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• W1914836626 abstract "Arbuscular mycorrhizal fungi (AMF) belong to a widely distributed, ecologically and economically important symbiotic fungal phylum, the Glomeromycota. These heterotrophic fungi take plant-assimilated carbon through an obligate symbiosis with most vascular plants, in exchange of enhancing their host's uptake of mineral nutrients (phosphate and nitrates) from the soil (Bonfante & Genre, 2010) and protecting them against pests and fungal pathogens (Bonfante & Genre, 2010; Corradi & Bonfante, 2012). In addition to being ecologically relevant, the AMF are also intriguing from a cellular and genetic perspective, as they produce coenocytic hyphae and spores that contain multiple nuclei in a common cytoplasm. The presence of co-existing nuclei has been suggested by some to facilitate the purge of deleterious mutations in these supposed ancient asexuals (Sanders & Sanders, 2003; Sanders & Croll, 2010), although recent studies have found indirect evidence that sex and more conventional mode of reproduction may exist in this lineage (Riley & Corradi, 2013; Riley et al., 2014). The genetic organization of coexisting nuclei in the Glomeromycota has been a major source of debate for the past 15 years, with two opposite hypotheses being supported within the research community. Specifically, the presence of an unusually high sequence variability in some AMF genes has been suggested by some authors to result from the presence of genetically divergent nuclei within AMF spores and hyphae (i.e. heterokaryosis hypothesis). This hypothesis has been backed by sequence analyses of cloned PCR products and fluorescence in situ hybridization (Kuhn et al., 2001), as well as by models based on the existence of hyphal fusions among genetically nonidentical individuals (Bever & Wang, 2005) and by quantitative real-time PCR approaches of a few selected genes (Hijri & Sanders, 2005). Though highly intriguing, the assumption that AMF could harbor a population of diverse nuclei has also faced challenges from other studies. In particular, PCR-based analyses of individually microdissected nuclei and approaches based on the distribution of genetic variation among and within field isolates of AMF, have both indicated that molecular diversity in AMF may be the result of polyploidy – that is, present within each AMF nucleus, as opposed to among nuclei (Pawlowska & Taylor, 2004; Pawlowska, 2005). Models supporting the possibility of heterokaryosis in AMF based on the nuclear exchange have also been disputed (Pawlowska & Taylor, 2005). More recently, the presence of heterokaryosis in AMF has been challenged by whole genome analyses of diversity, which revealed that the model AMF (Rhizophagus irregularis) harbors very little polymorphism (e.g. ranging from 0.06 to 0.43 SNP kb−1; Tisserant et al., 2013; Lin et al., 2014), in line with what is known for homokaryotic fungi. Although genome investigations appeared to have settled the debate on heterokaryosis in AMF for good, a recent study based on the analysis of four single copy genes is sparking the debate once again by revealing the presence of an atypically high polymorphism in three Rhizophagus isolates (DAOM197198, DAOM234328, DAOM229456) (Boon et al., 2015). At the extreme, allelic diversity along these small sequences was found to reach 103 alleles using a combination of pyrosequencing (Roche 454 Life Science Technology), cloning and Sanger sequencing. This is in stark contrast with the minute amount of diversity detected through genomic analyses, and begs the question: what is causing such enormous differences in single nucleotide polymorphism (SNP) count? To answer this question, we reassessed the occurrence of SNP along the entire coding sequence of these four genes (as opposed to < 100 bp in Boon et al., 2015) using Illumina technology and Sanger sequencing on five different isolates of the AMF R. irregularis. To ensure a fair comparison of our results, analyses were also performed on R. irregularis DAOM197198; a strain that is also included in the analysis of Boon et al. (2015) and whose genome is available (Tisserant et al., 2013). To confirm the presence of polymorphism in the four earlier mentioned genes, the published alignments from Boon et al. (2015) were used as queries in reciprocal tblastx searches to identify the corresponding orthologous sequences in the available genome of R. irregularis DAOM197198. Their respective paired-end Illumina reads (kindly provided by F. Martin, INRA, France) were mapped using BWA-MEM v0.7.10-r789 (Li, 2014) with default parameters (the gene 40S-riboprot could not be found in the assembly of DAOM197198 and was therefore not analyzed for this isolate), and SNPs were called using FreeBayes v0.9.18-3-gb72a21b (Garrison & Marth, 2012) with the following parameters: -K (e.g. output all alleles which pass input filters), excluding alignments with a mapping quality less than 20 (-m 20) and taking into account only SNPs with at least two alternate reads (-C 2). SNP were filtered to avoid the analysis of false positives (i.e. SNP originating from misalignment and/or paralogy) using vcffilter from the vcf-lib library according to (1) the read depth (maximum read depth: DP < 1.25 × genome mean coverage; minimum read depth: DP > genome mean coverage – (0.1 × genome mean coverage)), (2) the type of SNPs (only considering SNPs, not indels), (3) the reference allele observation (RO > 1). Blast procedures confirmed the single-copy status of these genes, but our mapping approaches failed to detect the presence of any SNP along their