Evolutionary and genomic comparisons of hybrid uninucleate and nonhybrid Rhizoctonia fungi

Li, Cheng; Guo, Zejian; Zhou, Shanyue; Han, Qingyue; Zhang, Manman; Peng, Youliang; Hsiang, Tom; Chen, Xujun

doi:10.1038/s42003-021-01724-y

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Published: 15 February 2021

Evolutionary and genomic comparisons of hybrid uninucleate and nonhybrid Rhizoctonia fungi

Communications Biology volume 4, Article number: 201 (2021) Cite this article

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Abstract

The basidiomycetous fungal genus, Rhizoctonia, can cause severe damage to many plants and is composed of multinucleate, binucleate, and uninucleate species differing in pathogenicity. Here we generated chromosome-scale genome assemblies of the three nuclear types of Rhizoctonia isolates. The genomic comparisons revealed that the uninucleate JN strain likely arose by somatic hybridization of two binucleate isolates, and maintained a diploid nucleus. Homeolog gene pairs in the JN genome have experienced both decelerated or accelerated evolution. Homeolog expression dominance occurred between JN subgenomes, in which differentially expressed genes show potentially less evolutionary constraint than the genes without. Analysis of mating-type genes suggested that Rhizoctonia maintains the ancestral tetrapolarity of the Basidiomycota. Long terminal repeat-retrotransposons displayed a reciprocal correlation with the chromosomal GC content in the three chromosome-scale genomes. The more aggressive multinucleate XN strain had more genes encoding enzymes for host cell wall decomposition. These findings demonstrate some evolutionary changes of a recently derived hybrid and in multiple nuclear types of Rhizoctonia.

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Introduction

Rhizoctonia was created as an anamorphic genus in the Basidiomycota. The best-known species, Rhizoctonia solani (also known as Thanatephorus cucumeris), causes severe damage to more than 200 plant species¹, while genetic breeding strategies have been limited because only minor- to moderate-effect quantitative trait loci related to resistance to this pathogen have been found thus far^2,3. The Rhizoctonia species reported are mostly multinucleate, but there have been increasing numbers of binucleate and uninucleate Rhizoctonia isolates discovered. Both binucleate and multinucleate isolates have been reported to cause stem canker and black scurf on potato plants^4,5. Uninucleate isolates incite root dieback in forest nurseries on Norway spruce and Scots pine⁶ and have also been isolated from brown patch disease samples of Festuca arundinacea⁷. However, Rhizoctonia spp. have also been found as endomycorrhizae from medicinal plants of Orchidaceae⁸. Rhizoctonia spp. are generally soil-borne phytopathogens with a necrotrophic lifestyle, and the diseases have elevated incidence and severity with increasing adoption of direct-seeding, no-till, and straw-turnover farming techniques⁹.

Somatic hybridization or anastomosis is a process commonly found in filamentous fungi¹⁰. The hyphal fusion between two different fungal mycelia results in the formation of heterokaryotic cells. Generally, this hyphal fusion triggers the vegetative incompatibility response (VIR) that leads to the death of the fused cell. However, the fusion cells occasionally overcome the VIR to undergo nuclear fusion to generate a single diploid nucleus, which can then form haploid or some aneuploid nuclei followed by mitotic recombination and chromosome loss¹⁰.

Accumulating information indicate that somatic hybridization of fungal pathogens has an important impact on genetic diversity and adaptation to new hosts. Verticillium longisporum, causing Verticillium stem stripe on Brassica species, contains a genome composed of two lineages and has conidia that are twice as long but with a narrower host range in comparison with its close relative, V. dahliae¹¹. Zymoseptoria pseudotritici is a recently emerged hybrid species causing disease on wild grasses in northern Iran¹². The oomycete pathogen Bremia lactucae, inciting downy mildew on lettuce, maintains a high incidence of heterokaryotic cells¹³. Genomic analyses of wheat stem rust (Puccinia graminis f. sp. tritici, Ug99) and wheat leaf rust (P. triticina, Pt64) demonstrated that the virulent isolates arose from somatic hybridization and had nuclear exchange between dikaryons^14,15. The results suggest that hybrid genomes of phytopathogens can provide evolutionary flexibility for the pathogen enabling rapid adaptation to different hosts and other environmental changes.

Classification of Rhizoctonia spp. and closely related binucleate species is based on a compatibility system of hyphal fusion into different anastomosis groups (AGs). Binucleate Rhizoctonia are grouped into 21 AGs (AG A-U), and multinucleate R. solani into 13 AGs (AG1–13)^16,17. However, the physiology, genetics, and genomics of these predominantly asexually reproducing pathogens are largely unknown. To date, there are draft genomes reports on five multinucleate Rhizoctonia isolates belonging to four AGs including AG1-IA and AG8 causing blight on cereal crops etc.^3,18,19,20. Variations among the genomes were remarkable from the assembly size to the predicted gene numbers, for instance, 10,489 genes predicted in GD118 (36.94 Mb)³, 13,952 genes in WAC10335 (39.82 Mb)¹⁹, and 11,897 genes in BBA69670 (56.03 Mb)²⁰. In previous genome sequencing and assembly efforts using short-reads data^19,20, the occurrence of multinucleate R. solani of primarily heterokaryotic and diploid features were hindrances to complete genome assembly. Therefore, we sequenced genomes of field isolates of Rhizoctonia of different nuclear types: uninucleate (JN, SM, and YR)^21,22, binucleate (LY and RW), and multinucleate (XN), among those JN, LY, and XN were assembled to chromosome-scale. Unexpectedly, each uninucleate isolate had a genome assembly that was twice the size of the binucleate isolate, LY. Genomic comparisons implied that uninucleate genomes (e.g., JN) were hybrids derived potentially from binucleate ancestors.

