Fast method for identifying inter- and intra-species Saccharomyces hybrids in extensive genetic improvement programs...

Area of Science:

• Microbiology
• Yeast Genetics
• Molecular Biology

Background:

• Spore-to-spore mating is crucial for generating improved homothallic wine yeast hybrids.
• Low spore viability and haplo-selfing often hinder successful hybrid selection.

Purpose of the Study:

• To develop a two-step molecular strategy for selecting inter- and intra-species Saccharomyces hybrids.
• To overcome common challenges in yeast hybridization, such as false positives from haplo-selfing.

Main Methods:

• Colony screening PCR (csPCR) using discriminative gene markers to select hybrids and discard homozygous diploids.
• Recursive streaking and conventional PCR to differentiate true hybrids from progenitor cell admixtures.
• Utilizing internal transcribed spacer (ITS) PCR with endonuclease digestion (HaeIII, RsaI) and minisatellite region analysis (PIR3, HSP150, DAN4) for hybrid validation.

Main Results:

• Successfully selected six inter-species Saccharomyces cerevisiae × Saccharomyces uvarum hybrids from 64 crosses.
• Validated six intra-species Saccharomyces cerevisiae hybrids from 34 crosses using cost-effective agarose gel electrophoresis.
• Demonstrated the efficiency of csPCR and subsequent analyses in identifying stable hybrids.

Conclusions:

• The proposed protocols significantly reduce the need for extensive DNA extractions.
• The method prevents misinterpretations arising from haplo-selfing, ensuring accurate hybrid identification.
• These easily implementable techniques offer a cost-effective, rapid, and reproducible approach for marker-assisted selection of yeast hybrids.