Screening of the White Button Mushroom (Agaricusbisporus) Homokaryons and Producing New Hybrid Strain by SSR Markers

Document Type : Research Article


1 Ferdowsi University of Mashhad

2 Iranaian Academic Center for Education, Culture and Research (ACECR), Mashhad branch, Iran


Introduction: Edible white button mushroom (Agaricusbisporus) is the most common edible mushroom in Iran and the world. The yield of this mushroom is less than the average of yield in the world because of strain degeneration and using strains with low yield. Most of the current hybrids are either identical or very similar to the first hybrids. Ongoing breeding programs are exploiting the variability in Agaricus germplasm to produce new varieties with better traits including higher yield and resistance to biotic and abiotic stresses. One of the breeding programs is F1 production from parental homokaryons crossing. These homokaryonsis were isolated among germinated basidiospores on the culture media. During the last decades, various molecular markers based on nucleic acid polymorphisms (such as Restriction Fragment Length Polymorphism, Random Amplification of Polymorphic DNA, Amplified fragment of Length Polymorphism, Inter Simple Sequence Repeat, Simple Sequence Repeat markers) have been used to differentiate homokaryons and heterokaryons. Microsatellites consist of short tandem repeat motifs distributed throughout the genome. Microsatellites are usually highly polymorphic due to a high degree of variation in the number of repeats among individuals. Microsatellite markers are multiallelic and co-dominant and thus tend to be more informative than other marker systems. Microsatellite markers have been widely developed in animals and plants and more recently in fungal species. The presence of microsatellites in the genome of A. bisporus was previously reported.
Materials and Methods: In this research, 160 germinated basidiospores were collected from commercially cultivated strain A15 and they were grown on compost extract agar (CEA). The mycelial growth rate of these160 isolates was evaluated at 25°C on CEA medium. 18 isolates with slow growing rate were selected from 160 isolates. In the next step, co-dominant SSR markers were used to homokaryons detection. Ten SSR primers showed polymorphism in parental control samples that were used to this experiment. The isolates were divided into two general homoallelic and heteroallelic groups and seven isolates from homoallellic group, which showed one-band pattern, characterized as putative homokaryon. Genetic similarity was calculated by NTSYSpc software version 2.02 e using UPGMA method. In the next step of experiment, the isolates (4 and 8) had minimum genetic similarity that was crossed to produce hybrid. In order to confirm the hybrid formation, PCR-SSR reaction with a primer (AbSSR 45) was performed.
Results and Discussions: Basidiospores were collected and allowed to germinate on CEA medium. Putative homokaryons were different in colony morphology and growth rate compared to the original heterokaryons. Mycelium samples showed different colony morphology including tomentose, apprised and strandy mycelium. Different growth rate can be affected by genetic factors in nucleus and mitoconderia. After four weeks, mycelium browning was appeared in liquid compost extract medium and created a disturbance in DNA extraction. To solve this problem, DNA was extracted from three-week old mycelium. Mycelium browning may cause by phenolic compounds produced by mycelium and enzymes that catalyze melanin biosynthesis reactions. Ten primers were used to homokaryon isolation. These primers were situated on the 9 linkage groups of 13 haploid chromosomes. Seven isolates were distinguished as putative homokaryon that showed one-band in all primers on the gel electrophoresis. The results of genetic similarity calculation showed that this index was variable between 0.17 to 0.67in 7 homokaryon isolates and the minimum genetic similarity (0.17) was observed between isolates 4 and 8. These two isolates were crossed and the result of this crossing was N1 hybrid. Also, other homokaryon isolates were crossed and mating incompatibility was observed in some of them. According to these observations, it is suggested that in future studies, in addition to genetic similarity, sexual incompatibility should also be considered. Hybrid N1 produced aerial mycelium and had higher growth rate in comparison to parental homokaryons and similar to heterokaryon control, had two-bands pattern. This two bands pattern indicates the presence of two non-sister nucleuse in each cells. Finally, the results showed that SSR marker can result to accurate detection of homokaryons.
Conclusions: The aim of the present study was screening homokaryon isolates of A.bisporus using SSR markers to obtain hybrid. Results showed that growth rate of homokaryon isolates were lower than the heterokaryons. Since, SSR markers were able to show high polymorphism in the isolates, thus it can be said that these markers are suitable to homokaryon screening. Final result of this study is N1 hybrid that can compare to commercially cultivated strains.


