [START_REF] Galluccio | Sociality-aided new adaptive infection recovery schemes for multicast DTNs[END_REF]. The population of agents is assumed well-mixed, i.e., for a given agent, the probability of meeting an agent with a given LoA θ is proportional to p θ .

Our aim in this paper, for each agent i, is to minimize the LoA estimation error considering a given number of PCT outcomes. For that purpose, during each meeting with an other agent, Agent i will have to decide whether it is worth performing a PCT. More precisely, considering the LoA estimate of the other agent, it has to determine whether a sufficient decrease of the estimation error of its LoA θ i is likely to be obtained from the PCT outcome.

III. MAP ESTIMATION OF AGENTS' LOA

Two cases are considered, depending on whether the LoAs of the opponents are known.

A. MAP estimator with unknown opponents' LoAs

Consider Agent i, which LoA θ i corresponds to a realization of the random variable Θ i . Agent i has performed n PCTs against different opponents without knowing their LoAs. The PCT outcomes are considered as realizations of n independent binary random variables Y , = 1, . . . , n with conditional probability given by [START_REF]Pairwise Ranking Aggregation in a Crowdsourced Setting[END_REF]. The MAP estimate of θ i using the vector of PCT outcomes y

(n) = (y 1 , . . . , y n ) T is θ i,MAP = arg max θ Pr Θ i = θ | Y (n) = y (n) . ( 4
)
In what follows, the random variables will be omitted when there is no ambiguity. Proposition 1: The MAP estimate of the LoA θ i of Agent i which has performed n independent PCTs with outcomes y 1 , . . . , y n against different opponents, without knowing their LoA, is

θ i,MAP = arg max θ s m θ (1 -s θ ) n-m p θ , (5)
with

s θ = k∈Θ q (θ, k) p k (6) and m = n =1 y . The proof of Proposition 1 is in Apprendix A.
Proposition 1 leads to a partition of the set {1, . . . , n} into K decision subsets

N (n) 1 , . . . , N (n) K defined as N (n) k = m ∈ {1, . . . , n} st s m k (1 -s k ) n-m p k > s m j (1 -s j ) n-m p j , ∀j = k (7)
and such that

n =1 y ∈ N (n) k ⇔ θ i,MAP = k.

B. MAP estimator with partly known opponents' LoAs

Here, one assumes that Agent i performs n = n u +n k PCTs against n different agents. The LoA of n u of them is unknown, the LoA of n k of them is known by Agent i. Without loss of generality, one assumes that the LoAs of the last opponents are known and are denoted as θ , = n u + 1, . . . , n. The PCT outcomes are gathered in y (n) = (y 1 , . . . , y n )

T .

With these assumptions, the MAP estimate of θ i is

θ i,MAP = arg max θ Pr Θ i = θ | y (n) , θ nr+1 , . . . , θ n . ( 8
)
Proposition 2: The MAP estimate of θ i from n = n u + n k PCT outcomes against n u opponents of unknown LoAs and n k opponents of known LoA θ nu+1 , . . . , θ n is

θ i,MAP = arg max θ s mu θ (1 -s θ ) nu-mu n =nu+1 y q θ, θ + (1 -y ) 1 -q θ, θ p θ , (9)
where

m u = nu =1 y . The proof of Proposition 2 is in Appendix A.
In this case, m = n =1 y is no more a sufficient statistic for the MAP estimator. Nevertheless, Proposition 5 still leads to partitions of the set {0, . . . , n u } into K subsets. The partitions depend on (y nu+1 , . . . , y n ) and (θ nu+1 , . . . , θ n ) and are expressed as

N (nu,n k ) k yn u +1, . . . , yn, θ nu+1 , . . . , θ n = {m ∈ {0, . . . , nu} st s m k (1 -s k ) nu-m n =nu+1 f y , k, θ p k > s m j (1 -sj) nu-m n =nu+1 f y , j, θ pj, ∀j = k . with f (y , k, θ ) = y q k, θ + (1 -y ) 1 -q k, θ .
Again, one has

nu =1 y ∈ N (nu,nk) k y nu+1 , . . . , y n , θ nu+1 , . . . , θ n ⇔ θ i,MAP = k. ( 10
)
IV. OPTIMAL OPPONENT SELECTION FOR PAIA Assume that Agent i has performed n PCTs. It aims at determining the LoA its n + 1-th opponent should have so as to minimize its probability of mis-classification once it has used the outcome of the n + 1-th PCT.