Results

Genome sequencing, assembly, and annotation

Uninucleate (JN, SM, and YR), binucleate (LY and RW), and multinucleate (XN) Rhizoctonia spp. were sequenced and assembled with 114- to 265-fold coverage for the six genomes using Illumina reads (Supplementary Tables 1, 2 and Supplementary Fig. 1). To generate more complete assemblies, about 11 GB of the long-reads (average length: ~17.5 kb for JN and LY, 11.5 kb for XN) from PacBio sequencing for each isolate were combined with the short Illumina reads to assemble JN, LY, and XN genomes separately (Table 1 and Supplementary Table 3). Contigs from PacBio reads were obtained independently using HGAP4²³, Canu v1.5²⁴, and MECAT v1.3²⁵. Scaffolds were constructed based on contig comparisons using MUMmer v3.23²⁶ and corrected using Pilon v1.22²⁷ with Illumina paired-end reads. In the final assembly, we obtained 36, 19, and 21 scaffolds for JN, LY, and XN, respectively (Table 1). The assembly size of each genome was at least 98% of the content estimated by k-mer analysis, and the total length of the chromosomes accounted for 99.0% or more of each genome assembly (Supplementary Table 4 and Supplementary Fig. 2). Among those scaffolds, 32 JN, 16 LY, and 16 XN scaffolds had putative telomeric sequence repeats 5′-(TTAGG)n-3′ at both F and R ends (Supplementary Tables 5–7), suggesting chromosome-level scaffolding. An orthologous set of 45 transfer RNAs (tRNAs) was found in each of the three genomes (Supplementary Table 8).

Table 1 Description of Rhizoctonia spp. genome assembly.

Full size table

The number of proteins annotated in XN (12,349) and LY (14,549) were comparable to R. solani WAC10335 of AG8 (13,964) and 7/3/14 of AG1-IB (12,268) (Table 1 and Supplementary Tables 9–10). The number of proteins predicted in uninucleate JN (29,028) was nearly double that in binucleate LY, but coincidently all of the three chromosome-level genome assemblies had a gene density of ~3.3 kb per gene. The average predicted gene size in XN was larger than that in LY and JN, due to more and larger introns in XN (Supplementary Table 10). The 6.95 exons per XN gene was more than other R. solani isolates sequenced²⁰. Protein functions were predicted using the eukaryotic orthologous group (KOG) database. In general, the distributions of different categories of genes were similar in the three strains, with about 45% of genes predicted as “poorly characterized” (Supplementary Table 11).

Whole-genome duplication of uninucleate Rhizoctonia genomes

In comparison with binucleate and multinucleate strains, the uninucleate JN isolate had a larger genome. It was first examined for the intragenomic collinearity, which may be generated by whole-genome duplication (WGD). There were 9,459 (65.2% of the total) gene pairs with synteny in JN (Fig. 1a) versus 496 genes in LY and 464 in XN genome (Supplementary Figs. 3 and 4). Furthermore, 7,919 of the two-copy genes in JN were remarkably larger than that of 151 ortholog groups in the LY genome (Supplementary Table 12). On the other hand, the percentage of single-copy genes was much higher in LY and XN compared with JN (Supplementary Fig. 5). The results imply the occurrence of WGD in the JN genome.

**Fig. 1: Genomic characterizations and evolutionary relationships of *Rhizoctonia* species.**

Next, to facilitate genome comparison, we tried to separate the JN genome into two subgenomes. The basic principle was that the protein identity of a JN chromosome as a whole that was closer to binucleate LY was designated as belonging to the JNa subgenome, and those of lower sequence identity were placed with JNb. Therefore, putative subgenome JNa included 16 chromosomes with a total length of 49.57 Mb containing 14,978 predicted coding genes whereas putative subgenome JNb was 46.73 Mb in size, with 14,043 predicted genes (Table 1). Syntenic blocks were determined by MCscan v1.1, setting a minimum stretch containing five genes²⁸. The syntenic genes in JN subgenomes against LY genome were 11,685 and 9,058 to JNa and JNb, respectively (Supplementary Fig. 6). As expected, the ratio of identical proteins was higher between LY and JNa in relation to its counterpart JNb (Fig. 1b). The data implied that LY was related to a progenitor of JNa.

Because of the high levels of synteny detected among JNa, JNb, and LY as mentioned above, synonymous substitution (Ks) rates were used to infer the occurrence of WGD events that occurred during the evolution of these genomes²⁹. The Ks distributions showed a paralogous peak at ~0.037 (between JNb and JNa) and an orthologous peak at ~0.044 (between JNb and LY), implying that the divergence time between the JNb progenitor and the LY ancestor was estimated to be around 21 million years (MY). The estimated diploidization of JN was 2.4 MY based on the Ks value of 0.005 between JNa and LY (Fig. 1c).

A phylogenetic tree was constructed with Rhizoctonia spp. and Ascomycota yeasts as outgroups using 637 single-copy orthologous genes. LY, JN, and SM genomes and subgenomes formed a monophyletic clade with RW, a binucleate Rhizoctonia from wheat (Supplementary Fig. 7). The sequence divergence across the hybrid subgenomes was estimated by using the well-characterized WGD in yeasts as a calibration point, as described by Sriswasdi³⁰. The sequence divergence across the hybrid subgenomes (Ks = 0.0475 for JN and 0.0481 for SM) relative to that of the post-WGD genomes (Ks = 1.5146) implied that the Rhizoctonia hybridization events were about 31 times more recent (Fig. 1d).

Differential expansions of LTR-retrotransposons in Rhizoctonia spp.

The repetitive sequences comprised around one-fifth of each assembly, in which long terminal repeat-retrotransposons (LTR-RTs) were the predominant transposable element (TE) (Fig. 2a and Supplementary Table 13). The estimated TE content was found to be higher in the genome assembly containing PacBio subreads than the Illumina-only assembly (Supplementary Table 14). The content of LTR-RTs in XN was slightly higher than JN or LY, and XN, in which XN contained less TE DNA and uncategorized repetitive elements. There was an uneven distribution of LTR-RTs and full-length LTR-RTs (FL-RTs) in JN subgenomes (Supplementary Tables 14 and 15). The relative ratio of FL-RTs to genomic read abundance of the total LTR-RTs was slightly less in LY, JNa, and JNb compared to XN (Fig. 2b and Supplementary Fig. 8a). The distribution of FL-RTs revealed periodical retrotransposition bursts (Fig. 2b). The median insertion age of FL-RTs in XN was older than that in LY, JNa, and JNb, due to fewer new amplification bursts of LTRs (insertion age 0 MY), indicating more active transposable activity in the uninucleate and binucleate isolates. We found that the number of RNA-Seq reads of LTR-RTs was much more in JNa than XN (Supplementary Fig. 8b). In addition, the number of FL-RTs with 0 MY age also showed a strong relationship with their own RNA-Seq read counts, as well as the RNA-Seq, read counts of total LTR-RTs, suggesting that the greater the number of new LTR-RT insertions, the greater the number of total active LTR-RTs in the genomes reflected in the number of matching RNA-Seq reads. Furthermore, approximately one-third of the JNa intact FL-RTs were syntenic to those of LY (40.4%), and the majority had congruent insertion ages, unlike those of JNb (Supplementary Fig. 9a–c). Phylogenetic analyses of FL-RTs revealed a recent huge expansion branch of LY (103) and JNa (136) in relation to JNb (3) (Supplementary Fig. 10a). In addition, LY and XN showed more independent evolution of FL-RTs (Supplementary Fig. 10b).