1- AdamsL. S., Chen S., Phung S., Wu X., and KiL. 2008. White button mushroom (Agaricusbisporus) exhibits antiproliferative and proapoptotic properties and inhibits prostate tumor growth in athymic mice. Nutrition and Cancer, 60(6): 744-756.
2- Alipoor M., Farsi M., and Mirshamsi Kakhki A. 2014. Improvement of white Button Mushroom Yeild by AFLP Marker-assisted Single Spore Selection Method.Journal of Horticultural Science, 28(3):327-337. (in Parsian)
3- Birati M., Malekzadeh Shafaroudi S., Malekzadeh Kh. and Mirshamsi Kakhki A. 2015. SSR markers for screening and estimating genetic distancr of homokaryotic single spores in the white button mushroom(Agaricus bisporus). Journal Of Agricultural Biotechnology,6(4):23-36.(in Parsian with English abstract)
4- Callac P., Spataro C., Caille A., and Imbernon M. 2006. Evidence for outcrossing via the Buller phenomenon in a substrate simultaneously inoculated with spores and mycelium of Agaricusbisporus. Applied and Environmental Microbiology, 72(4): 2366-2372.
5- Castle A. J., Horgen P. A., and Anderson J. B. 1987. Restriction fragment length polymorphisms in the mushrooms Agaricus brunnescens and Agaricus bitorquis. Applied and Environmental Microbiology,53(4): 816-822.
6- Castle A. J., Horgen P. A., and Anderson J. B. 1988. Crosses among homokaryons from commercial and wild-collected strains of the mushroom Agaricus brunnescens (A. bisporus). Applied and Environmental Microbiology,54(7): 1643-1648.
7- Cullings K.W. 1992. Design and testing of a plant‐specific PCR primer for ecological and evolutionary studies. Molecular Ecology, 1(4): 233-240.
8- De La Bastide P.Y., Sonnenberg A., Van Griensven L.J.L.D., Anderson J.B. and Horgen P.A. 1997. Mitochondrial Haplotype Influences Mycelial Growth of Agaricus bisporus Heterokaryons. Applied and environmental microbiology, 63(9): 3426-3431.
9- Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin, 19: 11-15.
10- Farsi M., Jalalzadeh B., and MalekzadehKh. 2009. The breeding and production of hybrid straians of white button mushroom. Proceeding of the 6th Iranian Horticultural Science Congress, 2-15 Jul. 2009, Rasht, and I. R. Iran. (in Parsian)
11- Farsi M., and Pourian Far H. 2011. Cultivation and breeding of white button mushroom. JahadDaneshgahi Publications, Mashhad. (in Parsian)
12- Foulongne-Oriol M., Spataro C. and Savoie J.-M. 2009. Novel microsatellite markers suitable for genetic studies in the white button mushroom Agaricus bisporus. Applied Microbiology and Biotechnology,84(6): 1125-1135.
13- Foulongne-Oriol M., Spataro C., Cathalot V., Monllor S. and Savoie J.-M. 2010. An expanded genetic linkage map of anintervarietal Agaricus bisporusvar. bisporus× A. bisporus var. burnettii hybrid based on AFLP, SSR and CAPS markers sheds light on the recombination behaviour of the species. Fungal Genetics and Biology,47(3): 226-236.
14- Foulongne-Oriol M., Spataro C., Moinard M., Cabannes D., Callac P. and Savoie J.-M. 2012. Development of polymorphic microsatellite markers issued from pyrosequencing technology for the medicinal mushroom Agaricus subrufescens. FEMS Microbiology Letters,334(2): 119-126.
15- Gordan H.R., MahmoudniaMeymand M., ZouAlali J., Khatamirad M., and Farsi M. 2008. A study of potential of AFLP markers to genetic fingerprinting of the button mushroom, Agaricusbisporus. Journal of Plant Protection (Agricultural Science and Technology), 22(1): 27-36. (in Parsian with English abstract)
16- Ghorbani FaalP., Farsi M., Pourianfar H.R., Mahmoodnia M. and Zolala J. 2009. Preparation of AFLP Mediated-Molecular Certificate for 12 Bred Strains of the Button Mushroom, Agaricus bisporus. Journal of Plant Protection, 23(1): 58-67. (in Parsian with English abstract)
17- Heath M.C., Li A., Horgen P.A. and Tam P.L. 1995. Hyphal morphology associated with strain instability in the commercial mushroom, Agaricusbisporus. Mycologia, pp.442-450.
18- Horgen P. and Anderson J. 1992.Biotechnology and edible mushrooms. Biotechnology and Filamentous Fungi, Finkelestein, D. and C. Ball (Eds.). Butter Worth, Boston, USA. ISBN, 750691158: 447-462.