Assume that the n first PCTs are with outcomes y (n) = (y 1 , . . . , y n ), and are done without knowing the opponents' LoA. In the case Θ i = θ i , the probability of LoA estimation error is

p (n) e,i = Pr n =1 Y / ∈ N (n) θi |θ i , y (n) = 0 if n =1 y ∈ N (n) θi 1 else, (11)
since, once n =1 y is known, the outcome of the MAP estimator is deterministic.

Assume now that the n + 1-th opponent is chosen such that its LoA is Θ n+1 = θ n+1 . The expected probability of error p (n+) e,i before performing the n + 1-th PCT, when y (n) as well as the LoA of the n + 1 opponent are known but not y n+1 is

p (n+) e,θ i θ n+1 = Pr n =1 Y / ∈ N (n,1) θ i Yn+1, θ n+1 |θi, θ n+1 , y (n) = y n+1 ∈{0,1} Pr n =1 Y / ∈ N (n,1) θ i yn+1, θ n+1 , yn+1|θi, θ n+1 , y (n) = y n+1 ∈{0,1} Pr n =1 Y / ∈ N (n,1) θ i yn+1, θ n+1 |θi, θ n+1 , y (n) , yn+1
Pr yn+1|θi, θ n+1 , y (n) Since Pr y n+1 |θ i , θ n+1 , y (n) does not depend on y (n) , once θ i and θ n+1 are known, one has

Pr y n+1 |θ i , θ n+1 = f y n+1 , θ i , θ n+1 .
The first term in the sum is evaluated as [START_REF] Graepel | Score-based bayesian skill learning[END_REF]. One observes that p (n+) e,θi θ n+1 for a given value of θ i and θ n+1 only depends on n =1 y = m. To get the average (over all possible values of Θ i ) expected probability of error before performing the n + 1-th PCT, once the n + 1-th opponent has been chosen, one has to evaluate

p (n+) e θ n+1 = Pr n =1 Y / ∈ N (n,1) θi Y n+1 , θ n+1 |θ n+1 , y (n) = K θi=1 Pr n =1 Y / ∈ N (n,1) θi Y n+1 , θ n+1 , θ i |θ n+1 , y (n) = K θi=1 Pr n =1 Y / ∈ N (n,1) θi Y n+1 , θ n+1 |θ i , θ n+1 , y (n) Pr θ i |θ n+1 , y (n) . ( 12
)
One has to determine Pr θ i |θ n+1 , y (n) . Knowing the LoA of the n + 1-th opponent, without knowing the outcome of the n + 1-th PCT does not bring more information on θ i than the outcomes of the last n PCTs. Thus Pr θ i |θ n+1 , y (n) = Pr θ i |y (n) may be evaluated using Proposition 1. As a consequence, to minimize the probability of LoA estimation error after n + 1 PCTs, an agent has to select the n + 1-th opponent which LoA θ n+1 minimizes

p (n+) e θ n+1 = K θi=1 p (n+) e,θi θ n+1 Pr θ i |y (n) . ( 13
)
In [START_REF] Kailkhura | Data falsification attacks on consensus-based detection systems[END_REF], the impact of y (n) is only through the sum of its components m = n =1 y . The decision on θ n+1 will thus only depend on m.

These derivations are easily extended to the selection of the n + 1-th opponent that minimizes the probability of LoA estimation error assuming that n = n u + n k PCTs have been performed against n u agents with unknown LoA and against n k agents selected depending on their LoA.

V. SIMULATION RESULTS

One considers K = 4 different LoAs and the following preference matrix

Q = ⎡ ⎢ ⎢ ⎣ 0.5
0.855 0.958 0.988 0.145 0.5 0.855 0.958 0.042 0.145 0.5 0.855 0.012 0.042 0.145 0.5

⎤ ⎥ ⎥ ⎦
The a priori information on the proportions of agents is p = (0.5, 0.25, 0.125, 0.125) T .

