Rhizophagus proliferus genome sequence reiterates conservation of genetic traits in AM fungi, but predicts higher saprotrophic activity

Arbuscular mycorrhizal (AM) fungi are ubiquitous endosymbionts of terrestrial plants. It helps plants to extract more nutrients from the soil and enhances the plant tolerance to various ecological stress factors. The AM fungal genome sequence helps to identify the gene repertoires that are crucial for adaptation to different habitat and mechanisms for interaction with host plant. The present work comprises the first draft of the genome sequence of Rhizophagus proliferus, which is an important AM species present in biofertilizer consortia for agricultural purpose. The estimated genome size of R. proliferus is ~ 110 Mbps and its genomic assembly is 94.35% complete. Genome mining was carried out to identify putative gene families important for biological functions. A total of 22,526 protein-coding genes were estimated in the genome, with an abundance of kinases and reduced number of glycoside hydrolases as compared to other fungal classes. A striking finding in the R. proliferus genome was higher number of carbohydrate esterases (CE), which may suggest towards presence of higher saprotrophic activity in this species as compared to the previously reported AM fungi, which may indicate towards its role as a link between plants and soil mineral nutrients. The genome sequence and annotation of R. proliferus presented here would serve as an important reference for functional genomics studies required for developing biofertilizer formulations in future. In addition, the findings from this work may also prove important in deciphering molecular mechanisms in AM fungi that govern the host-specific interaction and associated agriculture benefits.


Introduction
AM fungi have approximately 350 to 1000 molecularly defined species under the Division Glomeromycota. These fungi are obligate biotrophs and complete their life-cycle by developing symbiotic relationship with a host plant (Smith and Read 2008). AM fungi have mutualistic association with more than 80% of terrestrial plants (Schüßler et al. 2001) and benefit them by improving nutrient and water uptake efficiency, and also resistance to various abiotic and biotic stresses (Jung et al. 2012). These fungi are endosymbionts and form highly branched-hyphal structures called arbuscules inside the plant cortical cells. The arbuscules deliver mineral nutrients to the cortical cells and also function in carbon acquisition from the host. Mechanisms underlying obligate symbiotic relationship of AM fungi with plant are not completely understood. Knowledge regarding genome organization, genetic functions and reproductive mechanism of only a few species of AM fungi is available at present. These species include: Rhizophagus irregularis (Tisserant et al. 2013), Rhizophagus clarus (Kobayashi et al. 2018), Diversispora epigaea (Sun et al., 2018), Gigaspora rosea, Rhizophagus diaphanus, Rhizophagus cerebriforme (Morin et al. 2019) and Gigaspora margarita (Venice et al. 2020). Plant productivity in ecosystems are dependent on the species of AM fungi present in the soil ( Van et al. 1998). Lack of genetic information of AM fungi creates difficulty in their optimization for crop-yield enhancement. Association of AM fungi can increase net primary productivity of plants by improving Phosphorous (P) and Nitrogen (N) uptake in different ecosystems (Treseder et al. 2018). AM fungi enhance stress tolerance capability of plants and support plants to grow in heavy metal polluted soils (Leyval et al. 2002;Yang et al. 2016). Furthermore, gene repertoire coded in the AM fungal genomes are responsible for making host-specific interactions (Prasad et al. 2019). The crop-yield benefits are in part governed by host-specific interactions and different combinations of AM species associated with host-plants in different agro-climatic regions (Schütz et al. 2018). In line with this, it has been recently reported that the genotypes of R. irregularis and the associated host determine colonization efficiency in Cassava (Peña Venegas et al. 2021).
