Interlaboratory comparison of fig (Ficus carica L.) microsatellite genotyping data and determination of reference alleles

Abstract

Microsatellites have been identified as the marker of choice in plant genotyping projects. However, due to length discrepancies obtained between different laboratories for the same allele, interlaboratory comparison of fingerprinting results is often a difficult task. The objectives of this study were to compare genotyping results of two laboratories, to evaluate genetic parameters of microsatellite markers and to determine reference allele sizes for fig cultivars from the Istrian peninsula. Genotyping results of ninety fig (Ficus carica L.) accessions were comparable between the laboratories despite differences observed when comparing electropherograms of different capillary electrophoresis systems. Differences in lengths of the same alleles were detected due to different PCR methods and laboratory equipment, but the distances between alleles of the same locus were preserved. However, locus FSYC01 exhibited one allele dropout which led to misidentification of 28 heterozygotes as homozygote individuals suggesting this locus as unreliable. Allele dropout was assigned to the tail PCR technology or to a touchdown PCR protocol. Genotypes of twenty-four reference cultivars from the Istrian peninsula were confirmed by both laboratories. These results will contribute to the usage of markers with greater reliability, discrimination power and consequently, to more reliable standardization with other fig genotyping projects.Key words: microsatellite marker; reference genotype; interlaboratory comparison; Ficus carica L. IZVLEČEK MEDLABORATORIJSKA PRIMERJAVA REZULTATOV GENOTIPIZACIJE FIGE Z MIKROSATELITSKIMI MARKERJI (Ficus carica L.) IN DOLOČITEV REFERENČNIH ALELOVMikrosateliti so se izkazali kot zelo uporabni markerji pri genetskih raziskavah rastlin. Zaradi odstopanj dolžin enakih alelov v različnih laboratorijih, je primerjava rezultatov med laboratoriji pogosto težavna. Namen raziskave je bil primerjati rezultate genotipizacije dveh laboratorijev, ovrednotiti genetske parametre mikrosatelitskih markerjev in določiti dolžine referenčnih alelov za sorte fig istrskega polotoka. Rezultati genotipizacije devetdesetih vzorcev fige (Ficus carica L.) so bili primerljivi med laboratorijema, kljub razlikam, ki smo jih opazili pri primerjavi elektroferogramov različnih sistemov kapilarnih elektroforez. Razlike med dolžinami enakih alelov med laboratorijema so bile odkrite zaradi različnih metod PCR in analitske opreme, vendar pa so bile razlike med aleli istega lokusa ohranjene. Pri lokusu FSYC01 smo ugotovili izpad alela, kar je privedlo do napačne identifikacije; namesto 28 heterozigotov smo posameznike določili kot homozigote. Ugotovljena lastnost nakazuje na nezanesljivost lokusa FSYC01. Izpad alela smo pripisali uporabi ekonomične metode PCR ali uporabi protokola PCR s postopnim nižanjem temperature prileganja začetnih oligonukleotidov. Genotipi štiriindvajsetih referenčnih sort istrskega polotoka so bili potrjeni v obeh laboratorijih. Rezultati raziskave bodo prispevali k uporabi bolj zanesljivih mikrosatelitskih m...