J. Appl. Genet. 43(1), 2002, pp. 43-47
Short communication
Genetic relationship between Polish and Chinese strains of the mushroom Agaricus bisporus (Lange) Sing., determined by the RAPD method Miros³awa STANIASZEK1, Waldemar MARCZEWSKI2, Krystian SZUDYGA1, Jêdrzej MASZKIEWICZ1, Andrzej CZAPLICKI3, Guo QIAN4 1 Research Institute of Vegetable Crops, Skierniewice, Poland Plant Breeding and Acclimatization Institute, M³ochów, Poland 3 Plant Breeding and Acclimatization Institute, Radzików, Poland 4 Edible Fungi Institute, Shanghai Academy of Agricultural Sciences, China 2
Abstract. The genetic relationship between twenty-six strains of Agaricus bisporus were analysed by the RAPD (random amplified polymorphic DNA) method. DNA amplification was performed with the use of twelve arbitrary 10-mer primers. Four primers, which gave polymorphic band patterns were chosen for RAPD analysis. In total, they gave 24 distinguishable bands, of which nine were polymorphic. The conducted research showed that there is a great genetic similarity among the examined strains. Low polymorphism of the strains may be a proof of a limited genetic pool used in the cultivation of those strains. Key words: genetic diversity, mushroom strains, RAPD.
The interest in mushroom growing has increased considerably for the past few years. Fast progress in breeding research in the world resulted in development of a wide range of strains recommended by different spawn producers and it is sometimes hard to determine their genetic identity (GAPIÑSKI et al. 1997). Both breeders and producers of mushroom spawn need fast methods of strain identification and assessment of genetic diversity of the chosen genotypes, which are used in breeding programmes. Received: July 6, 2001. Accepted: September 26, 2001. Correspondence: M. STANIASZEK, Research Institute of Vegetable Crops, ul. Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland, e-mail:
[email protected]
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Identification of mushroom strains on the basis of morphological features (size, shape and colour of the cap, presence of scales) raises many objections because cultivation conditions have a significant effect on the phenotype and on fruit bodies. The isoenzymatic analysis is an alternative to morphological assessment. Isozyme markers have been used for confirmation of crosses between lines of Agaricus brunnescens and for identification of genotypic commercial and research-maintained lines (MAY, ROYSE 1982, ROYSE, MAY 1982). Variation in allozymes revealed that the wild population of Agaricus bisporus is a valuable source of genetic variability for creative breeding purposes (KERRIGAN, ROSS 1989). Introduction of DNA markers has caused a turn in genetic research on mushrooms. The RFLP (restriction fragment length polymorphism) method has been successfully used for the analysis of polymorphism of ”wild” mushroom species, identification of strains and evaluation of breeding material (CASTLE et al. 1987, LOFTUS et al. 1988). Then, RAPD (random amplified polymorphic DNA) was applied to determine the extent of genetic diversity in mushroom breeding materials intended for crossings (KHUSH et al. 1991, 1992). Here, we report on identification of the genetic diversity of twenty-six strains of Agaricus bisporus by RAPD analysis. The twelve Polish and fourteen Chinese strains of mushroom were received from the collections at the Research Institute of Vegetable Crops, Skierniewice, Poland, and the Edible Fungi Institute, Shanghai Academy of Agricultural Sciences (SAAS), China, respectively (Table 1). DNA was extracted from 10 samples of freeze-dried cap tissue of each strain, according to the procedure of DELLAPORTA et al. (1983). Bulked DNA samples were made by mixing equal amounts of DNA isolated from each of the 10 individuals. PCR reaction was performed in 20 ml volume containing: 10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1 mM of each deoxynucleotide, 3 mM MgCl2, 0.001% gelatin, 0.4 unit of Taq DNA polymerase (Sigma), 20 ng of primer, 20 ng of template DNA. The amplification reaction was carried out in an MJ Research PTC-200 thermal cycler. The cycle parameters were: 1 cycle at 94oC for 30 s, followed by 45 cycles of 15 s at 92oC, 25 s at 36oC, 74 s at 72oC, and final extension time of 5 min at 72oC. DNA amplification was performed with the use of twelve arbitrary 10-mer primers from Operon Technologies Inc. (Alameda, CA). The primers employed were: OPA02, OPA03, OPA04, OPA05, OPA06, OPA07, OPU11, OPU12, OPU13, OPU14, OPU18, OPU19. Amplification products were analysed by electrophoresis in 1.4% agarose gel after staining with ethidium bromide. For the diversity analysis, all polymorphic loci were scored as presence/absence of the band. The bivariate 1-0 data were used as the raw matrix. A square symmetric matrix of similarity was obtained using the Czekanowski-Sorensen coefficient. Similarity matrix was used to construct
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Table 1. Strains of Agaricus bisporus used in the study of genetic diversity by RAPD method Number in collection