**Fig. 2: Characterization of repeat elements in the *Rhizoctonia* LY, XN, and JN (JNa and JNb subgenomes).**

Transition hypermutations in Rhizoctonia spp.

Single-nucleotide polymorphism (SNP) and InDel mutations were estimated between LY and JN. The ratio of SNPs in the JNb subgenome was about 12.5-fold higher than that in JNa, and with slightly lower fold changes in the exonic regions (Fig. 3a and Supplementary Table 16). The pattern of SNP mutations was that the ratio of transition (Ti) to transversion (Tv) was higher in the exonic regions than intronic and intergenic regions (Fig. 3b). The Ti/Tv ratios of synonymous and nonsynonymous variants were very close to those of a set of selected genes observed by Freudenberg-Hua et al.³¹, implying that selection was a major contributor to the Ti to Tv substitution bias.

**Fig. 3: Comparisons of SNP and the heterozygous SNP (hSNP) mutation spectra in *Rhizoctonia* spp.**

The process of repeat-induced point (RIP) is one form of genome defense against TE expansion that is active during meiosis³². In R. solani WAC10335, the heterozygous SNP (hSNP) mutation is considered a RIP-like phenomenon¹⁹. The hSNP density in XN was the highest, 2.56‰ (i.e., 2.56 SNPs per kb) compared to 0.64‰ in LY, 0.22‰ in JNa, and 0.16‰ in JNb (Fig. 3c and Supplementary Table 17). Mutation patterns indicative of hSNPs were predominantly Ti conversions found in exonic, repetitive or non-repetitive regions (Supplementary Fig. 11a–c). Since RIP-affected genomic regions were often associated with elevated AT content concomitant with a decrease in GC abundance³³, the distribution of GC content was analyzed and found to be unimodal in the Rhizoctonia data sets (Fig. 3d). Furthermore, the RIP indices from the repeat element families of each Rhizoctonia genome did not meet the thresholds (Fig. 3e, f), whereas the positive control Parastagonospora nodorum met the conditions. The results implied that RIP was not prevalent in the Rhizoctonia genomes tested.

Gene loss, disruption, replacement, and exchange in JN subgenomes

Gene loss, insertion, and replacement in JNa and JNb were assessed in relation to synteny with LY. Gene losses were observed only in JNa (Fig. 4a and Supplementary Table 18), supporting the greater similarity between JNa and LY. However, we observed four-fold levels of gene losses in JNb over JNa, and also large numbers of gene insertions (904) and replacements in JNb (i.e., the gene difference in noncollinear regions of JNa and JNb). The gene-gain phenomenon was estimated to be early events that occurred in the JNb progenitor before the genome hybridization since about half of the genes in JNb had no close homologs in LY or JNa. The largest insertion occurred in chr. B11 of which genomes comprised of 742 kb and the largest deletion was found in chr. B10 affecting about 1.7 Mb (Supplementary Fig. 12a, b). A phenomenon of gene loss took place at one or both chromosome ends of JNb except chr. A15 (Supplementary Table 19). In most cases, TEs were close by the breakage sites, suggesting the association of TEs with the gene loss.

**Fig. 4: Comparison of gene loss and disruption in the JN subgenomes (JNa and JNb).**

There was a noticeable number of disrupted genes in JN including pseudogenized ones by SNPs and InDels (Fig. 4b). The rate of nonsynonymous to synonymous substitutions (Ka/Ks) in the SNPs of exonic regions was relatively higher in JNa than JNb estimated from the whole subgenome or the homeologous regions (Supplementary Table 16), suggesting stronger selection pressure on JNa than JNb. The InDel density in JNb was ~4.2-fold higher than JNa (Supplementary Table 16), however, the gene numbers disrupted by InDels were only slightly more in JNb than JNa, implying higher ratio of gene disruption in JNa (at least in the coding region) (Fig. 4b).

Gene exchanges might have taken place between JN subgenomes and possibly resulted in differences of SNP density in the syntenic fragments. We analyzed the SNP densities of the total 187 homeologous blocks that exhibited uneven distribution in each subgenome (Supplementary Fig. 13). In the case of gene exchanges, the SNP density of b4 in chr. B04 of JNb was lower than its syntenic a4 in chr. A04 of JNa, and exhibited modifications of Ks value and amino acid identity in the regions correspondingly (Supplementary Fig. 14a). The data indicated that gene exchanges occurred between the hybrid subgenomes, which were not caused by possible assembly errors since the linkages of the syntenic blocks had normal read coverage. Among the entire subgenomes, there were 245 gene exchange events between the syntenic regions of JNa and JNb, in which 46 exchanged fragments containing multiple genes (Supplementary Table 20). Both chrs. A14 and B14 of the JN subgenomes diverged rapidly from LY with high Ks values and low identity of amino acid sequences, whereas both chrs. A15 and B15 showed a high and close identity of protein sequence with LY (Supplementary Fig. 14b, c).

Differential evolutionary rates and expression dominance among homeolog gene pairs

Evolutionary rates of the preserved duplication genes in JN were calculated with orthologs from the nonhybrid XN genome. The median Ka/Ks value of the preservation genes in JNb was higher than LY orthologs and JNa paralogs, whereas the Ka/Ks values of the singleton genes in the hybrid genome were not significantly different from their counterparts in LY (Fig. 5a). Evolutionary rates of homeologous genes were analyzed by using the method described by Sriswasdi³⁰. The fold differences between the Ka/Ks of genes in JN subgenomes and the corresponding background Ka/Ks in LY were calculated and categorized using 1.5-fold as a threshold, due to low numbers of the changed genes at a twofold threshold, which showed the same evolutionary patterns (Fig. 5b and Supplementary Fig. 15a). The number of decelerated/decelerated (Dec/Dec) homeolog gene pairs, with decreased Ka/Ks values, was more than accelerated/accelerated (Acc/Acc) pairs (Fig. 5b, c). Enrichment of evolutionarily accelerated single homeolog copy (i.e., accelerated/neutral pairs) was consistent with the general evolutionary scenario in which one of the homeolog gene pairs was under less evolutionary constraint. Intriguingly, we did not observe gene pairs with divergent changes in evolutionary rates (accelerated/decelerated pairs).

