Comparison of DNA-based techniques for differentiation of production strains of ale and lager brewing yeast
Comparison of DNA-based techniques for differentiation of production strains of ale and lager brewing yeast
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DOI：
10.1111/jam.13116
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发表时间：
2016-06-01
影响因子：
4
通讯作者：
Matoulkova, D.
中科院分区：
文献类型：
--
作者：
Kopecka, J.;Nemec, M.;Matoulkova, D.
目的：啤酒酵母分为巴斯德酵母和酿酒酵母。大多数酿酒酵母菌株是天然的种间杂种，通常是多倍体，因此根据所使用的方法，它们的鉴定通常很难给出异质性结果。我们进行了遗传特性的一组酿酒酵母菌株来自几个酵母培养物收藏的各种DNA为基础的技术相结合。方法和结果：采用ITS-5.8S、NTS、HIS 4和COX 2基因的PCR-RFLP分析、多重PCR、基因组DNA的RAPD-PCR、mtDNA-RFLP和电泳核型分析等方法对40株酵母菌进行了鉴定。利用PCR-限制性片段长度多态性技术对酵母菌的HIS 4基因进行了可靠的种间鉴别。数值分析所获得的RAPD指纹图谱和核型揭示了种特异性聚类对应的技术分类的菌株。多重PCR证实了菌株的分类地位和部分杂交性质。利用ITS-5.8S和NTS区域的PCR-RFLP进行种间区分是不可靠的。结论：HIS 4基因PCR-RFLP和基因组DNA RAPD-PCR是快速鉴定酵母菌的可靠方法。引物21的RAPD-PCR是一种快速、可靠的方法，适用于啤酒酵母的区分，两个主要的工艺组（爱尔啤酒和拉格啤酒）之间的指纹图谱只有35%的相似性研究的意义和影响：证明了HIS 4基因的PCR-RFLP方法可以精确区分三个重要的技术酵母物种。利用RAPD-PCR技术可以实现酿酒酵母在菌株水平上的区分。
Aims: Brewing yeasts are classified into two species-Saccharomyces pastorianus and Saccharomyces cerevisiae. Most of the brewing yeast strains are natural interspecies hybrids typically polyploids and their identification is thus often difficult giving heterogenous results according to the method used. We performed genetic characterization of a set of the brewing yeast strains coming from several yeast culture collections by combination of various DNA-based techniques. The aim of this study was to select a method for species-specific identification of yeast and discrimination of yeast strains according to their technological classification.Methods and Results: A group of 40 yeast strains were characterized using PCR-RFLP analysis of ITS-5.8S, NTS, HIS4 and COX2 genes, multiplex PCR, RAPD-PCR of genomic DNA, mtDNA-RFLP and electrophoretic karyotyping. Reliable differentiation of yeast to the species level was achieved by PCR-RFLP of HIS4 gene. Numerical analysis of the obtained RAPD-fingerprints and karyotype revealed species-specific clustering corresponding with the technological classification of the strains. Taxonomic position and partial hybrid nature of strains were verified by multiplex PCR. Differentiation among species using the PCR-RFLP of ITS-5.8S and NTS region was shown to be unreliable. Karyotyping and RFLP of mitochondrial DNA evinced small inaccuracies in strain categorization.Conclusions: PCR-RFLP of HIS4 gene and RAPD-PCR of genomic DNA are reliable and suitable methods for fast identification of yeast strains. RAPD-PCR with primer 21 is a fast and reliable method applicable for differentiation of brewing yeasts with only 35% similarity of fingerprint profile between the two main technological groups (ale and lager) of brewing strains.Significance and Impact of the Study: It was proved that PCR-RFLP method of HIS4 gene enables precise discrimination among three technologically important Saccharomyces species. Differentiation of brewing yeast to the strain level can be achieved using the RAPD-PCR technique.