Throughout the history of agriculture, many new crop species (polyploids or artificial hybrids) have been introduced to diversify products or to increase yield. However, little is known about how these new crops influence the evolution of new pathogens and diseases. Triticale is an artificial hybrid of wheat and rye, and it was resistant to the fungal pathogen powdery mildew (Blumeria graminis) until 2001 (refs. 1,2,3). We sequenced and compared the genomes of 46 powdery mildew isolates covering several formae speciales. We found that B. graminis f. sp. triticale, which grows on triticale and wheat, is a hybrid between wheat powdery mildew (B. graminis f. sp. tritici) and mildew specialized on rye (B. graminis f. sp. secalis). Our data show that the hybrid of the two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts. We conclude that hybridization between mildews specialized on different species is a mechanism of adaptation to new crops introduced by agriculture.
The artificial hybrid triticale was introduced into commercial agriculture in the 1960s. The hexaploid triticale genome is composed of genomes A and B from wheat and the rye genome, R (AABBRR)3. Triticale was initially resistant to powdery mildew; however, this pathogen was first observed on triticale in 2001 and has since become a major disease in Europe1,2. We sequenced 46 isolates of B. graminis (including the 96224 reference isolate4) from different European countries and Israel (Supplementary Table 1) with host ranges corresponding to four different formae speciales, including the previously described rye (B.g. secalis) and wheat (B.g. tritici) powdery mildews. Our infection tests showed that B.g. secalis grows exclusively on rye, whereas B.g. tritici is able to grow on tetraploid (durum) and hexaploid (bread) wheat (Table 1, Supplementary Figs. 1–23, Supplementary Table 2 and Supplementary Note). On the basis of infection tests, we define the triticale forma specialis as being able to grow on triticale, on hexaploid and tetraploid wheat (with lower penetration efficiency; Supplementary Fig. 24 and Supplementary Note), and to a very limited extent on rye. Furthermore, we designate mildew growing exclusively on tetraploid wheat as new forma specialis, B. graminis f. sp. dicocci5.
Overall, the genomes of the 46 isolates were very similar to each other, allowing high-quality mapping of the 45 resequenced isolates to the 96224 reference isolate (Supplementary Note). We identified between 115,543 and 332,450 polymorphic nucleotide sites per isolate in comparison to the B.g. tritici reference genome (Supplementary Figs. 25 and 26, and Supplementary Note). Principal-component analysis (PCA) based on 717,701 polymorphic sites clearly distinguished four groups (Fig. 1) that corresponded to the four formae speciales identified in the infection tests. This grouping indicates that gene flow is restricted between formae speciales. Interestingly, the B.g. triticale isolates formed a group distinct from the B.g. tritici isolates. This contradicts the current hypothesis that the host range of B.g. tritici expanded to include triticale through mutation of a few genes2,6,7. Instead, our analysis shows that B.g. triticale isolates form a specific group with a distinct evolutionary history (Supplementary Figs. 27–30 and Supplementary Note).
From genome comparisons, we identified sets of polymorphisms that were fixed in the four formae speciales (polymorphisms shared by all isolates of a particular forma specialis). Such fixed polymorphisms between formae speciales (substitutions) will hereafter be referred to as the 'genotype' of a forma specialis. Interestingly, the B.g. triticale genome consisted of large genomic segments that had the same genotype as B.g. secalis alternating with segments with a B.g. tritici genotype (Fig. 2a,b and Supplementary Figs. 31–33). Such patterns are characteristic of recent hybrids, reflecting a limited number of recombination events between parental genotypes. Furthermore, analysis of nucleotide diversity in the genomes of the different formae speciales showed that B.g. triticale had a characteristic distribution with multiple peaks that is consistent with the hypothesis of a recent origin by hybridization (Fig. 2c,d and Supplementary Note). We tested this hypothesis on the 172,274 fixed polymorphisms that distinguish B.g. tritici from B.g. secalis. We found that, in B.g. triticale, between 11.9 and 21.4% of the polymorphic sites represented the B.g. secalis genotype. These polymorphic sites were located on genomic segments that made up between 6.6 and 17.3% of the genomes of the B.g. triticale isolates. In contrast, over 80% of these genomes had the B.g. tritici genotype. Thus, we conclude that B.g. triticale is a hybrid of B.g. tritici and B.g. secalis and that the hybridization happened very recently (Supplementary Table 3 and Supplementary Note).