**Fig. 5: Divergence of evolutionary rates and homeolog expression in JN subgenomes (JNa and JNb).**

Analyses of evolutionary rates revealed that both Dec/Dec and Acc/Acc homeologs evolved, respectively, slower and faster than the remaining homeolog pairs in the hybrid genome (Fig. 5d and Supplementary Fig. 15b). Approximately a quarter of Dec/Dec homeologs (21/86) were in the KOG category of information storage and processing, including helicases, transcription and translation initiation factors, and DNA repair proteins (Supplementary Table 21), implying that conserved proteins were prone to decelerated evolution. The protein identity of Dec/Dec homeolog pairs was markedly higher than the other genes (Fig. 5e), but a relevant change was not observed for Acc/Acc gene pairs (Supplementary Fig. 15c), suggesting that gene conversion may have a role in genome stability of young hybrids by creating evolutionary constraints³⁰.

To explore the transcriptional behavior of the hybrid subgenomes, we compared the genome-wide transcriptional levels of homeolog genes and found about half of the gene pairs displayed homeolog expression dominance (Fig. 5f and Supplementary Fig. 16a). The homeolog expression dominant genes were classified as dominant, subordinate, and neutral (i.e., higher, lower, and equal expression level in homeolog gene pair, respectively) as previously described³⁴. The median Ka/Ks and Ka values of dominant and subordinate genes were markedly higher than those of neutral genes (Fig. 5g and Supplementary Fig. 16b). The results revealed that the differentially expressed dominance genes evolved faster than the neutral genes, with subordinate genes even faster.

To examine whether sequence evolution had any relationship with the transcriptional evolution, we checked the distribution of the gene pairs (61%, 352) remaining in the expression data set and found no biased proportion of gene losses (Fig. 5b). The homeolog gene pairs in JN showing divergent evolutionary rates in relation to LY were significantly more common (chi-square test, P = 1.93e-4) in the dominance expression category (173 in 1,137 gene pairs) than in the neutral expression category (179 in 1,790) (Fig. 5b). The results suggested that homeolog gene pairs of evolutionarily diverged rates were also more divergent in expression levels.

Identification of mating-type genes in Rhizoctonia isolates

Basidiomycete fungi have evolved a mating (MAT) system based on two genetic loci, e.g., the pheromones and pheromone receptors (P/R) and the homeodomain-containing transcription factors (HD)^15,35,36. While the P/R system is for haploids to recognize a compatible mating partner, heterodimer formation between HD1 and HD2 originating from different mating types is responsive for postmating events such as the formation of dikaryotic mycelium. Information on mating-type genes in Rhizoctonia species is lacking, so we analyzed MAT loci across the genomes. The orthologs of P/R organization were present for Rhizoctonia Pra (RhiPra) and the adjacent RhiMfa genes (Fig. 6a), which encode for a pheromone receptor and a putative pheromone precursor, respectively. RhiHD1 and RhiHD2 genes existed as a duplicate set on the same chromosome, but different from that harboring P/R genes (Fig. 6b). Syntenic analyses around MAT loci revealed that both P/R and HD loci were syntenic orthologs between LY and JNa, but arrangements were observed at the one side of JNb_Pra and JNb_E-HD. Likely, XN had maintained highly co-linearity on one side of the XN_Pra or XN_HD locus, and the other side with a large fragment insertion and sequence inversion (Fig. 6a, b).

**Fig. 6: Organization of mating-type loci of *Rhizoctonia* spp. and phylogenetic relationship with other basidiomycetes.**

RhiMfa proteins were almost the same in binucleate LY and uninucleate strains with only one amino acid difference in SMb_Mfa and YRb_Mfa, but highly divergent with binucleate RW and multinucleate XN (Fig. 6c and Supplementary Fig. 17a). Similar divergences were detected among the RhiPra pheromone receptors, showing marked variations among LY, RW, and XN (Fig. 6d). In the RhiHD locus (Fig. 6e), the copies of W-HD1 and W-HD2 were apparently intact except for lack of RW_W-HD2, whereas E-HD1 and E-HD2 varied in the loss of XN_E-HD1, and an N-terminal shortening of LY_E-HD1 and JNa_E-HD2. The Rhizoctonia P/R and HD genes were phylogenetically closer to corresponding genes of the ink cap mushroom Coprinopsis cinerea than to plant pathogenic genera, Ustilago and Puccinia, in agreement with the phylogenetic tree of these species generated using 889 single-copy genes (Supplementary Fig. 18). Allelic-like variants of RhiPra were present in each (sub)genome; however, their adjacent RhiMfa-like genes were absent. In addition, the RhiPra and RhiHD variants were in (sub)clades different from the potential MAT genes (Supplementary Fig. 17b, c), implying divergent evolution of these genes. Taken together, Rhizoctonia spp. maintain the ancestral tetrapolarity of Basidiomycota.

The divergence of XN vs. LY or JN

Although XN and LY genomes remained high levels of synteny for orthologous genes, dynamic rearrangements of the genes were observed when comparing XN and LY genomes (Supplementary Fig. 19). For instance, the rDNA operons were assembled in two copies in chr. 14 of LY and JN (sub)genomes, but the rDNA repeats were in chr. 03 of XN (Supplementary Fig. 20). However, the number of rDNA operon repeats should be at least 20 times greater estimated from the read coverage. Also, rearrangements were identified around MAT loci as mentioned above, even though the regions were shown to have lower recombination rates in some fungal species¹⁵.

