Estimation of the Glomus intraradices nuclear DNA content

Abstract

The arbuscular mycorrhizal fungi (AMF) are able to establish a symbiotic relationship with 70–90% of land plant species including Angiosperms, Gymnosperms, Pteridophytes and some Bryophytes (Smith & Read, 2008). In plants, AMF improve the uptake of phosphorus and nitrogen (Bücking & Shachar-Hill, 2005; Govindarajulu et al., 2005), provide protection from root pathogens (Newsham et al., 1994; Liu et al., 2007; Hata et al., 2010), improve salt and drought tolerance (Marulanda et al., 2006, 2007; Evelin et al., 2009) and potentially contributes heavy metal tolerance (Ricken & Hofner, 1996; Zhang et al., 2005). Despite the positive impact of AMF fungi on plant nutrition and stress tolerance, only a small number of studies have been performed on AMF genetics. Basic information, such as a genome sequence, ploidy level, the number of chromosomes or the origin of genetic material in spore is currently not available; this lack of knowledge hampers investigations on genetic exchanges, recombination and segregation in the AMF. The aim of this study was to determine the nuclear DNA content of the Glomus intraradices (syn.: G. irregulare) industrial strain produced by AMykor GmbH (Germany) with G. intraradices DAOM197198 isolate (Pont Rouge, Canada) used as the reference. The decision to use G. intraradices DAOM197198 Schenck & Smith DAOM 197198 (recently reassigned to Rhizophagus irregularis (Blaszk., Wubet, Render & Buscot) C. Walker & A. Schüβler comb. nov.) isolate as the reference organism was based on the fact that both isolates are closely related (data not shown), and the genome size and ploidy level of DAOM197198 isolate has already been determined by Hijri & Sanders (2004). G. intraradices DAOM197198 was also chosen for the first complete genome sequencing project on AMF (Martin et al., 2008). Unfortunately, despite a large amount of sequencing data, sequence assembly is complicated by the high degree of polymorphism among nuclei (F. Martin, pers. comm.). There are only a few reports on the nuclear DNA content of AMF. The average genome size of AMF seems to be highly variable and ranges from c. 15.7 Mb in Glomus intraradices (DAOM197198, Hijri & Sanders, 2004) and c. 37 Mb in Glomus etunicatum (Hijri & Sanders, 2005), up to c. 250 Mb in Glomus versiforme (Bianciotto & Bonfante, 1992) and c. 740 Mb in Gigaspora margarita (Hosny et al., 1998). In most of these reports the DNA content was estimated by means of flow cytometry (FC). In this study, we present data on the nuclear DNA content of Glomus intraradices AMykor isolate and a reference, DAOM197198 isolate, measured by FC and Feulgen DNA image densitometry. The nuclear DNA content of in-vitro monoxenic cultures from both Glomus intraradices isolates (AMykor and DAOM197198) was determined by FC on six independent nuclei isolations using Arabidopsis thaliana as an internal reference standard (Fig. 1). The nuclei of A. thaliana were isolated from differentiated leaf tissue, which is characterized by distinct endopolyploidization resulting in a mixture of polyploid cells (Galbraith et al., 1991). Nuclei suspensions from endopolyploid cells are consequently composed of nuclei with different DNA content (2C, 4C, 8C and so on). Accordingly, FC histograms, representing the relative fluorescence intensities of stained nuclei, are comprised of the 2C peak used for the genome size estimation and additional peaks of the higher ploidy level (Fig. 1). Histograms of relative fluorescence intensities obtained after FC analysis of propidium iodide-stained nuclei of (a) Glomus intraradices DAOM197198, (b) G. intraradices DAOM197198 (grey) with the internal reference standard Arabidopsis thaliana‘Col-O’ (black), (c) G. intraradices AMykor and (d) G. intraradices AMykor (grey) with the internal reference standard A. thaliana‘Col-O’ (black). Due to endoreduplication occurring in A. thaliana leaf tissue nuclei (Galbraith et al., 1991), different ploidy levels are detectable. The first peak represents 2C nuclei with a DNA content of 0.32 pg. Fluorescence intensity of nuclei is expressed in arbitrary units (linear scale). Due to different nuclei isolation protocols used for fungi and plants, nuclei were isolated separately and subsequently mixed for FC measurements. We used the same fixation procedure and staining buffers for both type of nuclei to minimize inaccuracies caused by the different treatments of the sample and the standard. Note: the only difference in the procedure was in the mechanical disruption of the cells (further details on the methods used can be found in the Supporting Information Methods S1). Using this