啤酒酵母工业菌株单倍体的诱导、分离和鉴定（英文）

Induction,separation and identification of haploid strains from industrial brewer's yeast

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。

[ Objective] 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. [ Methods] First, we selected efficient sporulation medium to induce the sporulation of an industrial brewer＇s yeast strain G-03, and then 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. [ 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 MATs. 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. [ Conclusion] 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.