Thus, the genome derived information on genetic structure of AM fungi may provide clear direction for applying external inoculation of AM fungi-based bio-fertilizer products. Therefore, to unravel the genetics of AM fungi, researchers have started exploring genome sequences of different species of AM fungi (Tisserant et al. 2013;Kobayashi et al. 2018;Sun et al. 2018;Morin et al. 2019;Venice et al. 2020). Information obtained from these next generation sequencing projects suggested that the genomes of AM fungi lack some core eukaryotic genes such as those involved in synthesis of thiamine, invertase gene, glutathione metabolism, fatty acid synthase (FAS) and many other (Supplementary file: Table S9). An interesting finding reported by researchers involved in understanding the molecular exchanges between AM fungi and the host plant is import of plant synthesized lipids by AM fungi (Jiang et al. 2017). In agreement with this finding, the genome sequencing projects clearly showed absence of the multi-domain fatty acid synthase FAS-I gene in the genomes of AM fungi (Tisserant et al. 2013;Kobayashi et al. 2018;Sun et al. 2018;Morin et al. 2019;Venice et al. 2020).
R. proliferus is an important AM fungal species that was first described by Declerck et al. (2000) to possess distinguishing characteristics of spores, such as, small size, hyaline color, smooth wall surface, permanent fourlayered spore wall structure, and long hyphae that produced clusters of spores containing several hundred individuals. Most noticeably, anastomoses between hyphae and retraction septa, which are peculiar traits for spore germination in the absence of a host (Logi et al. 1998), were frequently observed in R. proliferus. We performed de novo genome sequencing and genome annotation of R. proliferus, and compared the genetic structure with previously reported AM fungi, Ecto-mycorrhizal (EM) fungi and a pathogenic ascomycetes fungi. These included Rhizophagus irregularis (Tisserant et al., 2013), Rhizophagus clarus (Kobayashi et al. 2018), Diversispora epigaea (Sun et al. 2018), Gigaspora rosea, Rhizophagus diaphanus, Rhizophagus cerebriforme (Morin et al. 2019) and Gigaspora margarita (Venice et al. 2020) among the AM fungi; Laccaria bicolor (Martin et al. 2008), Tuber melanosporum (Martin et al. 2010) among the ecto-mycorrhiza (EM) fungi; and Rhizophus oryzea (Ma et al. 2009) as a pathogenic ascomycetes fungi. R. proliferus (previously known as Glomus proliferum), is morphologically different from the model AM fungal species R. irregularis.
The isolate of R. proliferus sequenced here has been found to provide benefits to multiple crop-species such as tomato, carrot and sugarcane by improving P and N uptake.

Aim, design and setting of the study
The research work reported here was undertaken to understand the genome structure and function of R. proliferus. Genome sequencing was done using Illumina's next-generation sequencing method. A de novo assembly was created using the sequenced reads after quality control. This

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Page 3 of 12 105 was followed by in silico prediction of gene repertoire in R. proliferus followed by their annotation and estimation for important protein families. An additional exploration to identify presence of core eukaryotic genes in R. proliferus was also carried out.

Fungal isolate and DNA extraction
Primarily spores of R. proliferus were collected from the Palwal district, which is situated between the eastern bank of Yamuna River and the western flank of Aravalli mountain range of Haryana, India. Its habitat type is plain terrain, annual temperature is 8.33 °C-40 °C, and annual rainfall is 542 mm and 28° 09′N 77° 20′E latitude and longitude location, elevated at 198 m. The spores were purified and monoculture (AM-1901) was established by the Centre for Mycorrhizal Culture Collection (CMCC) of The Energy and Resources Institute (TERI), India. Spores were produced in mono-axenic cultures that were maintained on Agrobacterium rhizogenes-transformed roots of carrot (Daucus carota, Clone GP1). A total of 150,000 sterile spores were collected and high molecular weight (HMW) genomic DNA was extracted using Cetyltrimethylammonium bromide (CTAB) method. Quality of the isolated genomic DNA was checked using the Qubit 2.0 fluorometer (Thermo Fisher Massachusetts, USA).
