RESUMO
Meiotic recombination shapes the genetic diversity transmitted upon sexual reproduction. However, its non-random distribution along the chromosomes constrains the landscape of potential genetic combinations. For a variety of purposes, it is desirable to expand the natural repertoire by changing the distribution of crossovers in a wide range of eukaryotes. Toward this end, we report the local stimulation of meiotic recombination at a number of chromosomal sites by tethering the natural Spo11 protein to various DNA-binding modules: full-length DNA binding proteins, zinc fingers (ZFs), transcription activator-like effector (TALE) modules, and the CRISPR-Cas9 system. In the yeast Saccharomyces cerevisiae, each strategy is able to stimulate crossover frequencies in naturally recombination-cold regions. The binding and cleavage efficiency of the targeting Spo11 fusions (TSF) are variable, being dependent on the chromosomal regions and potential competition with endogenous factors. TSF-mediated genome interrogation distinguishes naturally recombination-cold regions that are flexible and can be warmed-up (gene promoters and coding sequences), from those that remain refractory (gene terminators and centromeres). These results describe new generic experimental strategies to increase the genetic diversity of gametes, which should prove useful in plant breeding and other applications.
Assuntos
Troca Genética , Endodesoxirribonucleases/genética , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Fusão Gênica , Marcação de Genes/métodos , Recombinação Genética , Reprodutibilidade dos Testes , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
[This corrects the article DOI: 10.1186/s13068-017-0837-6.].
RESUMO
BACKGROUND: The presence of low complexity and repeated regions in genomes often results in difficulties to assemble sequencing data into full chromosomes. However, the availability of full genome scaffolds is essential to several investigations, regarding for instance the evolution of entire clades, the analysis of chromosome rearrangements, and is pivotal to sexual crossing studies. In non-conventional but industrially relevant model organisms, such as the ascomycete Trichoderma reesei, a complete genome assembly is seldom available. RESULTS: The chromosome scaffolds of T. reesei QM6a and Rut-C30 strains have been generated using a contact genomic/proximity ligation genomic approach. The original reference assembly, encompassing dozens of scaffolds, was reorganized into two sets of seven chromosomes. Chromosomal contact data also allowed to characterize 10-40 kb, gene-free, AT-rich (76%) regions corresponding to the T. reesei centromeres. Large chromosomal rearrangements (LCR) in Rut-C30 were then characterized, in agreement with former studies, and the position of LCR breakpoints used to assess the likely chromosome structure of other T. reesei strains [QM9414, CBS999.97 (1-1, re), and QM9978]. In agreement with published results, we predict that the numerous chromosome rearrangements found in highly mutated industrial strains may limit the efficiency of sexual reproduction for their improvement. CONCLUSIONS: The GRAAL program allowed us to generate the karyotype of the Rut-C30 strain, and from there to predict chromosome structure for most T. reesei strains for which sequence is available. This method that exploits proximity ligation sequencing approach is a fast, cheap, and straightforward way to characterize both chromosome structure and centromere sequences and is likely to represent a popular convenient alternative to expensive and work-intensive resequencing projects.