Strains or group of strains
Strains derived from collection of Shanghai Academy of Agricultural Sciences, China A1
Portabello
A2
Portabello
A4
snow white
A5
off-white
A6
snow white
A7
snow white
A8
snow white
A9
white hybrid
A11
brown button
A12
hybrid of white
A13
brown button
A14
off-white
A16
Midcague
A17
intermediate hybrid
Strains derived from collection of Research Institute of Vegetable Crops, Skierniewice, Poland 1S
small hybrid
2S
small hybrid
4S
small hybrid
5S
intermediate hybrid
6S
intermediate hybrid
7S
small hybrid
8S
intermediate hybrid
9S
off-white
10S
intermediate hybrid
11S
off-white
12S
intermediate hybrid
13S
small hybrid
a dendrogram by the unweighted pair group methods with arithmetic averages (UPGMA), (Figure 1). Of the twelve RAPD primers tested, OPA03, OPA04, OPA07 and OPU11 were chosen for the polymorphism analysis. The first three primers were studied previously by KHUSH et al. (1992) to differentiate between various strains of the cultivated mushroom Agaricus bisporus. In total, they gave 24 distinguishable
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Figure 1. UPGMA dendrogram showing genetic relationship of 26 strains of Agaricus bisporus on the basis of 24 RAPD products
bands after PCR amplification, ranging in size from 350 to 2500 bp, of which 9 were polymorphic between the strains (results not shown). Differences in the RAPD profiles made it possible to cluster the strains into four groups. The first group consisted of strains A1, A2, A11 and A13, derived from the Chinese collection, and was clearly distinct from the other clusters. Among these strains A1, A2 and A13 showed amplification of the marker OPA03680, while this band was not identified in A11. These findings are consistent with the pedigree data, indicating that these strains are either identical or closely related. Strains A4, A6 and A7 constituted the second group and originated also from Chinese germplasm. They displayed the same RAPD fingerprinting within five polymorphic loci. The third major cluster represented the seven strains grown in Poland (4S, 5S, 6S, 9S, 10S, 11S, 12S) and related to the Chinese strains A5 and A14, both belonging to off-white strains (Table 1). Eight polymorphic products were identified in this group. Only OPA041180 was not revealed on agarose gels.
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All nine polymorphic DNA fragments were detected in the fourth group, containing the Chinese strains A8, A12, A16 and A17. Strains A9, 1S, 2S, 7S and 13S have not been clustered within the four indicated groups. Nevertheless, they were clearly differentiated by the presence or absence of specific DNA fragments. Strain 8S was very distinct from all other strains examined. This variation referred both to the polymorphic and common DNA fragments identified. The conducted research showed that there is a marked genetic similarity among the examined strains. Low polymorphism of the strains may be a proof of a narrow genetic pool used in the cultivation of those strains. Probably this is due to the fact that most mushroom strains in the world derive from the Dutch U1 and U3 strains. In conclusion, RAPD analysis has proved to be useful for distinguishing between the mushroom strains and for clustering them according to their origin. The DNA polymorphism analysis by RAPD method gives new possibilities of evaluation of the genotypes collected in gene banks of cultivated mushrooms, which will consequently make it possible to eliminate doubled genotypes and simplify the choice of components for breeding new strains. REFERENCES CASTLE A.J., HORGEN P.A., ANDERSON J.B. (1987). Restriction fragment length polymorphisms in the mushrooms Agaricus brunnescens and Agaricus bitorquis. App. Environ. Microbiol. 53: 816-822. DELLAPORTA S.L., WOOD J., HICKS J.B. (1983). A plant DNA minipreparation. Version II. Plant Mol. Biol. Rep. 1: 19-21. GAPIÑSKI M., WONIAK W., MAZELA A., LEWANDOWSKI M. (1997). Ocena wzrostu grzybni pieczarki (Agaricus bisporus (Lange) Sing.) [Growth assessment of the mycelium of Agaricus bisporus (Lange) Sing.]. Materia³y VII Ogólnopolskiego Zjazdu Hodowców Roœlin Ogrodniczych. Szczecin, 11-13.09.1997: 315-318. KERRIGAN R.W., ROSS I. K. (1989). Allozymes of wild Agaricus bisporus population: new alleles, new genotypes. Mycologia 81(3): 433-443. KHUSH R., MORHAN L., BECKER E., WACH M., GRIENSVEN L.J. (1991). Use of the polymerase chain reaction (PCR) in Agaricus bisporus. Proc. 1st Intern. Seminar on Mushroom Science, 14-17 May 1991, Netherlands: 73-80. KHUSH R., BECKER E., WACH M. (1992). DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus. Appl. Environ. Microbiol. 58: 2971-2977. LOFTUS M.G., MOORE D., ELLIOT T.J. (1988). DNA polymorphisms in commercial and wild strains of the cultivated mushroom, Agaricus bisporus. Theor. Appl. Genet. 76: 712-718. MAY B., ROYSE D.J. (1982). Confirmation of crosses between lines of Agaricus brunnescens by isozyme analysis. Exp. Mycol. 6: 238-292. ROYSE D.J., MAY B. (1982). Use of isozyme variation to identify classes of Agaricus brunnescens. Mycologia 74: 93-102.
