A method for quantifying sporulation efficiency and isolating meiotic progeny in non-GMO strains of Saccharomyces cerevisiae.
A method for quantifying sporulation efficiency and isolating meiotic progeny in non-GMO strains of Saccharomyces cerevisiae.
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DOI：
10.1002/yea.3802
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发表时间：
2022-06
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减数分裂作图是一种基于连锁的杂交重组后代分析方法，长期以来一直是遗传学研究的基石。酵母酿酒酵母是一个强大的系统，因为它可以分离和培养四个产品（孢子）的一个单一的减数分裂事件。然而，该方法的通量在历史上受到以下过程的限制：在营养细胞、四分体和二分体的异质群体中鉴定四分体，然后手动分离（解剖）四分体中所含的孢子。迄今为止，促进减数分裂后代的高通量表征和分离的方法依赖于基因工程。在这里，我们表征了荧光染料DiBAC 4（5）染色酵母四分体和二分体以及在大量四分体破坏后粘附于孢子的能力。应用包括通过流式细胞术对孢子形成速率和效率进行定量测定，以及通过FACS在未经遗传修饰的菌株中富集完整的四分体、二分体或破裂的孢子。
Meiotic mapping, a linkage-based method for analyzing the recombinant progeny of a cross, has long been a cornerstone of genetic research. The yeast Saccharomyces cerevisiae is a powerful system because it is possible to isolate and cultivate the four products (spores) of a single meiotic event. However, the throughput of this process has historically been limited by the process of identifying tetrads in a heterogeneous population of vegetative cells, tetrads, and dyads followed by manual separation (dissection) of the spores contained in a tetrad. To date, methods that facilitate high throughput characterization and isolation of meiotic progeny have relied on genetic engineering. Here, we characterize the ability of the fluorescent dye DiBAC4(5) to stain yeast tetrads and dyads as well as to adhere to spores following bulk tetrad disruption. Applications include quantitative assays of sporulation rates and efficiency by flow cytometry as well as enrichment of intact tetrads, dyads, or disrupted spores by FACS in strains that have not been genetically modified.