XN, LY, and JN also exhibited genomic differences such as genome size, GC content, and gene numbers (Table 1). GC contents of XN were lower than LY and JN in both exons and intergenic regions (Supplementary Fig. 21), in which exonic GC% was mainly contributed by the third codon (GC3) position (Fig. 7a). Analyses of the codon usage indicated that XN had similar patterns as LY and JN, but with less fluctuation (Supplementary Fig. 22a, b). The preferred codons for the three genomes, for example, CGC (Arg), ATC (Ile), CTC (Leu), TCG (Ser), and GTC (Val), all ended with C or G, and coincidently the codons at the troughs were the same amino acids but with lower GC composition ending with A or T. Intriguingly, there were no corresponding decoding tRNAs for these C-ending codons mentioned above (Supplementary Table 8). The data imply that the adenosine in the first position of the tRNA anticodon may be modified through deamination to inosine, which can wobble with codons ending in A, C, or T³⁷. Furthermore, we calculated the equilibrium GC3 content, GC3*, reflecting the AT to GC substitution rates of LY and JN compared with XN orthologs at the third codon positions. GC3 was strongly correlated with GC3* and distributed slightly above the diagonal lines for LY, JNa, and JNb (Fig. 7b and Supplementary Fig. 23), supporting the hypothesis that the Rhizoctonia lineages faced similar selection pressures for codon usage. However, LY and JN slightly favored fixation of GC over AT in comparison with XN, consistent with the resulting lower GC content in the XN genome.

**Fig. 7: Comparison of GC contents among JNa, JNb, LY, and XN.**

The abundance of LTR-RTs was another potential factor influencing genome GC content since the level of LTR-RTs was negatively related to gene density in a chromosome³⁸ (Supplementary Fig. 24a). Also, the chromosomal GC contents showed a high reciprocal correlation with the abundance of LTR-RTs or TEs, and a positive relationship with the gene density in the data sets (Fig. 7c and Supplementary Fig. 24b, c). LTR-RT content possibly posed a small contribution to decreased GC% due to the LTR sequences having lowered GC content than that of the genome or of exonic regions (Table 1 and Supplementary Fig. 8).

Gene expansions for degradation of cell wall components in XN genome

Multinucleate Rhizoctonia isolate was more aggressive than binucleate or uninucleate isolate²². To examine characteristics of Rhizoctonia spp. thriving on dead or dying plant cells and explore why the XN strain was more aggressive than LY or JN, CAZyme (carbohydrate-active enzyme) genes were investigated to see whether a particular set of enzymes was associated with host range and pathogenesis. As shown in Fig. 8a and Supplementary Fig. 25, the XN genome had experienced an expansion and diversification of polysaccharide lyases (PLs), which mainly degrade pectin and glycosaminoglycans. Compared with other Rhizoctonia genomes, XN had many fewer CAZyme members than BBA69670 of AG2-2IIIB²⁰ or WAC10335 of AG8¹⁹, but many more than GD118, which is of the same AG1-IA as XN³.

**Fig. 8: Comparisons of CAZyme genes in *Rhizoctonia* spp. and expression of effector candidate genes in XN.**

Secreted and effector proteins of plant pathogens are required for establishing successful infection and evading host defense responses during colonization. Secreted proteins in XN were fewer than those in LY and JN (Supplementary Table 22). Effector proteins in the secretome of XN (354) were slightly fewer than in LY (393) or JN (399/384, JNa/JNb). Expansion of effectors in XN (≥five than in LY) in relation to LY were PLs, polygalacturonases, and tyrosinases, on the contrary, LY had more alpha/beta hydrolases and hypothetical proteins (HPs).

Making use of the available RNA-Seq data from GD118-infected rice³, we found that most of the genes encoding CAZymes (93%), secreted proteins (87%), and effector candidates (73%) could be mapped by the Illumina reads from GD118, suggesting that both XN and GD118 maintained genes of fundamentally the same characteristics. A closer examination of the effector genes showed that the unexpressed genes in XN (97) related to GD118 reads were mainly HPs (57), PLs (21), tyrosinases (6), or polygalacturonases (5). A possible explanation is the expansion or gain of these genes after the split of the two strains since a high proportion of the unexpressed genes in GD118 had detectable expression in XN RNA-Seq. From the phylogenetic tree, clades of vii, viii, and including i with the genes of early high expression were enriched for HPs, but also contained several proteases of various kinds, tyrosinases, three chitin deacetylases (CDAs), and the only LysM domain protein of effector candidate (Fig. 8b). The LysM domain protein and CDAs may act on early pathogenesis through binding and modification of chitin oligomers to suppress chitin-induced immunity^39,40. In the subclades of ii and iii with the genes highly expressed in the later infection times, enrichments of the GH family in iii and PLs genes in ii revealed that the encoding enzymes may function on the degradation of the plant cell wall. The results indicate strongly that various effectors and secreted enzymes participate in different processes of Rhizoctonia infection host plants.

Discussion

Hyphal fusion is presumed to be common in Rhizoctonia since the anastomosis compatibility system is used to differentiate taxa. However, genomic information on hyphal fusion in Rhizoctonia spp. is incomplete. In this study, we identified naturally occurring uninucleate Rhizoctonia isolates as hybrid genomes which maintained in their diploid nuclei. Hybrid fungal species have been shown in Saccharomyces, Cryptococcus, Verticillium, and Puccinia^11,14,30,41. The fused fungal hyphae normally restore the haploid state from the hybrid diploid nucleus by chromosome loss¹⁰. Occasionally, the hybridized nuclei are stable, resulting in a hybrid species with a diploid or aneuploid nucleus. Epichloë polyploid Lp1 is a well-characterized asexual interspecific hybrid between sexual and asexual Epichloë species⁴². The endophyte Lp1 maintains most of both parental gene copies with the notable exception of uniparental rDNA repeats^42,43. Here, the uninucleate JN genome still contained both parental rDNA operons, suggesting recent hybridization or independent evolution of the rDNA loci in the hybrid lineage⁴³.

Mating phenomena of R. solani have been studied by pairing cultures of mycelia generated from single-basidiospore isolates or deduced from single-protoplast isolates because of difficulty in triggering sexual reproduction in this species complex⁴⁴. The mating system of R. solani is unclear because there are reports of homothallism or heterothallism with bipolar mating in several AGs, and both homothallic and heterothallic mating systems in an AG2-2 IV isolate⁴⁵. We found that the Rhizoctonia P/R and HD loci not only had a similar organization to those of C. cinerea, except for the absence of repeats but also the genes were phylogenetic closer than other species tested^35,46 (Fig. 6). Like in C. cinerea, additional non-mating-type-specific receptors and multiple alleles of HD genes also exist in the Rhizoctonia isolates (Supplementary Fig. 17). This information suggests that Rhizoctonia spp. may have a tetrapolar mating system similar to C. cinerea. In the hybrid uninucleate genomes, the presence of two sets of almost identical P/R and highly homologous HD1/HD2 genes revealed that the fungal fusions overcame the VIR and persisted into a parasexual cycle. The switch from sexual to asexual only requires a single-nucleotide change within a key gene of MAT locus, or some mutation involved in mating or meiosis⁴⁷. It is unclear what maintains the diploid state of uninucleate strains, which may be stable because the hyphae with diploid nuclei continue to grow. Nevertheless, our data provide valuable information to pursue the hyphal fusion and mating phenomena in the predominant asexual Rhizoctonia species.

