| Monascus spp. which is an important microorganism resource in China has been widely used for centuries to produce food fermentation starter, food colorant, food antiseptic and Chinese traditional medicine. Since Monascus spp. can produce various metabolites in the process of fermentation, such as Monascus pigment, Monacolin,γ-aminobutyric acid (GABA) and enzyme, researches of Monascus spp. and its metabolites have become a focus of attention in the world recently. However, the classification of Monascus spp. especially species classification, is still under debate. With the development of molecular technique, DNA molecular markers have been employed in the differentiation and genetic relationship analysis to be the supplement to traditional classification methods.In these study, methods in the aspects of morphology, biochemistry, molecular biology were used in the identification of 67 Monascus strains, to provide alternative methods for strains differentiation and genetic analysis of Monascus spp. Results are as follow:1. 53 isolates of the genus Monascus separated from 24 samples from different places and 14 purchased Monascus strains were used in the study. According to the investigation made by Zhongqing Li, based on the morphological characteristic of colony and micro-mophorlogy, 67 tested strains were identified as 10 different species primarily, including 39 strains of M. anka, 9 strains of M. purpureus, 9 strains of M. pilosus, 4 strains of M. barkeri, and one strain of each species as follow, M. aurantiacus, M. albidus, M. fuliginosus, M. rubber, M. lunisporas, M. argentinensis. The rest two strains (M-3, M-26) were not confirmed.2. On the basis of morphological observation, 51 Monascus strains selected from 67 tested strains (14 reference strains and 37 isolates) were in isozyme analysis. The results showed that Esterase patterns of strains in different species were polymorphic, and strains within one species were almost the same, which were similar with the identification results of morphological observation. The result of cluster analysis showed that 51 strains were divided into 4 groups at 70% similarity level. M. aurantiacus, M. albidus, M. fuliginosus, M. barkeri and M. pilosus were in a group, M. purpureus, M. serorubescens, M. anka and M. rubber were in the second group, M. lunisporas and M. argentinensis represented two groups, respectively.3. Seven primers (808~#,810~#,811~#,834~#,835~#,841~#,842~#) selected from 14 ISSR primers were used for ISSR amplification of 51 tested strains. A total of 106 bands were produced with 7 primers, among which 97.2% were polymorphic. Similarity coefficient ranged from 0.48 to 1.00. The result of cluster analysis showed that 51 strains were divided into 4 groups at 70% similarity level. M. albidus, M. fuliginosus, M. barkeri and M. pilosus were in a group. M. aurantiacus, M. purpureus, M. serorubescens, M. anka and M. rubber were in the second group. M. lunisporas and M. argentinensis represented two groups, respectively.4. Eight pairs of SRAP primers (me1-em2,me1-em4,me1-em5,me2-em4,me3-em2,me3-em6,me4-em4,me5-em6)screened from 30 SRAP primers were used for SRAP amplification of 51 tested strains. A total of 183 bands were produced with 8 primers, among which 94.5% were polymorphic. Similarity coefficient ranged from 0.46 to 1.00. The result of cluster analysis showed that 51 strains were divided into 4 groups at 70% similarity level. M. albidus, M. fuliginosus, M. barkeri and M. pilosus were in a group. M. aurantiacus, M. purpureus, M. serorubescens, M. anka and M. rubber were in the second group. M. lunisporas and M. argentinensis represented two groups, respectively.5. In the IGS amplification results of 51 tested strains, IGS2 could not be amplified, whereas IGS 1 could be amplified clearly and stably. Cluster results revealed that when similarity coefficient reached 70%, 51 tested strains were separated into 3 groups with the similarity coefficient from 0.42 to 1.00. OUT2011, OUT2014, M-8, M-10, M-17, M-18, M-19, M-20, M-21 were in a group, M. lunisporas was represented a group, and other strains were in a group。The results of this study showed that ISSR, SRAP and IGS techniques could validate the results of morphological identification and help to identify some strains which identities were controversial.
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