To investigate the morphological features of the species, microscopic analysis after PVLG (Polyvinyl-Lacto-Glycerol) and Melzer's staining of spores was carried out using a compound microscope (Carl Zeiss primostar) and a previously published protocol (Błaszkowski et al. 2014). Voucher specimen of R. proliferus spores was prepared using the methodology (https:// invam. wvu. edu/ metho ds) from INVAM [International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi]. Accordingly, to permanently mount either whole or broken spores on glass slides, spores with minimum quantity of water were put on a glass slide, added 1 drop of PVLG (Polyvinyl-Lacto-Glycerol) and Melzer's staining reagent, waited for 5 min to get the drop dried, then cautiously placed a coverslip. Molecular identification of R. proliferus was carried out using Internal Transcribed Spacer (ITS) region primer sequence in a nested PCR (Stockinger et al. 2010).

Genome sequencing, assembly and annotation
DNA was fragmented and library was constructed using the Nextera DNA Library Prep protocol. Sequencing (2 × 150 bp paired-end sequencing) was performed using the services of a commercial service provider (AgriGenome Labs Pvt. Ltd., Kerala, India) on a HiSeq 2500 sequencing platform. Quality control of the DNA library was done by analysis on an Agilent 2000 Bioanalyzer. Preprocessing of reads was carried out [adapter trimming and quality trimmed (Q > 20)] using AdapterRemoval version 2.2.0 (https:// adapt errem oval. readt hedocs. io/ en/ stable/). Homology search of preprocessed reads were done using Basic Local Alignment Search Tool. In the first step, sequences from the carrot genome were filtered out. Following this, Blastn suite against bacterial database was used and only the unaligned reads were considered. The mitochondrial sequences were removed from the bacterial unaligned reads by comparing the reads with the NCBI database mitochondrion.1.1.genomic.fna. gz. The filtered sequences were assembled into scaffolds by executing De novo assembly method Spades version 3.12.0 (https:// cab. spbu. ru/ softw are/ spades/). All scaffolds with length < 1000 bp were excluded in the final assembly. The scaffold sequences were also subjected to homology search in the NCBI nucleotide database and all the scaffolds with Identity > 90%, Query-coverage > 75%, GC content > 50% and from non-fungal origin were excluded from the assembly (Tisserant et al. 2013;Morin et al. 2019). The repeat sequences were masked using REPEATMASKER version 4.0.7 (http:// www. repea tmask er. org/) and the total survived scaffolds were considered for downstream analysis. The completeness of the R. proliferus draft genome assembly was searched against the core eukaryotic genes present in CEGMA (http:// korfl ab. ucdav is. edu/ datas ets/ cegma/) to evaluate genome completion. tRNAs were identified using tRNAscan-SE version 1.3.1 (http:// lowel ab. ucsc. edu/ tRNAs can-SE/). For annotation, gene prediction with taxonomybased parameters was conducted using AUGUSTUS version 3.1.0 (http:// bioinf. uni-greif swald. de/ augus tus/) with the gene model of Saccharomyces cerevisiae.

Phylogenetic analysis
To understand the phylogenetic relationship of R. proliferus, with respect to other species in Glomeromycota, a phylogenetic analysis was conducted. ITS (Internal Transcribed Spacer) gene sequences of six species of AM fungi were retrieved from NCBI. These species included R. irregula- The ITS gene sequence of the in-house sequenced R. proliferus was also included in the analysis. Sequences were aligned using MUSCLE alignment tool (https:// www. ebi. ac. uk/ Tools/ msa/ muscle/). Maximum Likelihood (ML) phylogenetic tree was constructed using MEGA X (https:// www. megas oftwa re. net/) and the reliability of the tree was estimated by bootstrapping with 100 bootstrap values.

Morphological and molecular characterization
The morphological details of the isolate of R. proliferus sequenced in this study are presented in Fig. 1. The spores were small in size with diameter ranging between 65 and 125 µm at different stages of life-cycle and had three distinct wall layers. The spores were observed to have hyaline color, smooth wall surface, and had long hyphae that held a bunch of spores. The identified morphological features, the sequence of the Internal Transcribed Spacer (ITS) gene (Accession No.: OK030706) (Supplementary file: Table S1) and phylogenetic analyses presented in Fig. 2 confirmed that the species under investigation is R. proliferus.

