Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8S rRNA gene and the two internal transcribed spacers

Abstract

Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.

Isolation and molecular identification of wine yeasts from a Brazilian vineyard

“…Sequence analysis of the ITS-5.8S region or the D1/D2 domains in the large 26S subunit of rDNA are also used in identification tests (Baleiras Couto et al 2005;Martorell et al 2006). These molecular techniques are useful for rapid identification of yeasts in wine ecosystems (Capece et al 2003;Sun et al 2009) and also in other foods (Heras-Vazquez et al 2003;Rómo-Sánchez et al 2010).…”

Screening and regulation of alkaline extracellular protease and ribonuclease production of Yarrowia lipolytica strains isolated and identified from different cheeses in Turkey

“…This rDNA region is sufficiently variable to allow reliable identification of yeast species. More recently, the differences in the rRNA internal transcribed spacer (ITS) have been used to identify yeast species (Las Heras-Vazquez et al 2003). The ITS region is relatively short (500-800 bp) and can be easily amplified by PCR using universal single primer pairs that are complementary to conserved regions within the rRNA subunit genes (White et al 1990), and the ITS region may be highly variable among morphologically distinct species and so ITS-generated RFLP restriction data can be used to estimate genetic distances and provide characters for systematic and phylogenetic analysis (Bridge et al 1998;Petersen et al 2001;Arias et al 2002;Alvarez-Martin et al 2007).…”

Taxonomical classification of yeasts isolated from kefir based on the sequence of their ribosomal RNA genes

“…unisporus has been previously characterized in kefir (Marquina et al 2002) and has also been found in naturally fermented fruit juices (Las Heras-Vazquez et al 2003). S. humaticus is a recently described species (Mikata et al 2001).…”