香菇单核体交配型及杂交后代的等位基因特异性PCR鉴定

Identification on mating types of monokaryons and their hybrids of Lentinula edodes by AS-PCR

摘要:

【背景】 食用菌交配型和单单杂交杂合子的鉴定通常采用显微镜检测观察是否具有锁状联合的方式进行，存在耗时长、工作量大且易出现误检等问题。【目的】 建立一种鉴定香菇单核体交配型和单单杂交后代的分子辅助育种技术，为提高育种效率提供技术支撑。【方法】 利用交配型因子保守序列单核苷酸多态性(single-nucleotide polymorphism, SNP)位点设计分型引物，建立等位基因特异性PCR (allele-specific PCR, AS-PCR)技术，鉴定香菇L808和YX7的单孢分离株交配型及其杂交后代。【结果】 AS-PCR鉴定结果表明，L808的38个单孢分离株中，交配型为A1B1、A2B2、A1B2和A2B1的单核体分别有6、13、8和11个；YX7的45个单孢分离株中，交配型为A3B3、A4B4、A3B4和A4B3的单核体分别有15、8、8和12个，交配型为A3A4B3B4的异核体2个；12个单单杂交菌株中，10个为真正的杂合子，2个为非杂合子。传统方法与AS-PCR分子鉴定结果完全一致，但前者容易将异核体误判为单核体。【结论】 基于SNP位点的AS-PCR技术能有效鉴别香菇单核体交配型和单单杂交后代，区分单核体与异核体，具有精准、高效的特点，是一种香菇分子辅助育种的理想工具。

Abstract:

[Background] The mating types and mon-mon hybrids of edible mushrooms are usually identified based on the presence of clamp connections or not as observed by microscopy, which is time-consuming, labor-costing, and prone to false results. [Objective] A molecular-assisted breeding technique was established for identifying the monokaryon mating type and mon-mon hybrids of Lentinula edodes, aiming to provide technical support for improving the breeding efficiency. [Methods] Typing primers were designed based on the SNP loci in the conserved sequence of mating-type factors, and then allele specific (AS)-PCR was employed to identify the mating type of the single spore isolates of L. edodes strains L808 and YX7 and their mon-mon hybrids. [Results] Among the 38 single spore isolates of L808, there were 6, 13, 8, and 11 monokaryons of mating types A1B1, A2B2, A1B2, and A2B1, respectively. Among the 45 single spore isolates of YX7, there were 15, 8, 8, and 12 monokaryons of mating types A3B3, A4B4, A3B4, and A4B3, respectively, and 2 heterokaryons of the mating type A3A4B3B4. The 12 mon-mon hybrids included 10 heterozygotes and 2 non-heterozygotes. The identification results obtained based on the conventional method were consistent with those obtained by AS-PCR, except that the former was prone to misjudging heterokaryons as monokaryons. [Conclusion] The AS-PCR based on SNP loci can identify the monokaryon mating type and mon-mon hybrids of L. edodes and distinguish between monokaryons and heterokaryons, with high precision and efficiency. It serves as an ideal tool for molecular-assisted breeding of L. edodes.