Recombination between homoeologous chromosomes of lager yeasts leads to loss of function of the hybrid GPH1 gene

Abstract

Yeasts used in the production of lagers contain complex allopolyploid genomes, resulting from the fusion of two different yeast species closely related to Saccharomyces cerevisiae and Saccharomyces bayanus. Recombination between the homoeologous chromosomes has generated a number of hybrid chromosomes. These recombination events provide potential for adaptive evolution through the loss or gain of gene function. We have examined the genotypic and phenotypic effects of one of the conserved recombination events that occurred on chromosome XVI in the region of YPR159W and YPR160W. Our analysis shows that the recombination event occurred within the YPR160W gene, which encodes the enzyme glycogen phosphorylase and generates a hybrid gene that does not produce mature mRNA and is nonfunctional due to frameshifts in the coding region. The loss of function of the hybrid gene leads to glycogen levels similar to those found in haploid yeast strains. The implications for the control of glycogen levels in fermentative yeasts are discussed.

FIG. 1.

(A) Types and numbers of chromosome XVI in CMBS-33. Gray regions are derived from S. cerevisiae, and white regions are derived from S. bayanus. The ends of all four types of chromosome XVI contain S. cerevisiae-like genes. The common white region encompasses genes YPR160W to YPR190C. Genomic DNA from S. cerevisiae (lane 1), lager strain 6701 (lane 2), and lager strain CMBS-33 (lane 3) were separated by pulsed-field electrophoresis. The gels were either stained with ethidium bromide (B) or probed with DIG-labeled DNA to ScYPR159W (C) or ScYPR160W (D). The arrow points to the hybridizing band.

FIG. 2.

Bacterial artificial chromosome clones (1 to 5) containing regions of chromosome XVI were hybridized to DIG-labeled DNA probes amplified from either S. cerevisiae (Sc) or S. bayanus (Sb) genomic DNA using the ORF-specific primers.

FIG. 3.

(A) Gene organization of YPR159W and YPR160W in S. cerevisiae (top panels) and the lager strain CMBS-33 (bottom panels). The gray regions are S. cerevisiae-like and the white regions S. bayanus-like. The coordinates for the S. cerevisiae genes on chromosome XVI are shown, and arrows indicate the direction of transcription. (B) Percentage sequence identity of the first 800 nucleotides of the CMBS-33 YPR160W gene to S. cerevisiae (gray) and S. bayanus (black) homologues. (C) Comparison of YPR160W DNA sequences of CMBS-33 (Lager), S. cerevisiae (S.c), and S. bayanus (S.b.) in the region where recombination has occurred. Nucleotides shown in black are common to all three species, those in red are identical in S. cerevisiae and CMBS-33, those in blue are identical in S. bayanus and CMBS-33, and those in pink are identical in S. cerevisiae and S. bayanus. The nine extra nucleotides at positions 450 to 459 present in CMBS and S. bayanus but absent in S. cerevisiae are shown. The in-frame stop codon in the CMBS sequence is underlined. Numbers on the right indicate the nucleotide position relative to the start codon (not shown) for the lager YPR160 gene. The full DNA sequence is shown in Fig. S1 in the supplemental material.

FIG. 4.

(A) Structure of the YPR160W gene in CMBS-33. The gray region is S. cerevisiae-like and the white region S. bayanus-like. The region where recombination between the homoeologous alleles occurred is shown in the bottom panel; the prerecombination (A), recombination (B), and postrecombination (C) regions are indicated, as are the locations of the primers used for the RT-PCR amplification of the regions. The expected sizes of the RT-PCR products are shown. The location of the primers in YPR160W are shown in Fig. S1 in the supplemental material, and the sequences of the primers are shown in Table 1. (B) Hybridization of RNA from S. cerevisiae (lane 1), S. bayanus (lane 2), and CMBS-33 (lane 3) with DNA probes to regions A, B, and C. Probe A was amplified from S. cerevisiae genomic DNA, probe B from CMBS-33 genomic DNA, and probe C from S. bayanus genomic DNA. (C) Percentage identity of probes A, B, and C to S. cerevisiae, CMBS-33, and S. bayanus YPR160W gene sequences. (D) Amplification of genomic DNA and cDNA corresponding to regions A, B, and C of the CMBS-33 YPR160W gene. Lane M, molecular weight marker hyperladder II (Bioline); lanes 1, 5, and 9, genomic DNA; lanes 2, 6, and 10, cDNA; lanes 3, 7, and 11, cDNA amplification in the absence of reverse transcriptase; lanes 4 and 8, blank. The primers used for amplification are indicated above the lanes. (E) Amplification of genomic DNA and cDNA from the CMBS-33 YPR160W gene. Lane M, molecular weight marker hyperladder I (Bioline); lanes 1 and 5, genomic DNA; lanes 2 and 6, cDNA; lanes 3 and 7, cDNA amplification in the absence of reverse transcriptase; lane 4, blank. The primers used for amplification are shown above the lanes.

FIG. 5.

(A) Glycogen levels in the S. cerevisiae (S.c), S. bayanus (S.b), and CMBS-33 strains. Cells were plated on YEP-dextrose (top panel) and stained with crystal violet (bottom panel). The levels of glycogen per cell are shown below the panels. Standard deviations (SD) were obtained from the results of three independent experiments. (B) Glycogen levels in isogenic polyploid strains of S. cerevisiae (thick black line). The level of glycogen in CMBS-33 is illustrated by a gray triangle. Thin black line, linear trend line.