Genome sequencing, assembly and structure
15 Mio reads and 7.8 Gb of primary sequences were received from the whole genome sequencing project. After quality control, the raw sequences were assembled into n = 12,903 scaffolds with an assembly size of ~ 102.4Mbps and average GC content 27.99%. N 50 scaffolds and L 50 values were 2126 and 13,544 bp, respectively (Table 1).

Genome annotation
A total of n = 22,526 protein-coding genes were estimated by specifying Saccharomyces cerevisiae as the model species in AUGUSTUS version 3.1.0. n = 187 tRNAs genes were predicted (Supplementary file: Table S3). n = 15,087 proteins shared homology with the NCBI nr database. n = 3988 genes that were identified by InterPro-Scan search were found to be distributed in 52 different domains (Supplementary file: Table S4). Two predicted domains were unique to R. proliferus: PPM-type phosphatase (IPR001932) and PTP-type proteins phosphatase (IPR000242), both of which have been found to influence, signal transduction and cell cycle (Supplementary file: Table S4). The terms protein phosphorylation, nitrogen compound metabolic process, cell communication, and signal transduction were frequent GO Biological process (Fig. 3a). Hydrolase, transferase and proteins involved in binding of different types of compounds and molecules were among the few top terms under GO Molecular function (Fig. 3b).
For core eukaryotic genes (CEG) NCBI blastp (1e-20), PFAM (protein family) homology and functional-domain search tools were employed to scan the genome sequence of R. proliferus. Distribution of CEG in R. proliferus was investigated by blastp analysis (1E-20) against the Saccharomyces genome database (Supplementary: Table S9) and n = 19 genes from the set of "missing ascomycete core genes (MACGs)" were identified. Furthermore, many important CEGs such as the fatty acid synthase (FAS), Thiamine biosynthetic pathway genes, Glutathione metabolism genes, and invertase gene were not found. Supplementary: Table 9 presents a status of these genes in other fungi, which were included for comparison in this study.
A total of n = 4569 genes were assigned to 321 KEGG pathways in R. proliferus (Supplementary file: Table S5) and n = 1200 genes were predicted under kinases gene families (Kinome) by search in Kinomer. Proportion of the genes belonging to the kinome family among AM, EM species and pathogenic ascomycetes species was compared (Tisserant et al 2013;Kobayashi et al. 2018;Sun et al. 2018;Morin et al. 2019;Venice et al. 2020;Martin et al. 2008Martin et al. , 2010Ma et al. 2009). An expansion of the protein kinase gene family was seen in R. proliferus as well as all other AM fungi. Particularly, the Tyrosine kinase-like proteins (TKL), exhibited 20 folds increase in size in R. proliferus and other AM species as compared to the ectomycorrhiza fungi L. bicolor and around 300 folds increase as compared to the ascomyetes R. oryzae (Table 2). Furthermore alpha protein kinases were  predicted in R. proliferus, which are similar to other AM fungi and are reported to be absent in ectomycorrhizal and pathogenic fungal species. n = 132 genes coding for carbohydrate active enzyme (CAZyme) (Supplementary file: Table S6) were identified by HMMSCAN searches in CAZy database. Expansins (EXPN) and polysaccharide lyases (PL) were not predicted in R. proliferus. Fig. 4 presents the distribution of important classes (AA, CBM, CE, GH, GT, PL and EXPN) under CAZyme in R. proliferus in comparison to the previously reported AM, EM and pathogenic ascomycetes fungi (Tisserant et al. 2013;Kobayashi et al. 2018;Sun et al. 2018;Morin et al. 2019;Venice et al. 2020;Martin et al. 2008Martin et al. , 2010Ma et al. 2009). In the present study, the Carbohydrate esterases (CE) were present in a significantly higher proportion, i.e.; n = 25 in R. proliferus in comparison to other AM fungi given in (Supplementary file: G-protein coupled receptors (GPCR) proteins with CFEM domain are involved in different biological processes in fungi such as cellular development and host-pathogen interaction. Only one GPCR-like protein coding gene was identified in R. proliferus, but CFEM domain was absent. Query for modular nonribosomal peptide synthetases (NRPS), and dimethylallyl diphosphate tryptophan synthases (DMATS) using the SMURF database did not yield any gene coding for polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS) or secondary metabolites/toxins.