The genome modifications of JN, especially in JNb, were composed of two parts: divergence between the unknown parents before the hybridization and post-genome hybridization events. For instance, a large proportion of the arranged JNb-genes had no homeologs in LY or JNa (Fig. 4a), implying that these genes did not come from JNa or LY but from the unknown parent. The faster evolutionary rate of JNb than LY or JNa (Fig. 5a) may be a source of this variation. Gene loss was expected to be high in the new hybrid genome. However, the number of gene loss events in JNa was markedly less than the number of genes disrupted by SNPs and InDels (Fig. 4b), indicating that sub- and neo-functionalization and pseudogenization of genes played active roles in the modification of the hybrid genome⁴⁸. The evolution of genome architecture is thought to be highly associated with TEs, particularly LTR-RTs, since TEs are the major driving force for genome evolution through their movement and proliferation³⁸. Increased copy number and transcriptional abundance of LTR-RTs especially in JNa subgenome are speculated to be genomic shock post the hybridization⁴⁹ (Fig. 2 and Supplementary Fig. 8), which can elevate mutation rates through activated TEs, as documented in hybrid fish and sunflower^50,51.

On the other hand, TEs impact the gene content of a genome by rearrangements through homologous recombination and by insertion or excision, which causes double-strand DNA breaks (DSBs)⁵². DBSs are repaired by homologous recombination and non-homologous end-joining (NHEJ) mechanisms, in which NHEJ is an error-prone process not only leading to nucleotide fixation but also causing genetic material deletions, inversions, and translocations^52,53,54. Bold speculation is that earlier expansion of LTRs in the XN genome is possibly one of the driving forces leading to variations of GC content and gene order between XN and LY. Also, chromosomal rearrangements have been found as a general mechanism for host adaptation of the asexual pathogen V. dahllium which established the lineage-specific genomic regions mediating aggressiveness⁵⁵.

In the hybrid JN genome, we observed not only a dominant relaxed selection on one copy of the homeologous gene pairs but also gene enrichment of Dec/Dec evolutionary rates (Fig. 5c–e), suggesting that gene conversion likely played a role in maintaining the functional integrity of redundant genes³⁰. Homeolog expression dominance is a consequence of eukaryotic hybrids and varies markedly across different hybrid species^34,56,57. We observe that the subordinate expression genes in the homeolog expression dominance were evolutionarily faster, in consistent with accelerated genes being mainly in the subordinate group of low expression levels (Fig. 5g). This phenomenon also agrees with that observed in the allopolyploid plant Brassica juncea³⁴. The evolutionarily diverged homeolog genes tended toward expression dominance (Fig. 5g), suggesting that the sequence diverged homeolog pairs would be divergent in their expression dominance. However, these findings and analyses were preliminary, and future work should focus on comparisons and more in-depth analyses to explore the evolution of Rhizoctonia and evaluate species boundaries.

The expanded gene families, such as PLs, polygalacturonase, and tyrosinases in XN possibly played important roles in its pathogenicity, since the tested strains contained similar kinds of secreted proteins and effectors with different virulence. Polygalacturonases, cleaving the linkage of galacturonic acid in pectin, have been characterized as virulence effectors in plants^58,59,60. The expanded PLs are mainly pectate lyases PL1 and PL3, predominantly degrading the poly-galacturonan regions of pectin. Tyrosinases, functioning in plant biomass decomposition and also in melanin biosynthesis, showed wide expression patterns probably associated with multifaceted roles. The early Rhizoctonia infection could be hemibiotrophic as previously suggested^61,62, whereas the late process of massive necrosis required more and active CAZymes and other enzymes to decompose host cell wall components. It is plausible that the more aggressive XN compared with LY and JN is due to XN evolving more genes or active enzymes for the late necrosis. Efforts are being made to facilitate molecular manipulation of Rhizoctonia, and to characterize effector candidates.

Methods

Collection of isolates

Rhizoctonia isolates JN, YR, LY, and XN were collected from maize as previously described^21,22,63, and isolate RW from wheat was kindly provided by Dr. W. Li⁶⁴. Isolate SM was obtained from a blighted maize sheath near Beijing, China. All Rhizoctonia isolates were further purified by a single-protoplast procedure and kept on potato dextrose agar slants covered with glycerol at 4 °C for further use. The rDNA-ITS sequences of each purified isolate were compared with those of standard strains for the determination of the anastomosis group (Supplementary Table 1). Nuclear status was assessed by 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) staining²¹.

Illumina and PacBio genomic DNA sequencing

Mycelia of each isolate were cultivated in potato dextrose broth at 28 °C for 72 h with shaking (150 rpm) and collected for genomic DNA isolation using CTAB. For Illumina sequencing of JN, multiple DNA libraries, paired-end (180 and 500 bp) and mate-pair (2, 5, and 10 kb), were used. Genome sequencing of LY, XN, YR, SM, and RW included two mate-pair sequencings (2 and 5 kb) and plus one 500 bp paired-end library for LY and XN or a 350 bp paired-end one for each of the remaining genomes. All DNA libraries were sequenced on an Illumina HiSeq 2000 system by BGI, Shenzhen, China. The raw reads were filtered using Trimmomaticv 0.32⁶⁵ by removing bases with a quality score of 25 or less, adapter sequences, possible contaminated reads, and reads less than 75 bp in length to obtain high-quality reads. For PacBio sequencing, ~20 kb libraries for JN, LY, and XN were prepared at Novogene Bioinformatics Technology Co., Ltd. After sequencing, the subreads were filtered using SMRTlinkv5.0 (-minReadScore = 0.8 and -minLength = 1000). SOAPec_v2.01⁶⁶ was used for genome size estimation with “Genome size = kmer_Number/Peak_Depth”.