Furthermore, phylogeographic analysis and the proportions of the parental genomes suggest that the initial hybridization event was followed by two backcrosses with B.g. tritici and that this process likely occurred in Europe (Supplementary Figs. 34–37 and Supplementary Note).
These findings raised the question of whether B.g. triticale originated from a single hybridization of B.g. secalis and B.g. tritici (followed by two backcrosses) or whether more individuals from the two formae speciales also contributed. If the first hybridization was a single event that involved only one B.g. secalis isolate, we would expect to observe no diversity in the B.g. triticale portion of the genome inherited from B.g. secalis. On the basis of phylogenetic analysis, we found 66 genes for which all B.g. triticale isolates inherited the B.g. secalis gene. We counted the number of haplotypes present in B.g. triticale for each of these 66 genes and found six genes that showed two different haplotypes. These different haplotypes were also present in the five sequenced B.g. secalis isolates. We conclude that the minimum number of B.g. secalis individuals that contributed to B.g. triticale is two, which is likely the result of two independent origins of B.g. triticale (Supplementary Fig. 38 and Supplementary Note).
B. graminis, as with most fungi, has two mating types4. Because the mating types of B.g. secalis and B.g. tritici have different genotypes, we could trace back the origin of mating types in B.g. triticale. We found that all B.g. tritici partners in the first hybridizations seemed to have been of the MAT1-1-3 mating type whereas the B.g. secalis partners were of the MAT1-2-1 mating type. The MAT1-2-1 locus of the B.g. tritici genotype was acquired by B.g. triticale through one of the backcrosses, whereas the MAT1-1-3 locus of the B.g. secalis genotype was not present in any of the sequenced B.g. triticale isolates (Supplementary Figs. 39 and 40, and Supplementary Note). These findings are in contrast to those on the plant pathogen Zymoseptoria pseudotritici, whose origin was traced back to a single hybridization event8. Unlike Z. pseudotritici, the new forma specialis B.g. triticale likely did not pass through a bottleneck, as the multiple isolates involved in hybridization passed on a considerable proportion of parental genetic diversity (Supplementary Table 4 and Supplementary Note).
On the basis of the number of observed recombination events between the B.g. tritici and B.g. secalis genotypes, we estimated that B.g. triticale isolates underwent between seven and 47 (depending on the isolate) sexual cycles from the origin of the forma specialis (Supplementary Fig. 41 and Supplementary Note). As B. graminis has a maximum of one sexual cycle per year4, we conclude that B.g. triticale originated after the introduction of triticale as a commercial crop in the 1960s, possibly multiple times, and since then different isolates have undergone a different number of sexual generations. Because B.g. triticale is a very recent hybrid, we studied the phylogenetic relationship of its two parents. We estimated the divergence time for the parental formae speciales of B.g. triticale—B.g. secalis and B.g. tritici—using 206 orthologous single-copy genes and found that they diverged between 168,245 and 240,169 years ago (Supplementary Fig. 42, Supplementary Table 5 and Supplementary Note). In contrast, their hosts—rye and wheat, respectively—had already diverged approximately 4 million years ago9. This incongruence in the divergence times of a host and pathogen can be due to host tracking: until a few hundreds of thousands of years ago, wheat and rye were still in the host range of a single forma specialis, and divergence into two distinct formae speciales occurred only relatively recently. This history would be consistent with previous findings that the formae speciales B.g. tritici and B. graminis f. sp. hordei diverged approximately 6 million years ago4, whereas their hosts, wheat and barley, respectively, diverged at least 2 million years earlier9. An alternative explanation could be a recent host jump from wheat to rye or vice versa, followed by the rapid emergence of barriers to gene flow.