For genes involved in sexual reproduction, a total of 89 HMG (high mobility group) box containing genes (Supplementary file: Table S7) and 47 meiosis-related genes (Supplementary file: Table S8) were identified through blastp search in SMART HMG-domain database. The genes also included the three meiosis-specific genes (Msh4, Dmc1 and Hop2) that have been reported to function exclusively in the meiosis process.

Discussion
AM fungi constitute an important group of fungi for sustainable agriculture benefits; however, the genome sequences and gene repertoire of most of the AM species are not yet explored. The information on genetic structure of these fungi could provide important information about molecular mechanisms underlying the host-specific interaction with different species of crop plants and associated agriculture benefits (Prasad et al. 2019). For majority of fungal classes and species, the information regarding their genetic structure and function has commonly been acquired by comparative   studies with the genomes of model species belonging to Ascomycota and Basidiomycota. However, it is difficult to achieve understanding about the genetic architecture of AM fungi by similar comparisons as Ascomycota and Basidiomycota are only distantly related with Glomeromycota and extensive divergence between them over the long evolutionary period has occurred (Sanders and Croll 2010). With such a background, the exceptional identifications regarding the lack of many genes constituting the basic machineries for eukaryotic metabolic pathways in Glomeromycota, expansion of kinome and reduction of CAZymes are being cautiously probed. The investigation reported here provided first draft of the genome sequence and genome annotation of R. proliferus, which is one of the important species of AM fungi known to provide benefits to multiple crops. The estimated size of genome is ~ 110 Mbps, which is the smallest of all the reported AM fungi till date. Like the previously reported AM fungi, conservation with respect to fewer carbohydrate active enzymes and higher number of protein kinases was predicted in R. proliferus in comparison to EM and Ascomycetes fungi. High proportion of protein classes representing "establishment of localization" were seen in GO classification in R. proliferus. Previous reports suggest that genes coding for "establishment of localization" play important role in the development of plant-microbial interactions in a symbiotic association (Voß et al. 2018).
The remarkable enlargement of protein kinase gene family and especially of tyrosine kinase-like (TKL) genes is supported by the previous reports in AM fungi (Tisserant et al. 2013;Lin et al. 2014;Salvioli et al. 2016;Tang et al. 2016). Protein kinases influence most cellular activities, especially cell signaling, by protein phosphorylation. The expansion of kinase gene family has been suggested to be crucial in signal transduction processes that are involved in establishment of symbiotic interaction between AM fungi and plant. Interestingly, a high number of TKL-containing proteins have been observed in germinating spores and intraradical mycelium in R. irregularis (Tisserant et al. 2013).
In R. proliferus, the conservation of alpha protein kinases, which is an ancient class of protein, is similar to the other species of AM fungi sequenced so far.