RNA sequencing and analysis

Mycelia of JN, LY, and XN were grown in 500-mL flasks containing 150 mL of potato dextrose broth at 28 °C for 48 h, and collected for RNA isolation. RNA Illumina sequencing was performed at BGI. The cleaned reads, after removing adaptor, empty tag, and low-quality sequences, were aligned to the respective assembled genome using TopHat v2.0.14⁶⁷. Transcript abundance (FPKM, fragments per kilobase of exon per million fragments mapped) was quantified using Cufflinks v2.0.0⁶⁸. The differentially expressed genes were examined using Cuffdiff within the Cufflinks program⁶⁸. Expression patterns of potential effector genes of XN were analyzed using the RNA-Seq data of GD118-infected rice plants at different time points post inoculation (BioProject, PRJNA147097).

Assembly and annotation of the genomes

The high-quality Illumina cleaned reads were assembled using SOAPdenovo v2.04⁶⁶ (parameters command line: SOAPdenovo-127mer all -s config.txt -F -K 23 -p 50 -o out_put.) and SSPACE v3.0⁶⁹ for scaffold construction, and GapCloser_v1.12⁶⁶ for gap filling. High-quality PacBio subreads of JN, LY, and XN were assembled using HGAP4²³ (parameters: algorithm options (--minMatch 12 --bestn 10 --minPctSimilarity 90.0), minimum concordance = 70, minimum length = 5 kb, seed coverage = 50, genome length = 95000000 (45000000 for LY and XN)), Canu v1.5²⁴ (parameters command line: canu -p canu_out genomeSize = 95 m useGrid = false -pacbio-raw subreads.fasta.gz gnuplotTested = true stopOnReadQuality = false maxThreads = 50, genomeSize = 45 m for LY and XN.), and MECAT v1.3²⁵ (Step 1, using mecat2pw (default parameters) of MECAT to detect overlapping candidates. Step 2, correct the noisy reads based on their pairwise overlapping candidates mecat2cns command (default parameters) of MECAT. Step 3, extract the longest 25X corrected reads using extract_sequences (default parameters) of MECAT. Step 4, assemble the longest 25X corrected reads using mecat2cacu of MECAT (mecat2canu ErrorRate = 0.02 maxMemory = 40 maxThreads = 50 useGrid = 0). The assembled PacBio genome contigs were corrected using Pilon v1.22²⁷ with Illumina paired-end reads. The telomeric repeats (TTAGGG/CCCTAA) were used to assess integrity at both ends of scaffolds⁷⁰.

Identification of SNP and heterozygosity

Genome analysis tool kit 1.6 (GATK) tools⁷¹ and Samtools⁷² were used for SNP and InDel analysis. SNPs and InDels of JN were called using the Illumina reads of LY, the results were filtered as “quality sites (QUAL) < 30, QualByDepth (QD) < 2.0, Fisher strand (FS) > 60.0, RMSMappingQuality (MQ) < 40.0, MQRankSum < −12.5, and ReadPosRankSum < −8.0” for SNPs and “QUAL < 30, QD < 2.0, FS > 200.0, and ReadPosRankSum < −20.0” for InDels. The genome heterozygosity was calculated using SOAPaligner v2.21 and SOAPsnp software v1.03⁷³, with the following filters: quality score of consensus genotype ≥20, rank-sum test P value >0.05, and minor allele count (supported by ≥ 5 reads).

Repetitive sequences and RIP analysis

Repeat sequences of the genome assemblies were identified using the RepeatModeler (URLs: http://www.repeatmasker.org/RepeatModeler.html), LTR-FINDER v1.0.6 and LTR_retriever v2.8.2^74,75. The insertion age of full-length LTR-RT was estimated by using the formula T = K/2r, where “T” is insertion time, “K” is the divergence level between the 5′- and 3′-LTRs, “r” is the fungal substitution rate (1.05 × 10⁻⁹ nucleotides per site per year)⁷⁶. Repeat-induced point (RIP) mutations were predicted with the RIPCAL program v1.0.5, setting indices of (TpA/ApT) ≥0.89 and (CpA + TpG)/(ApC + GpT) ≤ 1.03 as the indicative of RIP presence⁷⁷. A necrotrophic pathogen Parastagonospora nodorum was used as the positive control of RIP⁷⁸.

Analyses of mating-type loci

The mating (MAT) type genes of Rhizoctonia spp. were obtained by BLAST searches of their genomes against MAT proteins of basidiomycetes from NCBI, including the pheromones (Mfa) and pheromone receptors (STE3 or Pra), and homeodomain (HD) transcription factors. MCscan v1.1²⁸ was used for analyses of gene synteny of the MAT loci of Rhizoctonia isolates.

Gene prediction and functional annotation

Three de novo gene prediction programs, Augustus v2.7⁷⁹, GeneMark+ES v4.0⁸⁰ and SNAP v2013-02-16⁸¹, were used to predict the protein-coding regions of Rhizoctonia assemblies in combination with homology-based and RNA-Seq sequence mapping. Protein sequences from previously sequenced R. solani genomes were also used. These included GD118 of AG1-IA³, 7/3/14 of AG1-IB⁸², BBA69670 of AG2-2IIIB²⁰, WAC10335 of AG8¹⁹, and Rhs1AP of AG3¹⁸, which were mapped to our genome assemblies for homology-based gene prediction using Exonerate v2.2.0⁸³ (using options -percent 50 -showtargetgff -m protein2genome -n 1). All RNA-Seq reads were aligned to the genome assemblies with Tophat v2.0.14⁶⁷, and transcript assembly was conducted with Cufflinks v2.0.0⁶⁸. The final gene models were derived through EvidenceModeler v2012-06-25⁸⁴ integration. Functional annotations of the predicted genes were performed by BLAST and HMMER searches against the NCBI GenBank non-redundant, CAZy (carbohydrate-active enzymes), KOG, and Pfam databases. The tRNA genes were detected using tRNAScan-SE v1.3.1⁸⁵.

Secretome and effectors prediction

Secreted proteins and effector candidates of JN, LY, and XN were analyzed using TMHMM Server v. 2.0⁸⁶ and Phobius 1.01⁸⁷ for prediction of the transmembrane domains, SignalP 4.1 Server⁸⁸ and PrediSi⁸⁹, and TargetP 1.1 Server⁹⁰ for subcellular location and signal peptide cleavage sites. Glycosylphosphatidyl inositol anchor proteins were excluded by PredGPI⁹¹. For more comprehensive and accurate effector prediction, we took into account the predicted secretory proteins of those less than 400 amino acids and less than four cysteines using the Klosterman standard⁹², and we used the fungal effector predictor program, EffectorP 2.0⁹³.

