Laboratorio de Patología Vegetal, IIAF, Universidad Michoacana de San Nicolás de Hidalgo , Km. 9.5 Carr. Morelia-Zinapécuaro, Michoacán 58880 , Mexico.
Mycotoxin Prevention and Applied Microbiology Research Unit, Agricultural Research Service, United States Department of Agriculture , Peoria , Illinois 61604-3999.
Mycologia. 2019 Sep-Oct;111(5):772-781. doi: 10.1080/00275514.2019.1649956. Epub 2019 Sep 5.
We discovered that published polymerase chain reaction (PCR) assays for determining mating type () idiomorph failed to genotype some of the species complex (FFSC) isolates recovered from (mango), (big-leaf mahogany), (soursop), sp., and sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR-based diagnostic for typing within the FFSC. To accomplish this objective, we mined the or locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase () and apurinic endonuclease (), the genes that flank the locus in . As expected, three genes were identified within (, and ) and two in ( and ), using the prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting and successfully genotyped the entire 71-isolate validation panel, which included 56 FFSC and 4 FNSC phylospecies. By contrast, the published PCR assays we tested produced positive genotypes for only 46.5-59% of the 71-isolate validation panel, but only when they were run as a uniplex assay. Although only one-fifth of the FFSC/FNSC are known to reproduce sexually, our results suggest that if they possess a sexual cycle, it is heterothallic (self-sterile).
我们发现，用于确定交配型（）同态的已发表聚合酶链反应（PCR）检测未能对从墨西哥采集的某些物种复合体（FFSC）分离株进行基因分型，这些分离株来自（芒果）、（大果桃花心木）、（刺果番荔枝）、和 sp.，以及 sp.。因此，本研究的主要目的是设计和验证一种基于多重 PCR 的 FFSC 内基因分型诊断方法。为了实现这一目标，我们从代表 56 个种系的 60 个 FFSC 分离株的基因组中以及来自其姐妹群（FNSC）中的 4 个种的基因组中挖掘了 或 基因座。通过针对 DNA 裂合酶（）和脱嘌呤内切核酸酶（）进行靶向搜索，促进了生物信息学搜索，这两个基因侧翼在 基因座中。不出所料，在 （、和 ）中鉴定出了三个基因，在 （和 ）中鉴定出了两个基因，使用 预测工具 AUGUSTUS。在我们设计和测试的三个多重 PCR 检测中，针对 和 的检测成功地对整个 71 个分离株验证面板进行了基因分型，该面板包括 56 个 FFSC 和 4 个 FNSC 种系。相比之下，我们测试的已发表的 PCR 检测仅对 71 个分离株验证面板中的 46.5-59%产生了阳性基因型，但仅当它们作为单重检测运行时。尽管已知只有五分之一的 FFSC/FNSC 通过有性繁殖繁殖，但我们的结果表明，如果它们具有有性循环，那么它是异宗配合的（自不育的）。