A. Evolution of the decision regions N (n) k

Using [START_REF] Fagnani | A distributed classification/estimation algorithm for sensor networks[END_REF], one is able to plot the various decision regions N (n) k as a function of the number n of PCTs performed against agents with unknown LoA. Figure 1 (top) shows that as soon as n is large enough, the decision regions do not overlap.

B. Optimal LoA of next opponent

Using [START_REF] Kailkhura | Data falsification attacks on consensus-based detection systems[END_REF], one is able to evaluate the error probability p (n+) e θ n+1 as a function of m, see Table I in the case n = 10, i.e., when an agent has to select its most informative opponent after having performed n = 10 PCTs as a function of m, the number of PCTs won.

One observes that for several values of m, the next opponent may be chosen randomly, without any effect on the LoA estimation error. In what follows those values of m are indicated to belong to Class R (random agent). Such values of m belong to the middle of the decision regions N (n) k , where the next PCT will not affect the LoA estimate, see also Figure 1 (bottom).

In several cases, the most informative opponent should be with θ n+1 = 1. The corresponding values of m are indicated to belong to Class B (best agent). Finally, here, when m = 4, the most informative opponent is such that θ n+1 = 2. Values of m for which the most informative opponent is not such that θ n+1 = 1 and cannot be chosen randomly are indicated to belong to Class O (other agents). Values of m belonging to Class B and O are close to the boundaries of the decision regions.

Table II shows the proportions of values of m belonging to each class. One observes that when n gets large, the next opponent may be chosen at random without significantly affecting the performance of the LoA estimation algorithm. One may alternatively always select an agent such that θ n+1 = 1. Except for small values of n, this choice minimizes the LoA estimation error in 95% of the cases when n = 40 and in more than 97% of the cases when n = 80. This confirms the intuition found in [START_REF] Li | Peerassisted individual assessment in a multi-agent system[END_REF] that performing PCT always against opponents with LoA θ = 1 is a reasonable choice.

To evaluate the benefits of selecting the most informative opponents in terms of probability of LoA estimation error, one has considered N a = 1000 agents randomly moving in a unit square, with LoA randomly chosen according to the a priori probability vector p. Figure 2 represents the evolution of the proportion of agents having erroneously estimated their LoA as a function of the number of PCTs performed. Results are averaged over 100 simulations. Three cases are considered: in a first case, agents always perform a PCT when they come in vincinity to an other agent. In the second case, after n u PCTs against random agents, PCTs are only performed when the encountered agent estimates its LoA is θ = 1. The last case is similar to the second case, but now, the opponent with estimated LoA minimizing the probability of LoA estimation error is considered.

The estimation performance is much better in the last two cases. Selecting the agent with an estimated LoA θ = 1 provides slightly better results than selecting an opponent with LoA minimizing the probability of LoA estimation error. This may be due to the increased robustness of the second approach when the LoA is still not perfectly estimated.

VI. CONCLUSION

This paper presents two MAP estimation algorithms to determine the LoA of agents at doing some task in a crowsensing application . These estimators take as input the outcomes of PCTs performed with other agents. When the LoA of opponents is not known, the proportion of PCTs won against randomly chosen opponents is a sufficient statistic to estimate the LoA of the considered agent. When the LoA of some opponents is known, the expression of the MAP estimator is less straightforward.

This paper introduces a criterion to choose the LoA of the next opponent of a given agent to minimize its LoA estimation error. Using this criterion, one observes that in many cases, performing PCTs with opponents with the best LoA provides very good results, as already experienced in [START_REF] Li | Peerassisted individual assessment in a multi-agent system[END_REF]. Performing PCTs with opponents at random leads to an increased probability of error. One observes that the selection of an opponent with a specific LoA is important when the current LoA estimate remains uncertain. Significant work remains to be done to address situations where only the preference matrix is provided but no knowledge about the initial proportions of agents, or vice-versa. One has also to evaluate the impact of the selection of the LoA of the two or more next opponents on the LoA estimation error.

