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Abstract：
Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67-87 fragments), the values of polymorphism information content (PIC = 0.3345-0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788-60.3818), resolving power (Rp = 22.3152-34.3152), discriminating power (D = 0.5183-0.5553), expected heterozygosity (H = 0.4247-0.4443), and mean heterozygosity (H avp = 0.00002-0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651-0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.
摘要：
副球菌病（PCM）是一种威胁生命的全身性真菌感染，是从环境中吸入副球菌繁殖体后获得的。主要病原体包括巴西疟原虫复合体（系统发育定义的物种S1，PS2，PS3和PS4）和卢兹疟原虫的成员。蛋白质编码基因座的DNA测序(例如，GP43，ARF，和TUB1）是由于缺乏稳健的表型标记而识别副球菌物种的参考方法。因此，开发信息丰富且具有成本效益的新分子标记是提供高质量信息以探索副科植物遗传多样性的关键。我们报告了使用新的扩增片段长度多态性（AFLP）标记和交配型分析来对副球菌物种进行基因分型。生物信息学分析生成了144个计算机AFLP图谱，突出两个区分性引物对组合(#1EcoRI-AC/MseI-CT和#2EcoRI-AT/MseI-CT)。在体外使用组合#1和#2来对从南美洲广大地区回收的165个副球菌分离株进行基因分型。考虑到体外总体评分的AFLP标记（67-87个片段），多态性信息含量值(PIC=0.3345-0.3456)，标记指数(MI=0.0018)，有效复用率(E=44.6788-60.3818)，分辨能力（Rp=22.3152-34.3152），鉴别力(D=0.5183-0.5553)，期望杂合度(H=0.4247-0.4443)，和平均杂合度(Havp=0.00002-0.00004)，证明了AFLP标记可用于形成副球菌并解剖深层和精细遗传结构。分子方差分析（AMOVA）显示，总遗传变异（65-66％）是由于巴西疟原虫复合体和路兹疟原虫之间的变异性（PhiPT=0.651-0.658，P<0.0001），支持高度结构化的人口。异性恋是唯一的交配策略，巴西假单胞菌的MAT1-1或MAT1-2同态分布没有明显偏斜（比例为1：1）。str。(χ2=1.025;P=0.3113),委内瑞拉疟原虫(χ2=0.692；P=0.4054)，和P.lutzii（χ2=0.027；P=0.8694），支持每个物种内的随机交配。相比之下,美洲疟原虫(χ2=8.909；P=0.0028)和restrepiensis(χ2=4.571；P=0.0325)的分布呈偏态，MAT1-1占优势。地理分布证实了美洲疟原虫，P.restrepiensis，和P.lutzii比以前想象的更普遍。巴西P.s.str.是迄今为止拉丁美洲国家中出现最广泛的血统，发生在巴西的所有地区。我们新的DNA指纹分析被证明是快速的，可重复,高度歧视,为了深入了解分类法，生态学,和副球菌的流行病学，指导疾病控制策略以减轻PCM。
