Xu Weina, Wang Jinjing, Li Qi
Wei Sheng Wu Xue Bao. 2015 Jan 4;55(1):22-32.
Lager brewing yeasts (Saccharomyces pastorianus), the natural hybrids of S. cerevisiae and S. eubayanus, are usually heterothallic polyploidy or aneuploidy. Their intricate ploidy is a great challenge to genetic studies and strain improvement. Haploid breeding is an effective method to overcome these difficulties. Also, haploid strains play an important role in scientific research and breeding. However, lager brewing yeasts only divide asexually and hardly bear spores under normal conditions, so it is very difficult to get haploid strains from them. In this study, we established comprehensive methods to induce, separate and identify haploid strains of industrial brewer's yeast.
First, we selected efficient sporulation medium to induce the sporulation of an industrial brewer's yeast strain G-03, and ther isolated spores from vegetative cells and formed colonies on YPD plates. After that, flow cytometry was used to determine the ploidy types of the pre-judged haploid candidates. Ultimately, we analyzed the genotypes of the segregants by PCR reaction and mating test in order to get precise results.
Using this protocol, we obtained 26 yeast segregants by spore isolation, and 4 of them pre-judged as haploid candidates were finally confirmed as haploid by flow cytometric analysis. Two of them were MATa and others were MATα. By scanning electron microscope (SEM), the cells of 4 haploid segregants showed similar morphology to each other but had obvious differences compared with the parent strain. Pseudohyphal growth occurred in parent cells after long-period cultivation but none was found in haploid segregants.
Sporulation of industrial brewer's yeast and germination of their spores was difficult but not impossible. Nevertheless, the screening and identification of haploid segregants were more challenging.
拉格啤酒酿造酵母（巴氏酵母）是酿酒酵母和真贝氏酵母的天然杂交种，通常为异宗配合多倍体或非整倍体。其复杂的倍性对遗传研究和菌株改良构成了巨大挑战。单倍体育种是克服这些困难的有效方法。此外，单倍体菌株在科研和育种中发挥着重要作用。然而，拉格啤酒酿造酵母在正常条件下仅进行无性分裂，几乎不产生孢子，因此很难从中获得单倍体菌株。在本研究中，我们建立了诱导、分离和鉴定工业酿酒酵母单倍体菌株的综合方法。
首先，我们选择高效产孢培养基诱导工业酿酒酵母菌株G-03产孢，然后从营养细胞中分离孢子，并在YPD平板上形成菌落。之后，使用流式细胞术确定预先判断为单倍体候选菌株的倍性类型。最终，我们通过PCR反应和交配试验分析分离株的基因型，以获得精确结果。
使用该方案，我们通过孢子分离获得了26个酵母分离株，其中4个预先判断为单倍体候选菌株最终通过流式细胞术分析确认为单倍体。其中2个为MATa，其他为MATα。通过扫描电子显微镜（SEM）观察，4个单倍体分离株的细胞彼此形态相似，但与亲本菌株相比有明显差异。亲本细胞在长期培养后出现假菌丝生长，但在单倍体分离株中未发现。
工业酿酒酵母的产孢及其孢子的萌发虽困难但并非不可能。然而，单倍体分离株的筛选和鉴定更具挑战性。
