Third-generation sequencing-based mapping and visualization of single nucleotide polymorphism, meiotic recombination, illegitimate mutation and repeat-induced point mutation

Generation of new genetic diversity by crossover (CO) and non-crossover (NCO) is a fundamental process in eukaryotes. Fungi have played critical roles in studying this process because they permit tetrad analysis, which has been used by geneticists for several decades to determine meiotic recombination products. New genetic variations can also be generated in zygotes via illegitimate mutation (IM) and repeat-induced point mutation (RIP). RIP is a genome defense mechanism for preventing harmful expansion of transposable elements or duplicated sequences in filamentous fungi. Although the exact mechanism of RIP is unknown, the C:G to T:A mutations might result from DNA cytosine methylation. A comprehensive approach for understanding the molecular mechanisms underlying these important processes is to perform high-throughput mapping of CO, NCO, RIP and IM in zygotes bearing large numbers of heterozygous variant markers. To this aim, we developed ‘TSETA’, a versatile and user-friendly pipeline that utilizes high-quality and chromosome-level genome sequences involved in a single meiotic event of the industrial workhorse fungus Trichoderma reesei. TSETA not only can be applied to most sexual eukaryotes for genome-wide tetrad analysis, it also outcompetes most currently used methods for calling out single nucleotide polymorphisms between two or more intraspecies strains or isolates.

Tetrad analysis without tetrad dissection: Meiotic recombination and genomic diversity in the yeast Komagataella phaffii (Pichia pastoris)

Komagataella phaffii is a yeast widely used in the pharmaceutical and biotechnology industries, and is one of the two species that were formerly called Pichia pastoris. However, almost all laboratory work on K. phaffii has been done on strains derived from a single natural isolate, CBS7435. There is little information about the genetic properties of K. phaffii or its sequence diversity. Genetic analysis is difficult because, although K. phaffii makes asci with four spores, the spores are small and tend to clump together, making the asci hard to dissect. Here, we sequenced the genomes of all the known isolates of this species, and find that K. phaffii has only been isolated from nature four times. We analyzed the meiotic recombination landscape in a cross between auxotrophically marked strains derived from two isolates that differ at 44,000 single nucleotide polymorphism sites. We conducted tetrad analysis by making use of the property that haploids of this species do not mate in rich media, which enabled us to isolate and sequence the four types of haploid cell that are present in the colony that forms when a tetratype ascus germinates. We found that approximately 25 crossovers occur per meiosis, which is 3.5 times fewer than in Saccharomyces cerevisiae. Recombination is suppressed, and genetic diversity among natural isolates is low, in a region around centromeres that is much larger than the centromeres themselves. Our method of tetrad analysis without tetrad dissection will be applicable to other species whose spores do not mate spontaneously after germination.Author summaryTo better understand the basic genetics of the budding yeast Komagataella phaffii, which has many applications in biotechnology, we investigated its genetic diversity and its meiotic recombination landscape. We made a genetic cross between strains derived from two natural isolates, and developed a method for characterizing the genomes of the four spores resulting from meiosis, which were previously impossible to isolate. We found that K. phaffii has a lower recombination rate than Saccharomyces cerevisiae. It shows a large zone of suppressed recombination around its centromeres, which may be due to the structural differences between centromeres in K. phaffii and S. cerevisiae.

DeepTetrad: high-throughput analysis of meiotic tetrads by deep learning in plants

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate genetic dissection of mechanisms that control meiotic recombination.

DNA damage response clamp loader Rad24(Rad17) and Mec1(ATR) kinase have distinct functions in regulating meiotic crossovers

Crossover (CO) recombination is essential for chromosome segregation during meiosis I. The number and distribution of COs are tightly regulated during meiosis. CO control includes CO assurance and CO interference, which guarantee at least one CO per a bivalent and evenly-spaced CO distribution, respectively. Previous studies showed the role of DNA damage response (DDR) clamp and its loader in efficient formation of meiotic COs by promoting the recruitment of a pro-CO protein Zip3 and interhomolog recombination, and also by suppressing ectopic recombination. In this study, by classical tetrad analysis ofSaccharomyces cerevisiae, we showed that a mutant defective in theRAD24 gene(RAD17in other organisms), which encodes the DDR clamp loader, displayed reduced CO frequencies on two shorter chromosomes (IIIandV) but not on a long chromosome (chromosomeVII). The residual COs in therad24mutant do not show interference. In contrast to therad24mutant, mutants defective in the ATR kinase homolog Mec1/Esr1, including amec1null and amec1kinase-dead mutant, show little or no defect in CO frequency. On the other hand,mec1COs show defects in interference, similar to therad24mutant. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

The Factor Structure of Outcome Questionnaire–45.2 Scores Using Confirmatory Tetrad Analysis–Partial Least Squares

The researchers employed a confirmatory tetrad analysis (CTA) using partial least squares–structural equation modeling (PLS-SEM) with Outcome Questionnaire–45.2 (OQ-45) data, examining the measurement model of the OQ-45 scores with a sample of male adult clients ( N = 1,558) receiving individual therapy at a university-based community counseling and research center (UBCCRC). Using CTA-PLS, this study examined the reflective and formative nature of each of the OQ-45 items and dimensions. These results identified the innovative second-order formative–formative three-factor model as a best alternative measurement model to represent and calculate the scores of OQ-45 scale.