Studies on nuclear phase and genetic attribute of the basidiospores of Flammulina velutipes

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1 263369~ Mycosystema %7.5% 12.3% RAPD RAPD Q939.5 A Studies on nuclear phase and genetic attribute of the basidiospores of Flammulina velutipes XU Zhi-Yong 1 LIN Fan-Xue 1,2 XIONG Jie 1 MA Li 1 LIN Fang-Can 1* ( 1 The Institute of Applied Mycology, Huazhong Agricultural University, Wuhan , China; 2 Department of Biology, Jining College, Jining , China) AbstractNuclear phase and genetic attribute of the basidiospores were investigated for 3 strains of Flammulina velutipes. Fluorescence staining observation displayed that the majority of basidiospores were dikaryotic and the ratio of dikaryotic spores, monokaryotic ones and nuclear-free ones was 80.2%, 7.5% and 12.3% respectively. The mycelia derived from single spore isolates were confirmed as polykaryotic septate ones without clamp connection. In the mating test, mycelial protoplast pairs between single spore isolates of different strains formed colonies with clamp connection, while mycelial protoplast pairs between the same single spore isolates formed colonies without clamp connection, suggesting that the two nuclei of the spores possessed identical mating type factors. RAPD analysis indicated that the profiles of mycelial protoplasts obtained from same single spore isolates amplified by 10 random primers were entirely identical, corroborating that the two nuclei of a basidiospore were homogeneous. In addition, it is observed that there are 4 basidiospores growing on a basidium. Consequently, F. velutipes is a tetrapolar fungus possessing 4 basidiospores where there are two homogeneous No No * Corresponding author. linfangcan@mail.hzau.edu.cn

2 nuclei. Key wordsfluorescence staining, protoplast, RAPD, homogeneous nuclei, tetrapolarity Flammulina velutipesfr.singer Agaricales Tricholomataceae Flammulina RAPD F328 F2153 F PDAPDY MYG µg/mL Hoechst µg/mL Fluorescent Brightener primer12 1MgCl25mmol/LdNTP 2 10mmol/L Taq DNA 1U/µL MBI Leica Panasonic WV-CP470 Japan Nikon Edipse80iPCR PERKIN ELMER Gene Amp PCR System 9600 eppendorf BioPhotometerDYY11B Gene Genius Bioimaging System

3 3 371 Table1 Nucleotide sequence of 12 randomly selected primers tested Primer Sequence Primer Sequence S06 TGCTCTGCCC S24 AATCGGGCTG S08 GTCCACACGG S242 CTGAGGTCTC S10 CTGCTGGGAC S28 GTGACGTAGG S11 GTAGACCCGT S29 GGGTAACGCC S22 TGCCGAGCTG S30 CAATCGCCGT S23 AGTCAGCCAC S33 CAGCACCCAC PDA PDY 20d 20 CTAB 2004 DNA DNA RAPD 1 PCR buffer 150µmol/L dntp0.2µmol/l primer50ng DNA1.25U Taq DNA 2mmol/L MgCl 2 20µL 92 5min92 1min35 1min72 2min min 20055µL1.2 EB Leica 4 1-A 1-B

4 % 7.5% 12.3% 2-A Fig.1 Optical micrograph (400 ). A: Cut section of gill; B: Basidiospores. Fig.2 Photos of fluorescence staining (1000 ). A: Fluorescence staining of spores; B: Fluorescence staining of mycelia. 2.3 PDA Hoechst33258 Fluorescent Brightener28 2-B F d 12d PDA 4d

5 F % χ 2 25% Lentinula edodes Pleurotus ostreatus 25% F2153F5 F d 12d 27 PDA 4d F d PDA 7d F F d PDA 6d F328F5 F d Fig.3 The profile of mycelial protoplasts obtained from a single spore isolate of F328 amplified by 10 random primers.

6 DNA DNA RAPD F328F2153 F5 3 DNA 10 EB 3 DNA 3 4 F328 F5 3 DNA Fig.4 The profile of mycelial protoplasts obtained from a single spore isolate of F5 amplified by 10 random primers.1, 2 and 3 respectively stand for 3 protoplasts in the profiles. 3 Schizophyllum commune Coprinus cinereus 4 [REFERENCES] Chang ST, Lin FC, Genetic and Breeding of Mushroom. Chinese Agricultural Press, Beijing. 65~68(in Chinese) Huang NL, Guo MY, Wu SZ, Flammulina velutipes and Pleurotus sajor-caju. Shanghai Science and Technology Press, Shanghai. 1~52(in Chinese) Huang Y(ed.), Cultivation of Edible Mushroom. Higher Education Press, Beijing. 146(in Chinese) Lin FC, Chang ST, Analysis of incompatibility factors in cultivated strains of Lentinus edodes in China. Journal of Huazhong Agricultural University, 14 (5):459~466(in Chinese) Liu WX, Xie BG, Wang XQ, Jiang YG, Studies on RAPD makers of color gene in Flammulina velutipes. Chinese

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