单孢分离菌株的异核不对称特性揭示梯棱羊肚菌为假同宗结合真菌

本文以基因组数据显示只具有MAT1-1 idiomorph的单孢菌株YPL6-3和只具有MAT1-2 idiomorph的单孢菌株YPL6-1为材料,研究它们子代的子囊果、单孢菌株群体和同一子囊中8个单孢菌株的交配型分布情况。YPL6-3和YPL6-1菌株分别隔离栽培和互补混栽均能形成正常的子囊果,其子囊果菌柄交配型分布与亲本菌株有关。PCR扩增检测235株子代单孢菌株的交配基因出现有趣现象：一些菌株的MAT1-1-1基因电泳条带强,而MAT1-2-1基因的条带弱;另一些菌株则MAT1-1-1基因条带弱,而MAT1-2-1基因条带强。同时也有两基因条带都强或者一基因条带强,另一基因无条带的菌株。从3个子囊果中共挑取了10个子囊,并对每个子囊中的单孢进行独立分离,PCR扩增并电泳检测交配基因时也出现了上述相同的情况。若MAT1-1-1强,则MAT1-2-1弱或无,这样的菌株在同一子囊中不会超出4个,反之亦然。利用长片段PCR扩增YPL6-1和YPL6-3菌株的全长MAT idiomorph,利用Nanopore测序技术对扩增子进行单分子实时测序,2次重复实验的序列比对发现：菌株YPL6-1中存在99.63%和99.81%的MAT1-2 idiomorph分子及0.37%和0.19%的MAT1-1 idiomorph分子;菌株YPL6-3中存在99.45%和99.74%的MAT1-1 idiomorph 分子及0.55%和0.26%的MAT1-2 idiomorph分子。从而证实,这2个基因组测序和PCR扩增都只能检测到一种交配型的菌株,其实是异核菌株,只是2种交配型核的数量占比存在较大偏离。根据上述现象推断,梯棱羊肚菌的子囊孢子都是异核的,萌发后形成的单孢菌株具有异核不对称特点,从而推测梯棱羊肚菌是一种特殊的假同宗子囊菌,同时也揭示了梯棱羊肚菌单孢出菇的真相。

The asymmetrical distribution of opposite mating type nuclei in single-ascospore isolates revealed Morchella importuna is a pseudohomothallic fungus

The mating-types of F1 ascocarps, single-ascospore populations and eight single-ascospore strains in an ascus were analyzed based on the crossing of single-ascospore strains YPL6-1 and YPL6-3 of Morchella importuna which harbored MAT1-2 and MAT1-1 idiomorph respectively in their genome-sequencing data. Under the conditions of sowing separately and mix-sowing, strains YPL6-1 and YPL6-3 could fructify normally, and the distribution of mating type in the stipe was related to the parent strain. When the mating type tests were carried out by PCR amplification in 235 single-ascospore strains, something interesting happened: the electrophoretic bands of MAT1-1-1 gene in some strains were bright, but the bands of MAT1-2-1 were weak. In other strains, the MAT1-1-1 band was weak, while the MAT1-2-1 band was bright. Meanwhile, there were strains that two mating gene bands were bright, or strains that one mating gene band was bright while the opposite mating gene band unappeared. Ten asci were separated from three ascocarps and corresponding single ascospores were isolated from each ascus, and the mating types of these single-ascospore strains were analyzed. As a result, the same phenomenon occurred. The single-ascospore strains showing bright MAT1-1-1 band and weak or no MAT1-2-1 band originated from no more than four spores in an ascus, and vice versa. The PCR amplicons of MAT loci in the YPL6-1 and YPL6-3 were sequenced respectively by nanopore approach, and corresponding experiments were repeated twice. The alignment analysis indicated that there were 99.63% and 99.81% MAT1-2 idiomorphs, and 0.37% and 0.19% MAT1-1 idiomorphs in the strain YPL6-1, meanwhile, the strain YPL6-3 contained 99.45% and 99.74% MAT1-1 idiomorphs, and 0.55% and 0.26% MAT1-2 idiomorphs. The result confirmed that these two single-ascospore strains were actually heterokarytic, however, there was a great deviation in proportion of two mating type nuclei. It is speculated that all ascospores in M. importuna are heterokarytic, and the opposite mating type nuclei are asymmetrically distributed in mycelia germinated from single ascospore. Therefore, M. importuna is a pseudohomothallism ascomycete fungus, and the single-ascospore strain could be self-fertile.