Animal Genetics, SHORT COMMUNICATION

Equine plasminogen polymorphism: allelic frequencies in 23 breeds

1993,24,437-438

A T Bowling, M C T Penedo, L Gordon, K Bell

Summary 1 mm) for 30min approximately 1 cm from the

A T Bowling M C T Penedo L Gordon Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, California 95616- 8744, USA K Bell Australian Equine Blood Typing Research Laboratory, University of Queensland, Australia

A modified procedure for detection of the two alleles of equine plasminogen using Western blotting methods following polyacrylamide gel isoelectric focusing is described. Gene frequen- cies in 23 breeds and Equus przewalskii are pro- vided.

Keywords: horse genetics, plasma protein, plas- minogen, polymorphism, allelic frequencies, Equus przewalskii

Polymorphism for equine plasminogen (PLG), the proenzyme of plasmin (E.C.3.4.4.14), was first described by Weitkamp et al. (1983). The polymorphism was demonstrated in plasma by agarose isoelectric focusing (IEF) and immunofixation with a goat anti-human plas- minogen antibody. Analysis of mating data showed the autosomal codominant inheritance of the alleles, PLG' and PLG'. Using a polyacry- lamide gel technique and standard Western blot- ting methods we obtained allelic frequencies for 23 domestic horse breeds and the closely related species E. przewalskii.

Isoelectric focusing gels (280 mm x 130 mm x 0.5mm), pH 5-8, were cast between two glass plates in a manner similar to that described by Pollitt & Bell (1983). The composition of the solution to prepare one gel was 5.0ml acry- lamide (30%T, 3%C), 3ml glycerol, 0.6ml ampholine pH 5-7, 1.2ml ampholine pH 6-8, 20.3 ml distilled water, 200 p1 ammonium per- sulphate and 30 p1 TEMED. Isoelectric focusing was performed on a flatbed apparatus (LKB Ultraphor, model 2217) with three electrodes (common anode) at 10°C and 6 W constant power. The anolyte and catholyte were 0 . 5 ~ acetic acid and 0.5 M sodium hydroxide respec- tively and prefocusing was carried out for 20min. Sera or plasmas were applied on filter paper inserts (Whatman 3MM, 4 mm x 3 mm x

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anode. The electrophoresis was continued for a further 70min. The plasminogen bands were identified by print blotting of proteins onto nitrocellulose (Trans - Blot@ transfer medium 0.45 p, 45 min, 37OC) and immunoblotting with rabbit antihuman PLG (1:250 Atlantic Antibod- ies, Incstar) and goat anti-rabbit IgG alkaline conjugated antibody (Vector Laboratories).

Allelic frequencies (Table I) were calculated by direct allele counting from phenotypes. Observed and expected phenotypes did not show significant deviations from expected Hardy-Weinberg proportions by xz testing (data not shown).

Table 1. Allelic frequencies for PLG variants in horses ~ ~~

Breed n PLG' *

Andalusian Arabian Belgian Curly Dutch Warmblood Friesian Haflinger Hanoverian Icelandic Lippizan Miniature Morgan Norwegian Fjord Paso Fino Peruvian Paso Quarter Horse Saddlebred Shire Standardbred Tennessee Walking Horse Thoroughbred Trakehner Welsh Pony Equus przewalskii

141 106 108 109 39 31 18 54 196 58 114 133 77 113 130 236 109 116 102 167 284 85 21 40

0.96 0.93 0.56 0.91 0.91 0.87 1.00 0.90 0.69 0.59 0.64 0.84 0.74 0.94 0.97 0.91 0.76 0.81 0,87 0.98 0-85 0.86 0.81 1.00

Correspondence: Dr A.T. Bowling.

Accepted 4 August 1993

*Frequency of the alternative allele (PLG') is one minus the value given here. n: number tested.

437

438 Boding, Penedo. Cordon, Bell

Acknowledgements

Samples from E. Przewalskii Provided through courtesy of Dr O.A. Ryder, San Diego Zoological Society and Dr W. Zimmermann, Cologne Zoo.

tease inhibitory system in thoroughbred horse plasma by two dimensional (ISODALT) elec- trophoresis. I. Protein staining. Animal Blood Groups and Biochemical Genetics 14, 83-105.

Weitkamp L.R., Costello-Leary P. & Guttormsen S.A. (1 983) Equine marker genes: polymorphism for plasminogen. Animal Blood Groups and Biochemi-

References cal Genetics 14, 219-23.

Pollit C.C. & Bell K. (1983) Characterization of the pro-