VALIDATION OF YEAST IDENTIFICATION BY IN SILICO RFLP

Abstract

Reliability, high reproducibility and high throughput are advantages of genetic identification methods over conventional methods based on phenotypic characteristics. In our work, we showed that the PCR‐RFLP of internal transcribed spacer (ITS) of ribosomal DNA (rDNA) method can be a very powerful identification or validation method, if it is combined with computer‐based restriction analysis of sequences stored in broadening databases. We isolated 197 yeast samples from grape berries and fermenting grape must and identified 13 species. For identification conventional methods, D1/D2 sequencing and PCR‐RFLP of ITS regions were used. Because in our case, conventional methods were found insufficient for reliable and fast identification, in silico RFLP was applied; using appropriate software, enzyme restriction patterns on the agarose gel were simulated and compared with the restriction pattern obtained with PCR‐RFLP. With this in silico approach we managed to identify or validate the identification of all 13 species. Our results show that natural isolates of ascomycetous or basidiomycetous yeasts can be rapidly (in 3 days) and reliably identified by in silico RFLP. PRACTICAL APPLICATIONS In silico RFLP will be highly useful in the identification process of yeasts that could be reliably identified by conventional methods. For its speed, simplicity, high reproducibility and high throughput if compared to conventional identification methods, the method will be chosen when rapid validation or identification of yeast samples is needed. Another application, determining the enzymes, which distinguishes one species from another using RFLP, is obvious. Moreover, a database of ITS sequences will have great value at phylogenetic classification of new undescribed species.We believe that a program such as CandID with restriction databases of other yeast species can enrich the microbiological practice in the future.

The marula and elephant intoxication myth: assessing the biodiversity of fermenting yeasts associated with marula fruits (Sclerocarya birrea)

“…We sought to analyze the intra-species genetic variation of marula isolates, which showed a significantly high percentage of similarity of their ITS1-5.8S-ITS4 region. In-Silico PCR-RFLP restriction patterns are comparable to restriction patterns obtained in vitro (Raspor et al 2007 ). The precision of discrimination of PCR-RFLP has been considered to be parallel to sequencing analysis and therefore as an alternative method for species identification and delimitation (Pham et al 2011 , da Fonseca Meireles et al 2022 ) and the better option in instances where rapid validation or identification is needed considering its simplicity, speed, high reproducibility and high throughput (Raspor et al 2007 ).…”

“…In-Silico PCR-RFLP restriction patterns are comparable to restriction patterns obtained in vitro (Raspor et al 2007 ). The precision of discrimination of PCR-RFLP has been considered to be parallel to sequencing analysis and therefore as an alternative method for species identification and delimitation (Pham et al 2011 , da Fonseca Meireles et al 2022 ) and the better option in instances where rapid validation or identification is needed considering its simplicity, speed, high reproducibility and high throughput (Raspor et al 2007 ). While da Fonseca Meireles et al ( 2022 ) used one restriction enzyme to differentiate yeasts of different genera and species, here we further increase the precision by using multiple restriction enzyme to differentiate within strains .Single restriction enzymes often have poor resolution (similar restriction profiles) in differentiating closely similar sequences of strains, we further revealed that use of a combination of different restriction enzymes was sufficient to resolve the genetic differences.…”

The effect of fungicides on yeast communities associated with grape berries

“…AF075498); Fell et al, 2000], and for those, the sequencing of the ITS1-5.8S rRNA gene-ITS2 regions was required. Further, the species identities were confirmed by in silico RFLP of the ITS regions using the PDRAW 1.0 software (Kjeld, 2006) as suggested by Raspor et al (2007). We isolated 24 species belonging to 13 genera, of which nine belonged to basidiomycetous yeasts, mainly red-pigmented species of Rhodotorula and Sporidiobolus and nonpigmented or lightly pigmented Cryptococcus sp.…”

Minimizing enzymes to diferenciate between species

“…One is limited to the forecast of electrophoretic patterns [8], the other presents a program to assess the utility of a fixed set of endonucleases to distinguish between a given set of sequences [14]. However, the integration of all the available data in a comprehensive in silico approach, targeting optimality in identification by ARDRA is still to be proposed.…”