Whole-genome duplication and subgenome reconstruction

Syntenic blocks in each species and between JN and LY were identified by MCscan v1.1²⁸ using parameters of MATCH_SCORE 50, MATCH_SIZE 5, GAP_PENALTY -1, OVERLAP_WINDOW 5, E_VALUE 1e-15, MAX GAPS 5, IDENTITY 50%, and COVERAGE 70% and synteny distributions were plotted using Circos⁹⁴. Synonymous (Ks) and nonsynonymous (Ka) substitutions values of syntenic genes were calculated using the YN model⁹⁵ by MAFFT v7.221⁹⁶, ParaAT v1.0⁹⁷, and KaKs_Calculator v1.2⁹⁸. The Ks distributions were plotted to estimate speciation and whole-genome duplication events⁹⁹. The time was estimated at the peak value by using the “t = Ks/2r” formula, which was used to estimate the divergence time between two species genome, where “Ks” is the peak value of Ks distributions, “r” is the fungal neutral substitution rate (1.05 × 10⁻⁹)^76,99.

Subgenomes of JNa and JNb were divided from JN using LY genomic sequences as the reference, and the same for reconstructions of SM and YR subgenomes. Scaffolds of homeologs were compared to LY, and scaffolds of higher sequence identity with LY were assigned to subgenome a and the rest to subgenome b. Genomic modifications in JN subgenomes were analyzed through the syntenic genes among JNa, JNb, and LY. Given the synteny blocks of JNa, JNb, and LY having the same gene orders, changes of co-linear JNa and LY against JNb represented modifications in JNb, including gene loss, gene gain, and gene replacement, versus changes of co-linear JNb and LY against JNa as JNa variations. Amino acid identity and Ks value of syntenic gene pairs were used to find exchanged genes between the two subgenomes JNa and JNb using LY as the reference. Gene exchange between JN subgenomes was defined by comparison of amino acid identity values (Ks) among JNa, JNb, and LY, exchanges were considered to have occurred when the Ks of a JNb protein was closer to LY than that of JNa.

Evolution rate calculation

Multiple sequence alignment for each single-copy orthologs gene family of four different (sub)genomes (XN, LY, JNa, and JNb) were carried out using MAFFT v7.221⁹⁶, and the ML phylogenetic tree using RAxML v8.1.24 (randomized accelerated maximum likelihood)¹⁰⁰. Then, a codon multiple alignments was created from the protein sequence alignment result using ParaAT v1.0⁹⁷ for estimation of evolutionary rates (Ka, Ks, and Ka/Ks).

Identification of orthologous genes and phylogenetic reconstruction

OrthoMCL v2.0.9¹⁰¹ and all-versus-all BLASTP (E-value ≤1e-15, coverage ≥50%) were used to identify orthologous groups for JNa, JNb, SMa, SMb, LY, XN, RW, GD118³, Candida glabrata DSY562¹⁰², Saccharomyces cerevisiae YJM1078¹⁰³, and Lachancea waltii NCYC 2644¹⁰⁴, in which the Ascomycetous yeasts were used as references for whole-genome duplication³⁰. Single-copy orthologs were extracted using Perl script (command line parameters of Gblocks: Gblocks proteins.fasta -b4 = 5 -b5 = h.) and subjected to global alignment using MAFFT v7.221⁹⁶, in which the poorly aligned regions of concatenated sequences were removed by using Gblocks v0.91b¹⁰⁵. The final phylogenetic tree was constructed using RAxML v8.1.24¹⁰⁰.

Statistics and reproducibility

All statistical analyses and visualization were performed using R Project, online OmicShare tools (https://www.omicshare.com/tools), and MATLAB. Statistical significance was done with various tests, including Student’s t test, binomial test, chi-square test, and Mann–Whitney U test, as well as Kendall’s rank correlation.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The genome assemblies described in this paper are the first versions. The whole-genome assemblies and sequence data of Rhizoctonia spp. JN, LY, and XN described here are available and have accession numbers assigned in NCBI BioProjects PRJNA624246, PRJNA624247, and PRJNA624219, respectively.

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Acknowledgements

We thank Dr. Runmao Lin (Chinese Academy of Agricultural Sciences), Dr. Minfeng Xue (Hubei Academy of Agricultural Sciences), and Dr. Changfa Yin (China Agricultural University) for trouble-shooting during the genomic data analysis. We thank Prof. Vijai Bhadauria and Prof. Xin Zhou (China Agricultural University) for critical reading of the paper. Zhenghu Sun for technical support. This project was supported by the National Key Research and Development Program of China (grant no. 2016YFD0300703) and the National Natural Science Foundation of China (grant no. 31972253).

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Key Laboratory of Pest Monitoring and Green Management, MOA; Joint Laboratory for International Cooperation in Crop Molecular Breeding; Department of Plant Pathology, China Agricultural University, Beijing, China
Cheng Li, Zejian Guo, Qingyue Han, Manman Zhang, Youliang Peng & Xujun Chen
College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao, Shandong, China
Shanyue Zhou
Environmental Sciences, University of Guelph, Guelph, Ontario, Canada
Tom Hsiang

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Cheng Li
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Shanyue Zhou
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Manman Zhang
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Youliang Peng
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Xujun Chen
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X.C. and Z.G. conceptualized and supervised the project. S.Z. provided study materials. C.L., Z.G., and X.C. conducted data analysis. C.L. and Q.H. acquired experiments. Z.G., C.L., and X.C. drafted the paper. Z.G., C.L., X.C., T.H., Y.P., M.Z., S.Z., and Q.H. contributed to review and editing the paper.

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Correspondence to Xujun Chen.

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Li, C., Guo, Z., Zhou, S. et al. Evolutionary and genomic comparisons of hybrid uninucleate and nonhybrid Rhizoctonia fungi. Commun Biol 4, 201 (2021). https://doi.org/10.1038/s42003-021-01724-y

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Received: 21 May 2020
Accepted: 19 January 2021
Published: 15 February 2021
DOI: https://doi.org/10.1038/s42003-021-01724-y

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