The reduced presence of Glycoside hydrolases (n = 24) found in R. proliferus in comparison to other fungal division was in conformity with the other species of AM fungi. Expansins (EXPN) and Polysaccharide Lyases (PL) were absent in R. proliferus genome similar to the previous reports in AM species. Expansins (EXPN) functions in cell wall loosening and help the accommodation process of the fungus inside the cortical cells (Cosgrove et al. 2002). Expansins of fungal origin are supposed to function in the loosening of interfacial material. Polysaccharides lyases (PL) play a role in degradation of pectin layers of wood (Kristiina and Miia 2018). These observations in AM fungi, unlike the EM and the pathogenic fungi, has been proposed as "functional tradeoffs" in an obligate symbiont for achieving a stealth entry and colonization into root while evading plant immune response (Tisserant et al. 2012). In contrast to the previous reports in AM fungi (Tisserant et al. 2013;Tang et al. 2016;Kobayashi et al. 2018;Sun et al. 2018;Morin et al. 2019;Venice et al. 2020), presence of proteins belonging to AA4 family in R. proliferus was striking. AA4 codes for Vanilly-alcohol oxidase (VAO), which are intracellular FAD-dependent enzymes that act on activated aromatic alcohols like 4-hydroxybenzyl alcohols. AA4 is directly not involved in lignocellulolysis but in the metabolism of ligninderived compounds. Another noteworthy observation for CAZymes in R.poliferus is higher abundance of carbohydrate esterases (CE) in comparison to the other reported AM fungi. Larger number of carbohydrate esterases (CE) may suggest for its higher putative plant litter decomposing activity. AM fungi have been proposed to be involved in 'direct mineral cycling' and nutrient supply to plants (Bunn et al. 2019).Therefore, the higher number of CE genes in R. proliferus fungi in comparison to other species might suggest towards increased saprophytic activity leading to quicker and more extraordinary leaf litter decay and aid in transferring nutrients released by decomposing leaf litter to host. CE is a special class of enzyme which de-acetylate hemicellulose and pectin units of plant polysaccharides (Sista and Qin 2018). Deacetylation leads to breaking of glycosidic linkages and help in degradation of plant cell wall, which could further enable entry of microorganisms in plants. Furthermore, gene coding for Acetylxylan esterase was identified in many of the predicted CE families, i.e.; (CE1, CE2, CE3, CE4, CE6) of R. proliferus in comparison to other AM fungi. This enzyme plays a significant role in the complete hydrolysis process of lignocellulosic materials (Morley et al. 2006). Such observations emphasize that R. proliferus may have higher plant cell-wall degrading activity.
With regard to Vitamin B6 metabolism, a previously reported gene PDX1 (Pyridoxal), which is involved in vitamin B6 biosynthesis ) is also predicted in R. proliferus (Supplementary file: Table S5).
A widespread notion of the absence of sexual recombination in AM fungi was challenged by the contrasting observations made in the whole genome analysis of R. irregularis (Lin et al. 2014). An exploration of the sexual potential of R. irregularis identified a putative AM fungi mating-type locus with prominent similarities to the mating-type locus of Basidiomycota (Ropars et al. 2016). In addition, n = 76 HMG (high mobility group) box containing genes were identified in R. irregularis (Riley et al. 2014). Also, in G. rosea 48 meiosis-related genes were found . In agreement with these previous findings, n = 89 HMG (high mobility group) box containing genes and n = 47 meiosisrelated genes were identified in R. proliferus. Such a conservation of meiosis-related genes re-emphasized existence of a yet unknown sexual reproduction mechanism in Glomeromycota fungi and particularly in R. proliferus.
In context of presence of CEG in R. proliferus a total of n = 234/248 ultra-conserved CEGs were predicted in R. proliferus using CEGMA analysis. The absence of some core eukaryotic ascomycete genes were found. The fatty acid synthesis, type I multienzyme complex (FAS-I) was in agreement with previously reported species of AM fungi. Homology search-based prediction in R. proliferus revealed all components of the bacterial type FAS (type II FAS) genes only. The FAS-I complex is responsible for the cytosolic fatty acid synthesis, which produces the bulk of long-chain fatty acids in other fungi (Leibundgut et al. 2008). This gene has been reported missing in the gene repertoires of AM fungi (Tisserant et al. 2013;Tang et al. 2016;Sun et al. 2018;Kobayashi et al. 2018;Morin et al. 2019;Venice et al. 2020), which has motivated extensive exploration to understand how AM fungi may generate lipid reserves. Interestingly, in AM-colonized cells of plant roots intensive stimulation of genes involved in lipid metabolism occur, perhaps to provision the increased demand for lipids for the periarbuscular membrane. Based on these findings it has been suggested that AM fungi may receive fatty acids synthesized by plant cells. In this regard, recent studies have demonstrated that AM fungal lipids are, at least partially, derived from the plant host (Bravo et al. 2017;Keymer et al. 2018).