APPENDIX One has

Pr Θ i = θ | y (n) ∝ Pr y (n) | Θ i = θ Pr (Θ i = θ) [START_REF] Kantarci | Reputation-based sensing-as-a-service for crowd management over the cloud[END_REF] Since the PCT outcomes have been assumed as independent,

Pr Θ i = θ | y (n) ∝ n =1 Pr (y | Θ i = θ) p θ . ( 15
)
Let Θ be the random variable representing the LoA of the -th opponent of Agent i. One has

| Θ i = θ) = K k=1 [y q (θ, k) + (1 -y ) (1 -q (θ, k))] p k = y s θ + (1 -y ) (1 -s θ ) (16)
for any = 1, . . . , n, where one uses [START_REF] Chiuso | Gossip algorithms for simultaneous distributed estimation and classification in sensor networks[END_REF]. Finally, since y ∈ {0, 1}, introducing ( 6) in [START_REF] Li | Distributed faulty node detection in delay tolerant networks: Design and analysis[END_REF], one gets

Pr (Θ i = θ | y 1 , . . . , y n ) ∝ s m θ (1 -s θ ) n-m p θ . ( 17
)
The a posteriori probability in (8) may be rewritten as (

Pr Θ i = θ | y (n) ,
) 19
The first products are obtained from Proposition 1. A given term of the second product may be expressed as Pr y | θ, θ = y q θ, θ + (1 -y ) 1 -q θ, θ . (20) Finally, ( 9) is obtained combining [START_REF] Luce | Individual choice behaviour: A theoretical analysis[END_REF], [START_REF] Ren | SACRM: Social Aware Crowdsourcing with Reputation Management in mobile sensing[END_REF], and [START_REF] Nihar | A permutation-based model for crowd labeling: Optimal estimation and robustness[END_REF].

Fig. 1 .

 1 Fig. 1. Decision regions N (n) k (top) and optimal LoA of the next opponent (bottom) as a function of the number of PCTs and of the number of PCTs won

  Pr (y | Θi = θ) = K k=1 Pr y l , Θ = k | Θ i = θ = K k=1 Pr y | Θ i = θ, Θ = k Pr Θ = k | Θ i = θ .Since the -th opponent is randomly chosen, one hasPr Θ = k | Θ i = θ = Pr Θ = k =p k and Pr (y

18 )

 18 θ nu+1 , . . . , θ n ∝ Pr y (n) | Θ i = θ, θ nu+1 . . . , θ n Pr Θ i = θ|θ nu+1 . . . , θ n (The knowledge of the LoAs of the n k last opponents does not provide any information about the LoA of Agent i, thusPr Θ i = θ|θ nu+1 , . . . , θ n = Pr (Θ i = θ) .Moreover, since the PCT outcomes have been assumed as independent, one hasPr Θ i = θ | y (n) , θ nu+1 , . . . , θ n ∝ nu =1 Pr (y | Θ i = θ) n =nu+1Pr y | θ, θ p θ .

TABLE I LOA

 I ESTIMATION ERROR PROBABILITY AND OPTIMAL OPPONENT WHEN n = 10; * INDICATES THAT ALL LOAS ARE OPTIMAL Fig. 2. Proportion of erroneous LoA estimation as a function of the number of PCTs, when PCTs are against (i) random opponents, (ii) opponents estimating their LoA as the best (except for the first 10 PCTs), and (iii) opponents with estimated LoA minimizing the probability of LoA estimation error.

						Number m of PCTs won
	opponent's LoA	0	1	2	3 Percentage of LoA estimation error 4 5 6 7	8	9	10
	θ n+1 = 1	1.1	3.59	2.7	1.7	3.2	5.4	5.0	1.2	0.4	0.06	0.004
	θ n+1 = 2	1.1	3.6	3.1	3.5	3.1	5.4	8.5	1.2	0.4	0.06	0.004
	θ n+1 = 3	1.1	3.6	4.4	6.5	7.9	5.4	9.5	1.3	0.4	0.06	0.004
	θ n+1 = 4	1.1	3.6	5.7	8.0	8.0	5.4	9.9	1.3	0.4	0.06	0.004
	optimal LoA	*	*	1	1	2	*	1	1	*	*	*