entire alignments. Importantly, reducing the mapping stringency did not result in the detection of lower-frequency variants. These results are completely different from those reported by Boon et al. (2015) on the same isolate. To obtain independent confirmation for the absence of polymorphism at these four genes in Rhizophagus, we took advantage of Illumina sequencing 50 bp mate pairs and 100/125-paired-end reads recently acquired in our laboratory from five other isolates of R. irregularis (A1, A4, A5, B3 and C2; Croll et al., 2008). Reads were obtained using the HiSeq2500 platform available at Fasteris SA (Geneva, Switzerland), and mapped against their respective reference sequences using earlier mentioned procedures. Mapping resulted in high coverage for all genes in all isolates (above 66× in all cases), but failed, once again, to confirm the presence of polymorphism; a finding confirmed using Sanger sequencing (Table 1 and Supporting Information Table S1 and Notes S1; BAM files available on the NCBI website under the BioProject ID PRJNA277029). The only SNP identified across our entire dataset was restricted to the isolate A5 in the ARP gene, and its 0.5/0.5 allelic frequency indicates it probably originated from heterozygosity (i.e. intra-nuclear diversity). When the entire coding sequences are considered, another SNP is found in the same isolate in the RLi-ABC gene, also with a 0.5/0.5 allelic frequency. Here, we have shown that alternative methodologies to detect SNP can result in drastically different outcomes. One possibility is that the sequencing methodology plays a central role in the discovery of real SNPs. In our study, high coverage Illumina reads (coverage ranging from 66 to 84× for our genes) were used for SNP detection purposes, as opposed to the 454 pyrosequencing amplicon sequencing approach in Boon et al. (2015). It is noteworthy that the latter technology is notorious for producing sequence bias in homopolymers (Balzer et al., 2011). Interestingly, our manual inspections revealed that polyA/T were flanking the sequences analyzed by Boon et al. (2015) (one is only 15 bp upstream of the analyzed region), and these have been shown to increase the likelihood of both carry forward and incomplete extension in 454 pyrosequencing, resulting in false indels and ambiguous bases and substitutions (Huse et al., 2007). Another possibility is that the level of genetic polymorphism in AMF differs greatly among isolates of the same species. If that is true, then our strains would represent exceptionally monomorphic cases, possibly as a consequence of their decade-long cultivation under laboratory conditions. This, however, would not explain why we found no variation in DAOM197198; a strain found to be highly polymorphic by Boon et al. (2015). Whatever the cause for these discrepancies, the picture is clear: there is an urgent need to standardize approaches for the detection of SNP in AMF research. As an illustration, the observed difference of SNP density calculated in the two manuscripts on the R. irregularis genome should be considered as null and void as SNP density was not calculated with same data (RNAseq vs genomic data), same reference genes, same software and same threshold of criteria. As a consequence, these values cannot be compared as proposed by Limpens & Geurts (2014). One way to achieve this goal of standardization could be to focus on sequencing techniques/methodologies that are universally recognized as being more reliable (i.e. less prone to sequencing errors), such as Illumina. This could be complemented by analyses of (1) well-covered areas of the genome, and (2) appropriate filtering and (3) downstream confirmations of SNP calls using alternative approaches for a subset of detected polymorphic loci (e.g. using PCR and Sanger sequencing procedures). It is also important to note that our reassessment of four single genes does not suggest that polymorphism is absent in AMF. In fact, genome-wide analyses of variation, and the present study, have revealed that low-frequency polymorphic regions clearly exist in these organisms (Tisserant et al., 2013; Lin et al., 2014). These variations are, however, far lower than in true heterokaryotic fungi (Hane et al., 2014). What future studies should now elucidate is the nature and extent of such polymorphism, that is, are some regions/genes particularly affected by SNP, and does variation reflect single substitutions or more complex structural variability among co-existing nuclei (i.e. paralogy, variation in ploidy). Hopefully, future analyses of SNP diversity in AMF will shed light into this exciting aspect of AMF genetics and biology. To this end, large scale comparative analyses of many isolates and/or species may be required. The authors would like to thank three anonymous reviewers for their helpful comments on a previous version of this Letter. We are grateful to Ian Sander's group for providing the in-vitro cultures of the isolates used in this study. Nicolas Corradi is a Fellow of the Canadian Institute for Advanced Research. This work was supported by the Discovery program from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery) and an Early Researcher Award from the Ontario Ministry of Research and Innovation to N.C. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W1914836626 title "Homokaryotic vs heterokaryotic mycelium in arbuscular mycorrhizal fungi: different techniques, different results?" @default.
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• W1914836626 doi "https://doi.org/10.1111/nph.13448" @default.
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