Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.

Single nucleotide polymorphisms (SNP) are the ideal marker for characterizing genomic variation but can be difficult to find in nonmodel species. We explored the usefulness of the dog genome for finding SNPs in distantly related nonmodel canids and evaluated so-ascertained SNPs. Using 40 primer pairs designed from randomly selected bacterial artificial chromosome clones from the dog genome, we successfully sequenced 80-88% of loci in a coyote (Canis latrans), grey fox (Urocyon cinereoargenteus), and red fox (Vulpes vulpes), which compared favourably to a 60% success rate for each species using 10 primer pairs conserved across mammals. Loci were minimally heterogeneous with respect to SNP density, which was similar, overall, in a discovery panel of nine red foxes to that previously reported for a panel of eight wolves (Canis lupus). Additionally, individual heterozygosity was similar across the three canids in this study. However, the proportion of SNP sites shared with the dog decreased with phylogenetic divergence, with no SNPs shared between red foxes and dogs. Density of interspecific SNPs increased approximately linearly with divergence time between species. Using red foxes from three populations, we estimated F(ST) based on each of 42 SNPs and 14 microsatellites and simulated null distributions conditioned on each marker type. Relative to SNPs, microsatellites systematically underestimated F(ST) and produced biased null distributions, indicating that SNPs are superior markers for these functions. By reconstituting the frequency spectrum of SNPs discovered in nine red foxes, we discovered an estimated 77-89% of all SNPs (within the region screened) present in North American red foxes. In sum, these findings indicate that information from the dog genome enables easy ascertainment of random and gene-linked SNPs throughout the Canidae and illustrate the value of SNPs in ecological and evolutionary genetics.