19- Horgen P. A. and Castle A. 2002. Application and potential of molecular approaches to mushrooms. Agricultural Applications, Springer, 3-17.
20- Jolivet S., Arpin N., Wichers H.J. and Pellon G. 1998. Agaricusbisporus browning:a review. Mycological Research, 102(12): 1459-1483.
21- Kavousi H.R., Farsi M. and Shahriari F. 2008. Comparison of random amplified polymorphic DNA markers and morphological characters in identification ofhomokaryon isolates of white button mushroom (Agaricusbisporus). PakistanJournal of Biological Sciences, 11(14): 1771-1778.
22- Kerrigan R.W. 1990. Evidence of genetic divergence in two populations ofAgaricusbisporus. Mycological Research, 94(6):721-733.
23- Kerrigan R.W., Baller L.M., Horgen P.A. and Anderson J.B. 1992. Strategies forthe efficient recovery of Agaricusbisporushomokaryons. Mycologia, 84(4): 575-579.
24- Kerrigan R.W., Royer J.C., Baller L.M., Kohli Y., Horgen P.A. and Anderson J.B.1993. Meiotic behavior and linkage relationships in the secondarily homothallicfungus Agaricusbisporus. Genetics, 133(2): 225-236.
25- Kerrigan R.W. and Spear M.C., Sylvan Spawn Laboratory Incorporated.1997.Method for the production of high proportions of homokaryons in breeding stockof the mushroom Agaricusbisporus. U.S. Patent NO. 5684228.
26- Kerrigan R.W. 2000. A brief hislory of marker assisted selection in Agaricusbisporus. Science and Cultivation of Edible Fungi 2000, 1:183.
27- LiR. and Fang X. 2002. Analysis of POD and EST isozyme of three Agaricus species. Edible Fungi of China, 21(4): 34-36.
28- Liu M.M., Xing Y.M., Zhang D.W. and Guo S.X. 2015. Novel microsatellite markers suitable for genetic studies in Polyporusumbellatus (Polyporales, Basidiomycota). Biochemical Systematics and Ecology, (61):450-457.
29- Ma F.Y. and Luo X.C. 2002. PCR-based restriction analysis of internal transcribed spacers of nuclear ribosomal DNA in the genus.
30- Mahmud M. A., Kitaura H., Fukuda M. and Yamada A. 2007. AFLP analysis for examining genetic differences in cultivated strains and their single-spore isolatesand for confirming successful crosses in Agaricusblazei. Mycoscience, 48(5): 297-304.
31- May B. and Royse D. J. 1982. Confirmation of crosses between lines of Agaricusbrunnescensby isozyme analysis. Experimental Mycology, 6(3): 283-292.
32- Miles P.G. and Chang S.T. 2004. Mushrooms: Cultivation, Nutritional Value,Medicinal Effect, and Environmental Impact. CRC press.
33- Miller R. E. 1971. Evidence of sexuality in the cultivated mushroom, Agaricusbisporus. Mycologia,8: 630-634.
34- Miller R.E. and Kananen D.L. 1972. Bipolar sexuality in the mushroom. MushroomSci, 8:713-718.
35- Moore A., Challen M., Warner P. and Elliott T. 2001. RAPD discrimination ofAgaricusbisporus mushroom cultivars. Applied Microbiology and Biotechnology,55(6): 742-749.
36- Mohammadi M., Jahadi M. and Khosravi-Darani K. 2013. Agaricusbisporus:Enzymatic browning and its inhibition methods. Iranian Journal of NutritionSciences and Food Technology, 7(4):63-71. (in Parsian with English abstract)
37- Murovec J., Stajner N., Jakse J. and Javornik B. 2007. Microsatellite marker forhomozygosity testing of putative doubled haploids and characterization of Mimulusspecies derived by a cross-genera approach. Journal of the American Society forHorticultural Science, 132(5): 659-663.
38- Nazrul M. I., Lin F. X. and Yin-Bing B. 2010. Screening of homokaryoticprotoclones of Agaricusbisporus (J. Lge) Imbach by colony characters and ISSR markers. Bangladesh Journal of Botany, 39(1): 119-122.
39- Nazrul M. I. and Yin-Bing B. 2010. ISSR as new markers for identification ofhomokaryoticprotoclones of Agaricusbisporus. Current Microbiology, 60(2): 92-98.
40- Nazrul M. I. and YinBing B. 2011. Differentiation of homokaryons and heterokaryons of Agaricusbisporus with inter-simple sequence repeat markers. Microbiological research,166(3): 226-236.
