380 MOLECULAR GENETIC MARKERS

Ten new cosmid-derived bovine microsatellite markers J 1. Williams', A L G Morgan', G GuBrin', B G D Urquhart' 'Division of Molecular Biology, Roslin Institute (Edinburgh) Midlothian EH25 gPS, UK. 'Laboratorie de GenBtique Biochemique rt (IP CytogBn6tique. INRA-CRJ. 78352, Jouy-en-Josas, Cedex, France A ( w p t e d 20 Marrh 1996

Source/description: Microsatellite-containing cosmid clones were selected from a cosmid library by screening with pc~l!~(dAdC).poly(dGdTj (Pharmacia). Clones containing satellite sequence were eliminated by a secondary screen with the insert fumi a clone containing sequences similar to the bovine 1.709 satellite.' Sequelice flanking the microsatellite repeat was obtained i n one direction by priming from the repeat itself using a modification of the Browne & Litt (1991) method' and by cycle s tymi r ing with \'ent polymerase (New England Biolabs). A primer W;IS designed from the sequence obtained to allow sequencing through the microsatellite repeat and into the sequence beyond,

ond primer to be designed to PCR amplify the niit:rosatelli te containing region. ?%'R conditions: One hundred ng genomic DNA was amplified in a 13 ul reaction containing 10pmols each primer, 0.2 mM dNTPs, 1.25inM MgC1,. 50mK KCI, 10mM Tris C1, 0401% gelatin and ihZ,7 ?i Taq polymerase (Boehringer). A two-step PCR cycle of 94°C il min) denaturation. 60°C (5 min) annealing and elongation was used for 3 0 cyclits. Ilenrleliorr inheritance arid chromosomal location: Segregation of a!loles was confirmed by typing the 327 individuals comprising the 15 families of the International Bovine Reference Panel (IBRP).? kfarkers were assigned to bovine chromosomes by two-point linkage i t i l a l y ~ k ( ( . R ~ M A I ' ) with thti exception of JABS. which showed limited Iwterozygosity (Table 1). .Viirzibf:r of olleles: As genotyping was carried out on the IBW, which is made u p of full-sib famiIies with only a small number of iirirelated parents, the number of alleles observed in the IBRP is given, but not froquency or PIC (Table 1). S\wtPny mnpping: The markers were assigned to syntenic groups using the somatir. hybrid cell panel from INRA4.' Results are given

in Table 1 for the markers with the exception of JAB5 and JAB10. which gave ambiguous results. The linkage and synteny assignments are all consistent. Synteny mapping of JAB9 to U1 places this marker on chromosome 16.

Acknowledgements: This work was supported by the Ministry of Agriculture, Fisheries and Food (OC9414). We are grateful to Dr W. Barendse for carrying out linkage analysis using the Cattle Genotypic Database. References 1 Skowronski J. et al. (1984)JMol Bml 177, 399-416. 2 Browne D.L. & Litt M. (1991) NucIAcids Res 20,141. 3 Barendse W et al. (1994) Anim Genet 25(suppl. 2), 44. 4 Guerin G. eta!. (1994) Anini Genet 25,31-5. 5 Vaiman D. et al. (1994) Mamm Genome 5,OO-0.

Correspondence: J L Williams

Genetic mapping of 26 cosmid-derived bovine microsatellite markers I L Williams', B G D Urquhad, W Barendse*, L Ferretti3 Division of Molecular Biology, Roslin Institute (Edinburgh)

Midlothian EH25 SPS, UK; 'Molecular Animal Centre, University of Queensland, Brisbane QLD 4072, Australia; 31DVGA-CNR, via Celoria 10, 1-20133, Italy Accepted 26 June 1996

Source/description: Microsatellite-containing cosmid clones were selected from a cosmid library by screening with poly(dA.dC).poly(dG.dT). Clones containing satellite sequence were eliminated by a secondary screen with the insert from a clone containing sequences similar to the bovine 1.709 satellite.' Sequence flanking the microsatellite repeat was obtained in one direction using cycle sequencing primed from the repeat itself as described by Ferretti et a].' A primer was designed from the

'I'able 1

Chromosomal i ~1::lIs Repeat Size of Number Synteny location

type PCR product of alleles group linkage

G GCT GGG ATT CC (AC)i, 224 7 u19 BTA15 -

I ) 1 :TSJR 2 , G AGG CTC ?.CA GCd G

. TAC TTG GGC AC (AC], 27 bases (AC], 220 3 U3 BTAS ATC TSG AGA GGC

229 3 u11 BTA13

8 U16 BTAll

f4B111 1 - - _ < :Ti- b . G GAG GGC ATG AC [AC),_ 244 6 - BTAlO 11104ih L -i 5 A Z L . L G GGT TGC TC

B 1996 International Socirtv for Animal Genetics, Animal Genetlcs 27, 371-383