{"title":"New insights into the DNA extraction and PCR amplification of minute ascomycetes in the genus Laboulbenia (Pezizomycotina, Laboulbeniales).","authors":"Warre Van Caenegem, Danny Haelewaters","doi":"10.1186/s43008-024-00146-9","DOIUrl":null,"url":null,"abstract":"<p><p>Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.</p>","PeriodicalId":54345,"journal":{"name":"Ima Fungus","volume":null,"pages":null},"PeriodicalIF":5.2000,"publicationDate":"2024-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11167896/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Ima Fungus","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s43008-024-00146-9","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MYCOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.
关于拉布贝尼亚属(Pezizomycotina,Laboulbeniales)微小子囊菌 DNA 提取和 PCR 扩增的新见解。
由于 Laboulbeniales 目(子囊菌目,Pezizomycotina)真菌体型微小,无法在轴向培养中生长,而且缺乏可靠、经济的 DNA 提取方案,因此多年来对其进行的分子研究一直受到阻碍。尤其是拉布贝菌属,其 DNA 提取和聚合酶链式反应(PCR)扩增的成功率很低。这是因为细胞中存在一种已知会抑制聚合酶链反应的分子--黑色素。我们评估了基于单细胞的标准 DNA 提取方案的效果,将推荐试剂量减半以降低每次提取的成本,并在多重置换扩增步骤中加入牛血清白蛋白(BSA)以逆转黑色素的影响。共进行了 196 次提取,其中 111 次成功。我们发现,将单细胞提取试剂盒中使用的试剂减半并不会明显影响成功提取 DNA 的概率。使用减半方案可降低成本,减少资源消耗。此外,是否添加 BSA 对成功提取 DNA 的概率也没有明显影响,这表明存在于苔藓细胞中的黑色素数量对 PCR 没有重大抑制作用。我们从五个位点生成了 277 个序列,但内部转录间隔区、线粒体小亚基 rDNA 和蛋白质编码基因的扩增仍具有挑战性。标本保存方法也会影响成功提取拉布贝氏菌DNA的概率,与保存在70%乙醇中的标本和干燥标本相比,保存在95%以上乙醇中的标本成功率更高。我们强调了正确保存材料的重要性,并建议设计拉布氏菌特异引物,以克服引物错配和杂质的问题。我们的新见解不仅适用于拉布贝菌属;拉布贝菌属的研究普遍不足,绝大多数物种仍未测序。新的、易于理解的分子发展将有利于拉布贝菌属的研究,有助于阐明这些奇特的微真菌的真正多样性和进化关系。
Ima FungusAgricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)
CiteScore
11.00
自引率
3.70%
发文量
18
审稿时长
20 weeks
期刊介绍:
The flagship journal of the International Mycological Association. IMA Fungus is an international, peer-reviewed, open-access, full colour, fast-track journal. Papers on any aspect of mycology are considered, and published on-line with final pagination after proofs have been corrected; they are then effectively published under the International Code of Nomenclature for algae, fungi, and plants. The journal strongly supports good practice policies, and requires voucher specimens or cultures to be deposited in a public collection with an online database, DNA sequences in GenBank, alignments in TreeBASE, and validating information on new scientific names, including typifications, to be lodged in MycoBank. News, meeting reports, personalia, research news, correspondence, book news, and information on forthcoming international meetings are included in each issue