葡萄酒有孢汉逊酵母属分子指纹图谱分析

The molecular fingerprinting analysis of Hanseniaspora in wine

为分析中国本土野生有孢汉逊酵母属Hanseniaspora酵母菌的种间差异和种内差异,构建分子指纹图谱,为优良有孢汉逊酵母资源的遗传多样性分析及开发利用提供科学依据。采用依赖于培养的经典方法包含WL营养培养基、26S rRNA基因D1/D2区域序列分析和5.8S-ITS-RFLP分析进行118株贵州紫云县刺葡萄自然发酵过程中分离的有孢汉逊酵母属酵母菌种水平的鉴定。采用Tandem repeat-tRNA(TRtRNA)指纹图谱法研究有孢汉逊酵母属酵母在两对微卫星引物扩增下的分子指纹图谱,并利用DPS软件分析不同分子指纹图谱类别之间的遗传发育关系。结果表明,118株贵州紫云县刺葡萄自然发酵过程中分离的野生有孢汉逊酵母属酵母菌,经依赖于培养的经典方法鉴定为3个种,包括Hanseniaspora opuntiae、H.uvarum和H.occidentalis。TRtRNA指纹图谱法的引物对一TtRNASC和5CAG扩增出5类图谱,而引物对二TtRNASC和ISSR-MB扩增出10类图谱,可以表现出所测118株有孢汉逊酵母属的种间差异和种内差异。其中种内差异主要表现在H.uvarum中。总体而言,贵州紫云县刺葡萄自然发酵过程中分离的有孢汉逊酵母属主要为H.opuntiae、H.uvarum和H.occidentalis,其中H.opuntiae和H.uvarum内部表现了不同的TRtRNA分子指纹图谱,表征了种内遗传多样性。

Interspecies and intraspecies differences of indigenous Hanseniaspora in China are analyzed, and molecular fingerprinting profiles of the Hanseniaspora strains are built. Classical culture-dependent methods including WL nutrient agar, D1/D2 sequencing of 26S rRNA gene and 5.8 S-ITS-RFLP analysis were used to identify 118 Hanseniaspora strains isolated from spontaneous fermentation of Vitis davidii in Ziyun of Guizhou province at species level. Tandem repeat-tRNA(TRtRNA) fingerprinting method was used to study molecular fingerprinting profiles of Hanseniaspora strains amplified with two pairs of microsatellite primers. The DPS software was further applied to analyze the phylogenetic correlation among different molecular fingerprinting profiles. The results showed that 118 Hanseniaspora strains identified by classical culture-dependent methods included H.opuntiae, H.uvarum and H.occidentalis. Five profiles were obtained by amplification with primer pair one TtRNASC and 5 CAG, and ten profiles were obtained with primer pair two TtRNASC and ISSR-MB.Therefore, TRtRNA fingerprinting method could exhibit the interspecies and intraspecies differences of 118 Hanseniaspora strains. The intraspecies differences were largely indicated in H.uvarum strains. Both H.opuntiae and H.uvarum strains showed different TRtRNA fingerprinting profiles within species, manifesting their intraspecific genetic diversity.