Data_Sheet_1_Heat Shock Improves Random Spore Analysis in Diverse Strains of Saccharomyces cerevisiae.PDF

Random spore analysis (RSA) is a classic method in yeast genetics that allows high-throughput purification of recombinant haploid spores following specific crosses. RSA typically involves a number of steps to induce sporulation, purge vegetative cells that fail to sporulate, and disrupt the ascus walls of sporulated cells to release haploid spores. These steps generally require expensive chemicals and/or enzymes that kill diploid cells but have few effects on spores. In the fission yeast Schizosaccharomcyes pombe, heat shock has been reported as an effective addition to RSA protocols, but to our knowledge heat shock has not been used for this purpose in the budding yeast Saccharomyces cerevisiae. Here, we evaluate the effects of heat shock on vegetative and sporulated cultures of four diverse yeast strains: a European wine strain (DBVPG6765), a Japanese sake strain (Y12), a West African palm wine strain (DBVPG6044) and a North American strain isolated from the soil beneath an oak tree (YPS128). We characterize this phenotype under multiple combinations of temperature and incubation time, and find specific conditions that lead to the exclusion of vegetative cells and an enrichment in spores, which differ by strain. We also collected genome sequence data from a recombinant population that experienced multiple rounds of RSA, including one round with a heat shock treatment. These data suggest that when incorporated into an RSA protocol, heat shock leads to increased genetic diversity among the cells that survive and mate. Ultimately, our work provides evidence that short heat treatments can improve existing RSA protocols, though in a strain-specific manner. This result informs applications of high-throughput RSA protocols, such as QTL mapping and experimental evolution research.

随机孢子分析（RSA）是酵母遗传学中的一个经典方法，它允许在特定杂交之后进行高通量纯化重组单倍体孢子。RSA通常涉及诱导孢子形成、清除未能形成孢子的营养细胞以及破坏已形成孢子的子囊壁以释放单倍体孢子的一系列步骤。这些步骤通常需要昂贵的化学物质和/或酶，这些物质和酶能够杀死二倍体细胞，但对孢子的作用甚微。在裂殖酵母Schizosaccharomcyes pombe中，已有报道称热休克是RSA方案的辅助措施之一，但据我们所知，热休克尚未在酿酒酵母Saccharomyces cerevisiae中用于此目的。在本研究中，我们评估了热休克对四种不同酵母菌株（包括欧洲葡萄酒菌株DBVPG6765、日本清酒菌株Y12、西非棕榈酒菌株DBVPG6044以及从橡树下土壤中分离的北美菌株YPS128）的营养细胞和孢子形成文化的效应。我们在多种温度和培养时间的组合下表征了这一表型，并发现了一些特定条件，这些条件可以排除营养细胞并丰富孢子，且这些条件因菌株而异。我们还从经过多轮RSA，包括一轮热休克处理的重组种群中收集了基因组序列数据。这些数据表明，当热休克纳入RSA方案时，能够增加存活并交配的细胞间的遗传多样性。最终，我们的研究提供了证据，证明短时间的热处理可以改进现有的RSA方案，尽管这种改进具有菌株特异性。这一结果为高通量RSA方案的应用提供了信息，如QTL定位和实验进化研究。