Identification of Cryptic Allele for Merle Patterning in Dogs by Molecular Genetics Methods / Identifikacija Skrivenog Alela Za Mramoriranu Šaru Pasa Pomoću Metoda Molekularne Genetike

Abstract

Merle patterning in dogs, caused by the insertion of a short interspersed element (SINE) in the genetic structure of SILV gene, is characterized by patches of diluted pigment intermingled with normal melanin. Sequencing analyses of SINE element localized in the canine SILV gene discovered a variability of the poly (A)-tail length which is responsible for the different expression of merle pattern. The SINE element with the length of poly(A)-tail between 91 - 101 nucleotides is responsible for the merle phenotype with all characters of merle pattern. On the contrary the dogs which have SINE element with the shorter length of poly(A) tail between 54-65 nucleotides are referred as cryptic merles without expresion of Merle pattern. The aim of this study was to improve molecular genetics method for the detection of cryptic allele for merle patterning in dogs. A total of 40 dogs of four breeds - Border collie, Shetland sheepdog, Australian Shepherd dog, and Chihuahua were used in this study. Canine genomic DNA was isolated from samples of whole blood and buccal cells by commercial column kit. Detection of merle (M), cryptic merle (Mc) and non-merle (m) alleles was done using M13-tailed primer protocol and two different allele-sizing methods for the verification of the electrophoresis result. In the analyzed population of dogs were detected 20 dogs with non-merle genotype mm, 17 dogs with merle genotype Mm, 2 dogs with double merle genotype MM and one dog with merle phenotype but with the presence of cryptic merle allele Mc with the consequential genotype MMc.Mramorirana šara pasa, nastala umetanjem kratkih ponovaca elemenata (SINE) u genetsku strukturu SILV gena, karakteriše se prisustvom slabije pigmentisanih polja pomešanih sa poljima normalne pigmentacije. Analizom SINE elementa lokalizovanog u SILV genu pasa sekvencioniranjem otkrila je varijabilnost poli(A) repa koji je odgovoran za različitu ekspresiju mramorirane šare. SINE element sa poli(A) repom dužine od 91 do 101 nukleotida je odgovoran za mramorirani fenotip sa svim karakterima mramorirane šare. Nasuprot tome, psi koji poseduju SINE element sa kratkim poli(A) repom dužine od 54 do 65 nukleotida su opisani kao prikriveni mramorirani bez ekspresije mramorirane šare. Cilj ove studije je poboljšanje metoda molekularne genetike za detekciju skrivenog alela za mramoriranu šaru pasa. Ukupno 40 pasa četiri rase: border koli, šetlandski ovčar, australijski ovčar i čivava bilo je uključeno u studiju. Genom DNK pasa bio je izolovan iz uzoraka pune krvi i ćelija sluznice usne duplje bio je izolovan pomoću komercijalnih kitova. Detekcija mramoriranog (M), prikriveno mramoriranog (Mc) i ne-mramoriranog (m) alela je izvršena pomoću „M13-tailed“ prajmera protokola i dve različite metode određivanja veličine alela za verifi kaciju rezultata elektroforeze. U analiziranoj populaciji pasa otkriveno je 20 pasa sa ne - mramoriranim genotipom mm, 17 pasa sa mramoriranim genotipom Mm, 2 psa sa duplim mramoriranim genotipom MM i jedan pas sa mramoriranim fenotipom, ali sa prisustvom prikrivenog mramoriranog alela Mc sa posledičnim genotiom MMC.