Despite our large sequencing effort, the molecular basis for the host range expansion of B.g. triticale remains obscure. Genetic determinants of the expansion of host specificity to triticale are likely located on genomic segments that were inherited from B.g. secalis. Among these genes might be the genetic determinant for the host range expansion of B.g. triticale. Six of the 66 B.g. secalis genes inherited by all B.g. triticale isolates encoded putative effectors (proteins that are secreted into the host cell to facilitate pathogen proliferation) (Supplementary Table 6 and Supplementary Note). In B. graminis, effectors are typically small proteins with short, characteristic motifs that can be identified bioinformatically4 (through the presence of signal peptide and lack of homology with the protein domains of other organisms). However, transcriptome profiling of B.g. triticale grown on wheat and triticale showed that none of these six genes were differentially expressed by the fungus in the two different hosts. In general, the transcriptome profile of B.g. triticale was mostly independent of the host species (Supplementary Figs. 43–47 and Supplementary Note). Alternatively, host specificity could be a quantitative trait that requires a certain number of genes with partially overlapping and/or complementary functions. Effector genes in particular are thought to have overlapping or partially redundant functions4,10.
It is intriguing that the host of B.g. triticale (triticale) is a hybrid of rye and bread wheat, which are the hosts of the ancestors of B.g. triticale. Interestingly, bread wheat (the host of B.g. tritici) is itself the result of a hybridization that occurred approximately 10,000 years ago between domesticated tetraploid (emmer) wheat and the diploid wild grass Aegilops tauschii11. Moreover, the host range of B.g. tritici (hexaploid and tetraploid wheat) encompasses the host range of B.g. dicocci (tetraploid wheat). This is reminiscent of the expanded host range of B.g. triticale that encompasses the host range of one of its parent species, B.g. tritici (wheat), in addition to triticale. We therefore hypothesized that B.g. tritici could also be a hybrid between a pathogen of tetraploid wheat (B.g. dicocci) and one that infects A. tauschii. However, in contrast to B.g. triticale in which we found genomic segments of the B.g. tritici genotype, we did not find large genomic segments in B.g. tritici isolates that could be assigned to a B.g. dicocci genotype. This absence could be explained by multiple rounds of recombination that have eroded the characteristic pattern of sequence segments. A different approach to test hybridization that is more resistant to the action of recombination makes use of gene genealogies12, which have been used to identify hybridization in different organisms13,14. We used coalescent-based methods to calculate the probability of the evolutionary model in which both B.g. tritici and B.g. triticale are hybrids and four alternative models in which there is no hybridization or only one of the two formae speciales is a hybrid15,16,17,18. The model depicted in Figure 3 with two hybridizations seems to be the most likely (Supplementary Fig. 48, Supplementary Tables 7 and 8, and Supplementary Note). However, we cannot completely rule out the possibility that B.g. tritici originated through a more complex untested scenario with multiple past admixture events.
We conclude that B.g. tritici arose sometime after the emergence of bread wheat, probably several thousand years ago, through hybridization of a B.g. dicocci strain and a different, yet unknown mildew strain. Hybridization has been reported to be important for adaptation to new hosts in several fungal and oomycete pathogens19,20,21,22,23,24. Our data now indicate that hybridization was the causal step in host range expansion to a newly bred or evolved species. It is apparent that co-evolution based on hybridization is a likely evolutionary pathway for B. graminis that infects wheat and other grasses, which themselves evolve predominantly through hybridization25. It is particularly fascinating that pathogen evolution mirrors evolution on the host side and that the hybrid of two mildews specialized on two different hosts can infect the hybrid plant species originating from those two hosts (Fig. 3). It is possible that our findings define a more general evolutionary pattern: stem rust (Puccinia graminis) was reported to infect triticale at approximately the same time as B. graminis26. Also, in P. graminis, hybridization between formae speciales has been reported to be important for host range determination27. It is noteworthy that this pattern of evolution is observed in pathogens of agricultural species. It is possible that agricultural ecosystems increase the possibility that different pathogens co-occur in large populations, thus making hybridization more likely than in natural ecosystems. Many agricultural crops are polyploids and/or contain resistance genes introgressed from wild relatives. Little is known about what determines the durability of such introgressed genes, but hybridization of pathogens of the wild relative with the crop pathogen should be considered in resistance breeding approaches. In addition, in the future production of man-made crops, the genetic resistance of parents should be carefully selected to make resulting hybrids more durably resistant in the field.
Host specificity tests.
