Cravero Melissa, Robinson Aaron J, Hilpisch Patrick, Chain Patrick S, Bindschedler Saskia, Junier Pilar
Laboratory of Microbiology, Institute of Biology, University of Neuchâtel, 2000, Neuchâtel, Switzerland.
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, 87545, USA.
IMA Fungus. 2022 Aug 22;13(1):14. doi: 10.1186/s43008-022-00101-6.
Morels are highly prized edible fungi where sexual reproduction is essential for fruiting-body production. As a result, a comprehensive understanding of their sexual reproduction is of great interest. Central to this is the identification of the reproductive strategies used by morels. Sexual reproduction in fungi is controlled by mating-type (MAT) genes and morels are thought to be mainly heterothallic with two idiomorphs, MAT1-1 and MAT1-2. Genomic sequencing of black (Elata clade) and yellow (Esculenta clade) morel species has led to the development of PCR primers designed to amplify genes from the two idiomorphs for rapid genotyping of isolates from these two clades. To evaluate the design and theoretical performance of these primers we performed a thorough bioinformatic investigation, including the detection of the MAT region in publicly available Morchella genomes and in-silico PCR analyses. All examined genomes, including those used for primer design, appeared to be heterothallic. This indicates an inherent fault in the original primer design which utilized a single Morchella genome, as the use of two genomes with complementary mating types would be required to design accurate primers for both idiomorphs. Furthermore, potential off-targets were identified for some of the previously published primer sets, but verification was challenging due to lack of adequate genomic information and detailed methodologies for primer design. Examinations of the black morel specific primer pairs (MAT11L/R and MAT22L/R) indicated the MAT22 primers would correctly target and amplify the MAT1-2 idiomorph, but the MAT11 primers appear to be capable of amplifying incorrect off-targets within the genome. The yellow morel primer pairs (EMAT1-1 L/R and EMAT1-2 L/R) appear to have reporting errors, as the published primer sequences are dissimilar with reported amplicon sequences and the EMAT1-2 primers appear to amplify the RNA polymerase II subunit (RPB2) gene. The lack of the reference genome used in primer design and descriptive methodology made it challenging to fully assess the apparent issues with the primers for this clade. In conclusion, additional work is still required for the generation of reliable primers to investigate mating types in morels and to assess their performance on different clades and across multiple geographical regions.
羊肚菌是备受珍视的可食用真菌，有性生殖对于其子实体的产生至关重要。因此，全面了解它们的有性生殖备受关注。其中的核心是确定羊肚菌所采用的生殖策略。真菌中的有性生殖由交配型（MAT）基因控制，羊肚菌被认为主要是异宗配合的，有两种不同的形态，即MAT1-1和MAT1-2。对黑色（高羊肚菌分支）和黄色（尖顶羊肚菌分支）羊肚菌物种进行基因组测序后，开发出了PCR引物，旨在扩增来自这两种不同形态的基因，以便对来自这两个分支的分离株进行快速基因分型。为了评估这些引物的设计和理论性能，我们进行了全面的生物信息学研究，包括在公开可用的羊肚菌基因组中检测MAT区域以及进行电子PCR分析。所有检测的基因组，包括用于引物设计的基因组，似乎都是异宗配合的。这表明最初利用单个羊肚菌基因组进行引物设计存在固有缺陷，因为要为两种不同形态设计准确的引物，需要使用具有互补交配型的两个基因组。此外，还为一些先前发表的引物组确定了潜在的非靶标，但由于缺乏足够的基因组信息和详细的引物设计方法，验证工作具有挑战性。对黑色羊肚菌特异性引物对（MAT11L/R和MAT22L/R）的检测表明，MAT22引物能正确靶向并扩增MAT1-2形态，但MAT11引物似乎能够扩增基因组内错误的非靶标。黄色羊肚菌引物对（EMAT1-1 L/R和EMAT1-2 L/R）似乎存在报告错误，因为公布的引物序列与报告的扩增子序列不同，并且EMAT1-2引物似乎扩增的是RNA聚合酶II亚基（RPB2）基因。引物设计中缺乏参考基因组和描述性方法，使得全面评估该分支引物存在的明显问题具有挑战性。总之，仍需要开展更多工作来生成可靠的引物，以研究羊肚菌的交配型，并评估它们在不同分支和多个地理区域的性能。