41- Pandey M. and Tewari R. 1994. Strategies for selection and breeding of edible mushrooms. Advances in Mushrooms Biotechnology. Edited by: MC Nair, C. Gokulapalan and L. Dass. Scientific Publishers, Jodhpur, India: 61-69.
42- Ramirez L., Muez V., Alfonso M., Barrenechea A.G., AlfonsoL. and Pisabarro A.G. 2001. Use of molecular markers to differentiate between commercial strains of the button mushroom Agaricusbisporus. FEMS Microbiology Letters, 198(1): 45-48.
43- Raper C. A., Raper J. R. and Miller R. E. 1972. Genetic analysis of the life cycle of Agaricusbisporus. Mycologia, 1088-1117.
44- Richard G.F., Hennequin C., Thierry A. and Dujon B. 1999. Trinucleotide repeats and other microsatellites in yeasts. Research in Microbiology, 150(9): 589-602.
45- Roupas P., Keogh J., Noakes M., Margetts C. and Taylor P. 2012. The role of edible mushrooms in health: Evaluation of the evidence. Journal of Functional Foods, 4(4): 687-709.
46- Royse D.J. and May B. 1982. Use of isozyme variation to identify genotypic classes of Agaricusbrunnescens. Mycologia, 93-102.
47- Sanchez-Perez R., Ruiz D., Dicenta F., Egea J. and Martinez-Gomez P. 2005. Application of simple sequence repeat (SSR) markers in apricot breeding: molecular characterization, protection, and genetic‌relationships. ScientiaHorticulturae, 103(3): 305-315.
48- Savoie J. M., Minvielle N. and Largeteau M. L. 2008. Radical scavenging properties of extracts from the white button mushroom, Agaricusbisporus. Journal of the Science of Food and Agriculture, 88(6): 970-975.
49- Selkoe K.A. and Toonen R.J. 2006. Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers. Ecology Letters, 9(5): 615-629.
50- Shehata A.I., Al-Ghethar H.A. and Al-Homaidan A.A. 2009. Application of simple sequence repeat (SSR) markers for molecular diversity and heterozygosity analysis in maize inbred lines. Saudi Journal of Biological Sciences, 16(2): 57-62.
51- SingerR. and Harris B. 1987. Mushrooms and truffles.Koeltz Scientific Books.Germany.389pp.
52- Soltis Lab CTAB DNA extraction protocol. 2002. The soltis lab, florida museum of natural history, on line at:
53- SongS., Su W., Zeng W. and Wang Z. 2000. RAPD analysis of species diversity and genomic differences in Agaricusbisporus. Science and Cultivation of Edible Fungi 2000, 1: 191.
54- Sonnenberg A. S. M., Johan J. P. B., Hendrickx P. M., Lavrijssen B., Wei G., Weijn A., Mes J. J., Savoie J. M., Foulongne-Oriol M., Largeteau M. and Barroso G. 2011. Breeding and strain protection in the button mushroom Agaricusbisporus. In Mushroom biology and mushroom products. Proceedings of the 7th International Conference on Mushroom Biology and Mushroom Products, Arcachon, France, 4-7 October.
55- Terashima K. and MatsumotoT. 2004. Strain typing of shiitake (Lentinulaedodes) cultivars by AFLP analysis, focusing on a heat-dried fruiting body. Mycoscience, 45(1): 79-82.
56- WangY., ChenM., Wang H., Wang J.-F. AndBao, D. 2014. Microsatellites in the Genome of the Edible Mushroom, Volvariellavolvacea. Biomed Research International 2014.
57- XiaoY., LiuW., DaiY., FuC. and BianY. 2010. Using SSR markers to evaluate the genetic diversity of Lentinulaedodes’ natural germplasm in China. World Journal of Microbiology and Biotechnology, 26(3): 527-536.
58- YanP.-S. AndJiang J.-H. 2005. Preliminary research of the RAPD molecular marker-assisted breeding of the edible basidiomyceteStrophariarugoso-annulata. World Journal of Microbiology and Biotechnology, 21(4): 559-563.
59- ZhangR., HuD., Zhang J., Zuo X., Jiang R., Wang H. and Ng T.B. 2010. Development and characterization of simple sequence repeat (SSR) markers for themushroom Flammulinvelutipes. Journalof Bioscienceand Bioengineering, 110(3): 273-275.
60- Zhao J. and Chang S.T. 1993. Monokaryotization by protoplasting heterothallic species of edible mushrooms. World Journal of Microbiology and Biotechnology, 9(5):538-543.