To test the host specificity of the isolates used in this work, we infected six cultivars of triticale, five cultivars of wheat (three hexaploid and two tetraploid) and three cultivars of rye with 46 Blumeria isolates (Supplementary Table 2). The plant cultivars used in the host specificity tests were chosen for the absence of known race-specific resistance genes and known high susceptibility to many tested mildew isolates. In this way, we avoided race-specific resistance as a confounding factor in the determination of host specificity. We infected 10-d-old detached leaf segments with fresh spores, and the infected leaf segments were kept on benzimidazole agar plates at 20 °C and 70% humidity with a 16-h light/8-h dark cycle. We scored the phenotypes 10 d after infection using three categories: virulent, intermediate and avirulent (Supplementary Figs. 1–23 and Supplementary Note).
Staining for assessment of penetration efficiency.
Infected leaf segments were collected 2 d after infection and incubated for 4 d in destaining solution (8.3% lactic acid and 16.7% glycerol in ethanol). Leaves were stained for 45 s with neutral red (0.1%), which was used as a contrasting agent to observe haustoria inside plant epidermal cells. Aerial fungal structures were stained for 45 s using 0.25% Coomassie blue.
Sequencing, mapping, SNP calling, assembly and principal-component analysis.
DNA extraction was performed following the methods of Bourras and colleagues28. 101-bp paired-end libraries were created and sequenced with the Illumina HiSeq platform at the Functional Genomic Center of Zurich or at the Genome Analysis Centre at Norwich Research Park (TGAC), obtaining between 31,039,416 and 62,834,815 reads (mean of 41,810,764 reads) (Supplementary Table 1). Reads were mapped on the B.g. tritici reference genome4 using Bowtie 2.1.0 (ref. 29) with option —score-min L, -0.6, -0.25. We used the following command in SAMtools 0.1.19 (ref. 30) to convert formats and collect information about single genomic positions: view, sort, mpileup -q 15 (only reads with mapping quality greater than 15 were considered). Finally, we used bcftools to generate a VCF file that was parsed with in-house Perl scripts (available upon request). We considered as high-confidence SNPs only positions with a minimum mapping score of 20, a minimum coverage of 20× and a minimum frequency of the alternative call of 0.95. Polymorphisms were considered to be fixed if they were found to be present in all isolates of a forma specialis and were different in the other formae speciales.
De novo assembly of the genomes for all isolates was performed with CLC Genomic Workbench 7.5 using standard parameters. PCA was performed with the R package GAPIT31,32.
Identification of orthologous genes.
We used GMAP (version 2013-07-20)33 to annotate genes in the assembly of isolate S-1201 (B.g. secalis reference genome), using the 7,186 genes of the B.g. tritici reference genome (isolate 96224) as a template. We found 6,864 genes in the genome assembly of isolate S-1201. The protein and CDS databases for B.g. hordei and Neurospora crassa were downloaded from BluGen and the Broad Institute, respectively (accessed 1 March 2014; see URLs). After the elimination of genes with homology to the Blumeria repeat database (BRD) and the Triticeae transposable elements (PTREP12) databases, we retained 9,733 genes for N. crassa and 6,011 genes for B.g. hordei. To cluster genes in families, we used all-against-all BLAST searches34 and grouped together genes that were reciprocal hits with a minimum alignment length of 150 bp and a BLASTN e value ≤1 × 10−10. We then defined as single-copy gene families all families with four genes, one for each of the species used (N. crassa, B.g. tritici, B.g. hordei and B.g. secalis), that were reciprocal top BLAST hits. With these criteria, we obtained 208 orthologous gene families. We used GMAP (version 2013-07-20)33 to retrieve these genes in all the B.g. tritici and B.g. secalis isolates, using as a template the genes from the same forma specialis (isolates 96224 and S-1201, respectively). Assemblies for the B.g. hordei isolates AOIY01 and AOLT01 were obtained from Hacquard and colleagues35. We annotated the 208 genes on these two assemblies with GMAP (version 2013-07-20)33 using the genes of the B.g. hordei reference isolate as the template. We could not find two genes in all B.g. hordei isolates, and we therefore excluded them. The final data set was composed of 206 orthologous single-copy genes from N. crassa, three from B.g. hordei isolates, five from B.g. secalis isolates and 13 from B.g. tritici isolates. This data set was used to infer a species tree phylogeny and to estimate divergence time (Supplementary Note).