Thiamine is a cofactor for enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and therefore it is an essential constituent of all cells. The biosynthetic pathway for thiamine has been reported missing in AM fungi. In congruence with the previous reports, thiamine biosynthetic pathway genes were not predicted in R. proliferus.
In exchange for mineral nutrients, the plant supplies the fungal partner with reduced carbon (Shachar et al. 1995). Although the nutrient transfer mechanism from AM fungi to plant has been well investigated, transfer of carbon from host to fungus and its subsequent metabolism are less understood. Sucrose is the major sugar form produced during photosynthesis in plants. The invertase gene which is involved in the cleavage of sucrose into fructose and glucose, was found missing in the recently published Glomeromycota genomes (Tisserant et al. 2013;Lin et al. 2014;Salvioli et al. 2016;Tang et al. 2016).
Glutathione metabolism genes are involved in nutrient metabolism and regulation of cellular events. Presence of Glutathione metabolism genes in R. irregularis and its absence in R. proliferus species was striking and probably indicated towards species specific differences in core eukaryotic gene sets.
Proteins, uridine permease, uracil permease and dihydroorotate dehydrogenase support uracil metabolism, transport and maintain the intracellular level of uracil. Tight control of the intracellular uracil has been suggested important to reduce the rate of uracil incorporation into DNA (Sun et al. 2013). Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11), which is the fourth enzyme of the pyrimidine de novo biosynthesis pathway, was the only gene from the pathway that was present in both the R. irregularis and R. proliferus genomes. Genes for glutamate metabolism and glutathione metabolism were predicted in R. proliferus, which indicated for its potential for the metabolism of nucleic acids and proteins (Yelamanchi et al. 2016) and detoxification of xenobiotics and the oxidative stress response (Shen et al. 2015), respectively, similar to other AM fungi. Transporters and channels for potassium transport from the soil to the host by the AM fungi are still not completely deciphered in AM fungi. Seven sequences from an EST library of R. irregularis were annotated as K + transport systems (Casieri et al. 2013), which coded for SKC-type channels and KT/KUP/ HAK transporter. Noticeably, no Trk and TOK members were identified in either the EST library or the sequenced nuclear genome (http:// genome. jgi. doe. gov/ Gloin1/ Gloin1. home. html). In congruence with the previous reports, no homologue gene for the yeast TOK1 was identified in R. proliferus. However, a Trk-type K + transport system in R. proliferus was predicted. Probable ferric reductase transmembrane component 8, which is expected to function in the assimilation of iron, was identified only in R. proliferus by the conserved domain analysis and comparison with the proteins coded by yeast. In our comparative analysis, the absence of several CEG in R. proliferus was mostly in confirmation with the previously reported AM fungi, which suggested high conservation in genetic features among all species belonging to Glomeromycota.

Conclusions
The genome of R. proliferus shared several conserved features with previously reported AM species with respect to the genetic structure and functions. This included absence of several eukaryotic genes, prominently the type I FAS gene, abundance of protein kinases and reduced number of glycoside hydrolases. A unique finding was higher proportion of carbohydrate esterases, which might suggest for presence of higher plant litter decomposing activity in R. proliferus as compared to other AM fungi. The present first draft of R. proliferus genome would serve as a reference for all future genetics and functional genomics analysis of the species. It would also provide information for comparative genomics analysis required for developing comprehensive understanding about structure and function of AM fungi in future.