RAPD analysis of Aspergilli and its application in brewing industry

Abstract:

Phylogenetic analysis of sixteen Aspergilli was done by RAPD technology，using Aspergillus oryzae AS3.951，Aspergillus flavus GIM3.18 and Aspergillus sojae AS3.495 as controls. First，genome DNA of the sixteen test strains were prepared by improved extraction method，and their quality was verified by electrophoresis and spectrophotometry. They displayed an identical band (approximately 20kb) in agarose gel electrophoresis，which conformed to the fact that these strains all belong to Aspergillus. OD₂₆₀/OD₂₈₀ of the prepared DNA ranged from 1.80 to 1.90，illustrating that they were good enough to be used as templates in the following RAPD-PCR experiment. Then，three appropriate primers (Primer1，Primer2，Primer5) for RAPD-PCR were screened from nine random primers，and repetitive experiments demonstrated that the RAPD-PCR polymorphic patterns of the sixteen test strains based on these three primers were stable. There were usually 8～14 bands in their RADP-PCR patterns，where the number of the main bands was 4～9 and the secondary bands were abundant. There were totally 181 bands in their RAPD-PCR patterns，where the percentage of polymorphic bands reached to 40.9% (74 bands). The similarity coefficient between the strains was calculated based on their RAPD-PCR patterns，ranging from 8.0% to 96.6%. All these data suggests that the genetic polymorphism of the strains is abundant and they have evident genetic differentiation. The phylogenetic tree of the sixteen test strains was reconstructed according to their RAPD-PCR patterns with Primer1，Primer2 and Primer5. It basically corresponded to traditional morphological taxonomy，demonstrating that the application of RAPD molecular marker in the phylogenetic analysis of these Aspergilli is feasible. Besides，the aflatoxin-producing strains (GIM3.17，CICC2219，CICC2357，CICC2390，CICC2402，CICC2404) could be easily discriminated by RAPD molecular marker，whereas it is difficult to distinguish them by conventional morphological taxonomy. Consequently，RAPD molecular marker provides a novel clue to discriminating aflatoxin-producing strains in brewing industry.