We used analogous methods to define single-copy orthologous genes in B. graminis (without N. crassa). We used all-against-all BLAST searches (between B.g. hordei (reference isolate), B.g. tritici (96224), B.g. secalis (S-1201) and B.g. dicocci (220)). We grouped together genes that were reciprocal BLAST hits with a minimum alignment length of 150 bp and a BLASTN e value ≤1 × 10−10. We then defined as single-copy gene families all families with four genes, one for each of the formae speciales used (B.g. dicocci, B.g. tritici, B.g. hordei and B.g. secalis), that were top reciprocal hits. This resulted in 4,556 single-copy genes. We then retrieved these genes in all isolates, using as a template the genes from the same forma specialis (96224 for B.g. tritici, S-1201 for B.g. secalis and 220 for B.g. dicocci); the reference isolate of B.g. hordei was used as the outgroup. Multiple-sequence alignments were generated with Muscle36, and we inferred maximum-likelihood trees for all alignments with RAxML 8.0.22 (ref. 37) using a generalized time-reversible (GTR) + Gamma model38,39,40. We used Newick Tools41 to identify particular topology patterns. This data set was used in phylogeographic analysis, in the identification of genes inherited from B.g. secalis in all B.g. triticale isolates, in the estimation of the minimum number of isolates that contributed to the first hybridization and in the coalescent-based estimation of the most likely evolutionary network (Supplementary Note).
Alignment and phylogeny for divergence time estimation.
Protein multiple-sequence alignments were generated with Muscle 3.8.31 (ref. 36) and reverse translated into nucleotide sequences with TranslatorX v1.1 (ref. 42). The concatemer of all the alignments was 410,189 bp long. Phylogenetic inference on the partitioned data set was performed with MrBayes 3.2.2 (ref. 43). We ran two independent replications of 10,000,000 generations. Variation in substitution rates across sites was modeled with a discretized (four categories) gamma distribution39,40. The chains have been let free to sample all models of the GTR model family using reversible-jump Monte Carlo Markov chain44. The divergence time between B.g. hordei and B.g. tritici was used as the calibration point (5.2–7.4 million years ago4 or 10,000–14,000 years ago45) (Supplementary Note) under the independent gamma rate relaxed-clock model (white noise model)46.
RNA extraction and transcriptome analysis.
Leaf segments of the wheat variety Chinese Spring and the triticale variety Timbo were infected with two different B.g. triticale isolates (THUN-12 and T3-8). Leaves were left on benzimidazole agar plates as described in Parlange et al.47 and collected 2 d after infection. Each pathogen-host combination was replicated three times. RNA extraction was performed with the miRNeasy mini kit from Qiagen according to the manufacturer's recommendations. 125-bp single-end libraries were created and sequenced with the Illumina HiSeq platform at the Functional Genomics Center Zurich. RNA sequencing (RNA-seq) reads were mapped with STAR48 (allowing four mismatches per 100 bp). Read counts were determined with featureCounts 1.4.6 (ref. 49). The R package edgeR was used for statistical analysis, and genes were tested for differential expression with a generalized linear model and tagwise estimation of dispersion50.
Testing of evolutionary networks with PhyloNet.
The probabilities of five different evolutionary hypotheses (networks a–e; graphically represented in Supplementary Fig. 35) were evaluated with a coalescent-based method implemented in PhyloNet16 (Supplementary Note). Because of computational limitations, we generated gene trees using two subsets of isolates. We tested two different, non-overlapping sets of isolates (one B.g. hordei, two B.g. secalis and three each of B.g. tritici, B.g. dicocci and B.g. triticale, for a total of 12 isolates for each data set), each of them with ten different sets of 300 randomly selected single-copy genes for which trees were inferred with RAxML37. The composition of the first data set was as follows: B.g. hordei reference isolate; 96224, 97 and 103 (B.g. tritici); S-1201 and S-1400 (B.g. secalis); THUN-12, T3-8 and COPP-2C (B.g. triticale); and 58, 66 and 220 (B.g. dicocci). The composition of the second data set was the following: B.g. hordei reference isolate; 94202, 8 and 70 (B.g. tritici); S-1203 and S-1459 (B.g. secalis); T1-23, T6-6 and HO-101 (B.g. triticale); and 63, 207 and 209 (B.g. dicocci). To compare the likelihoods of models with a different number of parameters, we used three information criteria—the Akaike information criterion51 (AIC), the corrected AIC (AICc) and the Bayesian information criterion52 (BIC). These measures give increasing penalties to models containing more parameters.
