Mitochondrial Inheritance During Outcrossing In a Substrate Simultaneously Inoculated With Spores And Mycelium Of Agaricus bisporus

PR9.34 available on https://www.fgsc. net//ECFG9/ecfg_9_poster_session_3. htm Mitochondrial Inheritance During Outcrossing In a Substrate Simultaneously Inoculated With Spores And Mycelium Of Agaricus bisporus Philippe Callac1, Rana Kabalan1, Cyril Férandon2, Gérard Barroso2 ECFG9 5th-8th April 2008 Edinbrugh In Agaricus bisporus var. bisporus most of the basidiospores are heterokaryotic (predominant pseudohomothallic life cycle). From this, the outcrossing classical method (confrontation of homokaryons in vitro) is hampered by the difficulty to recover homokaryotic strains. However, it was recently shown that numerous hybrid sporocarps are easily produced in vivo by simultaneously inoculating a standard compost of culture with a homokaryon from one parent and the spores from a second parent (Callac et al. 2006). In this case, it was indirectly shown that these crosses occurred between the heterokarotic spores (or mycelia issued from them) and the inoculated homokaryon (Buller phenomenon). Our aim was to determine from which parent the mitochondria are inherited during this in vivo process, in comparison to an in vitro confrontation of homokaryons. For this, a SCAR mitochondrial marker derived from a previously reported (Xu et al. 1998) RFLP marker was developed to distinguish the two parental haplotypes. The PRM1 marker : a mitochondrial SCAR marker based on the polymorphism of the introns of the Cox1 gene Aknowledgements to J.B. Anderson and J.P. Xu who provided RFLP probes; to M. Moinard, C. Spataro and P. Castant for technical assistance References Calllac P., Spataro C. , Caille A. et Imbernon M. , 2006. Appl. Env. Microbiol. 72:2366-2372 . Xu J., Kerrigan R. W. , Sonnenberg A. , Callac P., Horgen P. A. et Anderson J.B. , 1998. Molecular Ecology 7: 19-33. Mitochondria inheritance in hybrid strains is uniparental and appears to depend on the used outcrossing method. When two homokaryons are mated in vitro, one of the two parental haplotypes is preferentially inherited. When crosses are performed in vivo, Buller phenomenon occurs and data show that one nucleus from the heterokaryotic spores (or the mycelium issued from them) migrates alone in the homokaryotic mycelium. In a simple model where mycelia from spores (6% homokaryotic) would cross at random, 97% of the crosses would occur between homokaryons and heterokaryons. This would lead to hybrids without preferential haplotype inheritance (when homoA x heteroB and heteroA x homoB equally occurred). This agrees with the numerous different haplotypes found in wild populations. DISCUSSION sporeprint PARENT A strain Bs 518 (brown cultivar C9) haplotype mt1 (genotype Prm1-1) Agaricus bisporus is amphithallic: among the offspring, about 94% of the single spore isolates were heterokaryotic, while 6% were homokaryotic. Spore suspension (about one billion) most of them are heterokaryotic (n+n) PARENT B strain Bs 548 (white hybrid cultivar S608) haplotype mt2, (genotype Prm1-2) Homokaryon Bs 548-2 protoplastes Compost tray  Spores and homokaryon are simultaneously inoculated in the compost of 4 culture trays  Fructification of hybrid sporocarps  Isolation of the AxB hybrid strains by tissue culture of the sporocarps 20 different hybrids (AxB) were isolated. Hybrid status was confirmed by using nuclear SCAR markers OUTCROSSING IN VIVO OUTCROSSING IN VITRO Outcrossing between parents A and B, by using two different methods 12 homokaryotic single spore isolates (n)  Homokaryons are confronted in pairs in 12 compost agar plates Positive reaction (fluffy mycelium) between sexually compatible homokaryons  Isolation of the AxB hybrid strains from the fluffy mycelium 12 different hybrids (AxB) were isolated. Hybrid status was confirmed by using nuclear SCAR markers genotype PRM1 The 20 hybrids possessed the Prm1-2 allele The 12 hybrids possessed the Prm1-1 allele CONCLUSION Mitochondria were inherited from the parent B Mitochondria were inherited from the parent A A B 1-INRA UR1264 Mycologie et sécurité des aliments BP 81,33883 Villenave d’Ornon, France email: callac@bordeaux.inra . fr 2– Université Victor Ségalen Bordeaux 2 Two primers: Mu and Mr Allele Prm1-1 : one intron I1 (≈ 1kb) Allele Pm1-2 : two introns I1’+I1’’ (≈ 3kb) 8,01 kb 2,9 kb B6 B15 E8 (6,7kb) E17b (2,5kb) E11 (4,2kb) E19 (2kb) 2,9 kb B6’ B15 E8’(11,3kb) E11’ (3,8kb) E19 Bs518 Bs548