GetPrimers: A generalized PCR-based genetic targeting primer designer enabling easy and standardized targeted gene modification across multiple systems.

Genetic targeting (e.g., gene knockout and tagging) based on polymerase chain reaction (PCR) is a simple yet powerful approach for studying gene functions. Although originally developed in classic budding and fission yeast models, the same principle applies to other eukaryotic systems with efficient homologous recombination. One-step PCR-based genetic targeting is conventionally used but the sizes of the homologous arms that it generates for recombination-mediated genetic targeting are usually limited. Alternatively, gene targeting can also be performed via fusion PCR, which can create homologous arms that are orders of magnitude larger, therefore substantially increasing the efficiency of recombination-mediated genetic targeting. Here, we present GetPrimers (https://www.evomicslab.org/app/getprimers/), a generalized computational framework and web tool to assist automatic targeting and verification primer design for both one-step PCR-based and fusion PCR-based genetic targeting experiments. Moreover, GetPrimers by design runs for any given genetic background of any species with full genome scalability. Therefore, GetPrimers is capable of empowering high-throughput functional genomic assays at multipopulation and multispecies levels. Comprehensive experimental validations have been performed for targeting and verification primers designed by GetPrimers across multiple organism systems and experimental setups. We anticipate GetPrimers to become a highly useful and popular tool to facilitate easy and standardized gene modification across multiple systems.

Telomere-to-telomere assemblies of 142 strains characterize the genome structural landscape in Saccharomyces cerevisiae.

Pangenomes provide access to an accurate representation of the genetic diversity of species, both in terms of sequence polymorphisms and structural variants (SVs). Here we generated the Saccharomyces cerevisiae Reference Assembly Panel (ScRAP) comprising reference-quality genomes for 142 strains representing the species' phylogenetic and ecological diversity. The ScRAP includes phased haplotype assemblies for several heterozygous diploid and polyploid isolates. We identified circa (ca.) 4,800 nonredundant SVs that provide a broad view of the genomic diversity, including the dynamics of telomere length and transposable elements. We uncovered frequent cases of complex aneuploidies where large chromosomes underwent large deletions and translocations. We found that SVs can impact gene expression near the breakpoints and substantially contribute to gene repertoire evolution. We also discovered that horizontally acquired regions insert at chromosome ends and can generate new telomeres. Overall, the ScRAP demonstrates the benefit of a pangenome in understanding genome evolution at population scale.

Genome-Wide Association Study and Fine Mapping Reveals Candidate Genes for Birth Weight of Yorkshire and Landrace Pigs.

Birth weight of pigs is an important economic factor in the livestock industry. The identification of the genes and variants that underlie birth weight is of great importance. In this study, we integrated two genotyping methods, single nucleotide polymorphism (SNP) chip analysis and restriction site associated DNA sequencing (RAD-seq) to genotype genome-wide SNPs. In total, 45,175 and 139,634 SNPs were detected with the SNP chip and RAD-seq, respectively. The genome-wide association study (GWAS) of the combined SNP panels identified two significant loci located at chr1: 97,745,041 and chr4: 112,031,589, that explained 6.36% and 4.25% of the phenotypic variance respectively. To reduce interval containing causal variants, we imputed sequence-level SNPs in the GWAS identified regions and fine-mapped the causative variants into two narrower genomic intervals: a ∼100 kb interval containing 71 SNPs and a broader ∼870 kb interval with 432 SNPs. This fine-mapping highlighted four promising candidate genes, SKOR2, SMAD2, VAV3, and NTNG1. Additionally, the functional genes, SLC25A24, PRMT6 and STXBP3, are also located near the fine-mapping region. These results suggest that these candidate genes may have contribute substantially to the birth weight of pigs.