Estimation of the number of sexual generations from hybridization in B.g. triticale.
We estimated the number of sexual generations after hybridization for each B.g. triticale isolate using the following formula modified from Stukenbrock et al.8
where NR is the number of recombination breakpoints, BGSS is the average length of B.g. secalis segments after two backcrosses (3.3 Mb) and BGSG is the amount of B.g. secalis genome in the B.g. triticale isolate in base pairs.
The original formulation in Stukenbrock et al.8 was
where ALRWFH is the average length of recombinant windows after the first hybridization and ALORW is the average length of the observed recombinant window.
We used equation (2) for the calculation of BGSS. We estimated the recombination rate from the genetic consensus map of B.g. tritici, which has a length of approximately 1,800 cM (ref. 28). Thus, one expects one recombination event every 10 Mb in a sexual cycle. In turn, after the first hybridization event, we expect segments of B.g. secalis genome sequence with an average length of 10 Mb. In the first backcross, these segments recombine once on average, resulting in parental segments of 5 Mb in length. With the second backcross, each window recombines 0.5 times on average. This results in BGSS being on average 3,333,333 bp for B.g. secalis segments in B.g. triticale after two backcrosses.
In each following generation, B.g. triticale isolates mate with each other, and the probability that a new recombination event can be observed corresponds to the probability that recombination occurs in a region that was inherited from B.g. secalis in one isolate and B.g. tritici in the other. Assuming a proportion of 12.5% for the B.g. secalis genome in B.g. triticale isolates, this is equal to 0.22 (that is, 0.125 × 0.875 × 2).
Adding these terms, equation (2) becomes
Because the B.g. tritici reference genome sequence is fragmented (owing to its extremely high repeat content) (Supplementary Note), we cannot use the average length of B.g. secalis windows (most of them would be delimited by the end of the contig). However, the detection of recombination breakpoints (NR) is not affected by contig size. The relationship between the average length of B.g. secalis windows and NR is given by
If we plug equation (4) into equation (3), we obtain equation (1).
We defined putative recombination breakpoints as the borders between regions that contained fixed substitutions inherited from the B.g. secalis parent and regions that contained fixed substitutions from the B.g. tritici parent. Here we excluded stretches of fixed polymorphisms that are likely the result of gene conversion events. Gene conversions typically affect fragments of less than 1,000 bp in size. Thus, we ignored groups of polymorphisms of a different parental genotype if they were clustered in a region of less than 1,000 bp in size or single substitutions of one genotype in a region otherwise originating from the other genotype.
The proportion of the genome that was contributed by B.g. secalis (BGSG) was calculated by adding up the sizes of sequence contigs that only contained polymorphisms of the B.g. secalis genotype (Supplementary Note).
All genomic and transcriptomic sequences used for this study can be accessed at the Sequence Read Archive (SRA) under accession SRP062198 and at the Gene Expression Omnibus (GEO) under accessions GSE73399, respectively.
B.g. f. sp. hordei genome (BluGen), http://www.blugen.org/; Neurospora crassa genome (Broad Institute), http://www.broadinstitute.org/annotation/genome/neurospora/; Triticeae transposable elements (PTREP12) databases, http://www.botinst.uzh.ch/en/research/genetics/thomasWicker/TREP.html.
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We want to thank S. Brunner for advice and support, A. Dinoor, V. Troch and F. Mascher for mildew isolates, C. Aquino and H. Rehrauer for their help with sequencing and bioinformatic analysis, and A. Widmer for reading and commenting on the manuscript. This work was supported by the University Research Priority Program (URPP) 'Evolution in Action' of the University of Zurich, Swiss National Science Foundation grant 310030B_144081/1, a grant from the Swiss Federal Office for Agriculture and an Advanced Investigator Grant from the European Research Council (ERC-2009-AdG 249996, Durableresistance).
The authors declare no competing financial interests.
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Menardo, F., Praz, C., Wyder, S. et al. Hybridization of powdery mildew strains gives rise to pathogens on novel agricultural crop species. Nat Genet 48, 201–205 (2016). https://doi.org/10.1038/ng.3485
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