procedure, the DNA content per nucleus was determined to be 0.156 pg for the Glomus intraradices AMykor isolate and 0.157 pg for the G. intraradices DAOM197198 isolate. According to Hijri & Sanders (2004), and the genome sequencing consortium (Martin et al., 2008; F. Martin, pers. comm.), G. intraradices is a haploid organism. Thus, the genome size of both isolates can be estimated to be c. 150 Mb which is similar to the genome size of Arabidopsis thaliana (157 Mb/1C). The reliability of our data was confirmed by additional FC measurements using a second internal reference standard, as well as Feulgen densitometry measurements. FC measurements of two Glomus isolates using Raphanus sativus (543 Mb/1C; Dolezel et al., 1998) as a reference revealed a nuclear DNA content of 0.146 pg for the Glomus AMykor isolate and 0.147 pg for the Glomus DAOM197198 isolate (Table 1). Although, the values obtained from measurements with two different reference standards are quite similar, they differ from each other by factor of 1.07. Since it is generally accepted that the genome size of the reference standard should be not too different from the unknown sample to avoid instrumental problems with linearity (Vindeløv et al., 1983), we consider the values estimated using Arabidopsis thaliana as reference to be more precise. In addition, Feulgen densitometry analyses of 20 nuclei per Glomus intraradices isolate with 10 telophase nuclei of Glycine max as a reference standard (Dolezel et al., 1998) gave an estimated DNA content for Glomus AMykor of 0.185 and 0.170 pg for DAOM197198 isolate (Table 1). These values are in agreement with the FC data and indicate that the genome of Glomus intraradices is 10 times bigger than previously postulated (Hijri & Sanders, 2004). FC has been successfully used for evaluating the nuclear DNA content of different Glomeromycota species (Hosny et al., 1998). In this study, we have improved the nuclei isolation method, which allowed rapid analysis of a large number of nuclei and reliable estimation of the DNA content. We calculated the average genome size per nucleus to be 153.0 ± 3.6 Mb for the Glomus intraradices AMykor isolate and 154.8 ± 6.2 Mb for the G. intraradices DAOM197198 isolate based on the conversion formula of Dolezel et al. (2003). Feulgen densitometry measurements resulted in very similar values to the FC analysis. These values are in contrast to data published by Hijri & Sanders (2004) who reported a genome size of 15.74 ± 1.69 Mb for the G. intraradices DAOM197198 isolate. The differing values may be explained at least partially by methodologicaly differences in the nuclei isolation procedure. In comparison to Hijri & Sanders (2004), we used Arabidopsis thaliana and Raphanus sativus as internal reference standards instead of Saccharomyces cerevisiae as an external reference. Although the isolation of nuclei was, due to technical reasons, performed separately for fungal and plant material, fixation, staining and measurements were done under identical conditions. Use of unsuitable or external standards for FC measurements performed separately from the analyzed samples can cause imprecise or erroneous results. Additionally, the initial results from a genome sequencing project of Glomus intraradices DAOM197198 (Martin et al., 2008; F. Martin, pers. comm.), confirm that the genome is much larger than estimated by Hijri & Sanders (2004) and is in agreement with our findings. Knowledge of the genome size of Glomus intraradices will be useful in a genome sequencing project and the success of this project will lead to better and faster recognition of the genes involved in plant–fungi symbiosis which will result in an improvement of the application of this relationship in agriculture and industry. Although there are still a number of questions concerning the genetics of AMF to be investigated, the data presented here will be useful in future investigations focused on understanding the genetics of AMF. The authors are grateful to Professor Ian Sanders (University of Lausanne, Switzerland) and Professor Mohammed Hijri for comments on the manuscript and provision of a sample from the Glomus intraradices DAOM197198 (Att1192-44) isolate. Thanks to Mrs G. Oswald for an excellent technical assistance. The research work was supported by grants from AMykor GmbH in Bitterfeld-Wolfen, Germany, the ‘Deutsche Bundesstiftung Umwelt’ (Grant No. 20005/803) and by Funds from the Chemical Industry (G.K.). Methods S1 All the techniques used to obtain results shown in this paper, including established method for AMF nuclei isolation and their analysis by FC and FISH. 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.