Protocols for quantitative trait loci mapping in saccharomyces cerevisiae based on mass selections and backcrossings

• Grant number: 21/13906-4
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: March 01, 2022
• End date: April 02, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Ana Paula Jacobus
• Grantee: Lucas Souza de Bem
• Host Institution: Instituto de Pesquisa em Bioenergia (IPBEN). Universidade Estadual Paulista (UNESP). Campus de Rio Claro. Rio Claro , SP, Brazil
• Associated research grant: 17/24453-5 - Collaborative network: modern genetic approaches to bust yeast tolerance to inhibitor-rich lignocellulosic hydrolysates, AP.BIOEN.JP
• Associated scholarship(s): 23/06203-2 - ReMaSSing meets SCRaMbLE: using Cre/Lox recombination, selection, and backcrossing to evolve the synthetic yeast Sc2.0, BE.EP.DD

Abstract

The second-generation ethanol (2G) is derived from lignocellulosic hydrolysates (LCHs). These LCHs carry several toxic substances to Saccharomyces cerevisiae, lowering the fermentation efficiency for 2G ethanol production. However, some S. cerevisiae are naturally resistant to LCH inhibitors. One way to analyze the complex phenotype of LCH-tolerant strains is through the mapping of Quantitative Trait Loci (QTL). To facilitate the study of genetic differences between tolerant and sensible strains to LCH, we are developing innovative QTL mapping procedures, applied for haploid or diploid recombinants. In the new protocols, after crossing a sensible strain with the tolerant one, all resulting recombinants are selected in an LCH-rich medium. Next, the tolerant pool is mixed with cells of the sensible parent for a mass backcrossing. After each backcrossing, by using specific antibiotics, it is possible to select only recombinant cells and also to control the population ploidy state. In this way, it is feasible to perform several rounds of mass selection and backcrossing in an easier way. Currently, we have completed the genomic sequencing of a diploid pool resulting from five rounds of backcrossing and selection in 35% of LCH of sugar cane bagasse. That allowed the mapping of several QTL, which will be validated by their reverse engineering via CRISPR/Cas9 into the sensible strain. In this Ph.D., these data, combined with the mapping resulting from the haploid protocol, will allow an in-depth analysis of the LCH tolerance phenotype in S. cerevisiae. (AU)