摘要：

香菇单核菌丝的杂交受到A和B等2种交配型因子的影响。基于全基因组测序可以对香菇单核菌丝进行交配型鉴定,但存在技术复杂和成本较高的局限。为实现对香菇单核菌丝未知交配型基因的快捷鉴定,以香菇H31和BJ4菌株为材料,采用对交配型位点区域的特异性扩增和重测序的方法,确定了可用于未知交配型基因鉴定的PCR扩增引物。利用菌丝杂交结果确定能够杂交的2种单核菌丝,将其交配型分别记为A1B1和A2B2。根据NCBI获得的A和B交配型位点基因序列的比对分析,在其保守区设计引物,对A1B1和A2B2单核菌丝进行PCR扩增。通过对扩增产物的重测序信息进行比对,获得A1和A2、B1和B2不同交配型基因序列中的非保守区,然后在此区域设计4种交配型基因特异性扩增引物,实现对H31和BJ4香菇单核菌丝交配型基因的鉴定。根据分子水平的交配型鉴定结果,对H31和BJ4香菇单核菌丝分别进行杂交验证。结果表明,交配型基因的分子鉴定结果与单核菌丝杂交结果一致。以香菇为试验材料确立的交配型基因鉴定方法不再依赖于全基因组测序,该方法同样适用于其他食用菌交配型基因的鉴定,因此,研究结果对食用菌的遗传育种具有广泛的应用价值。

关键词: 香菇, 杂交育种, 交配型鉴定, 单核菌丝, 引物设计

Abstract:

The hybridization of Lentinula edodes mononuclear mycelium is influenced by A and B two mating type factors.Based on whole genome sequencing,mating type identification of Lentinula edodes mononuclear hyphae can be carried out,but there are limitations in terms of technical complexity and high cost.In order to achieve rapid identification of unknown mating type genes in Lentinula edodes mononuclear mycelium,we used H31 and BJ4 strains as materials,and determined PCR amplification primers that can be used for identifying unknown mating type genes through specific amplification and resequencing of mating type site regions.The specific method was to use the hyphae hybridization results to determine the two mononuclear hyphae that could be hybridized,and record their mating types as A1B1 and A2B2,respectively.Based on the alignment analysis of the A and B mating type site gene sequences obtained from NCBI,primers were designed in the conserved region of their mating type sites to amplify the different mating type mononuclear hyphae A1B1 and A2B2.By comparing the resequencing information of the amplified products,we obtained nonconserved regions in gene sequences of different mating types A1 and A2,B1 and B2.Then,four mating type gene specific amplification primers to identify mating type genes in H31 and BJ4 Lentinula edodes mycelium were designed in this region.Based on the molecular level mating type identification results,hybridization validation was conducted on H31 and BJ4 Lentinula edodes mononuclear mycelium,and the results showed that the molecular identification results of mating type genes were consistent with the hybridization results of mononuclear mycelium.The mating type gene identification method established in this study no longer relies on whole genome sequencing.This method uses Lentinula edodes as experimental material,but it is also applicable to the identification of mating type genes in other edible mushrooms.Therefore,the results of this study have broad application value for genetic breeding of edible mushrooms.

Key words: Lentinula edodes, Cross breeding, Mating type identification, Mononuclear mycelium, Primer design