Draft genome sequence of Venturia carpophila, the causal agent of peach scab

Venturia carpophila causes peach scab, a disease that renders peach (Prunus persica) fruit unmarketable. We report a high-quality draft genome sequence (36.9 Mb) of V. carpophila from an isolate collected from a peach tree in central Georgia in the United States. The genome annotation is described and a phylogenetic analysis of the pathogen is presented. The genome sequence will be a useful resource for various studies on the pathogen, including the biology and ecology, taxonomy and phylogeny, host interaction and coevolution, isolation and characterization of genes of interest, and development of molecular markers for genotyping and mapping.

Fusicladium carpophilum Thüm (Oudem.); Cladosporium americanum H.C. Greene) is an important fungal pathogen in the family Venturiaceae and is the causal agent of scab in peach (Prunus persica) and other species of Prunus [1]. Typical symptoms on fruit are black freckles, spots, and/or scabs of variable size, distribution and density ( Fig. 1 a and b) that render the fruit unfit for market [2]. The pathogen also infects shoots and overwinters in lesions on 1-year-old twigs, which are thought to be the source of primary inoculum in the form of conidia that are both airborne and splashborne, resulting in infection of young fruit during the spring and early summer [3]. Peach scab generally develops on the most exposed, easily wetted, uppermost portion of the fruit surface, which is consistent with the splash-borne nature of conidia [4]. Multiple fungicide sprays are required to control peach scab at variable costs, which impose a risk of the pathogen developing resistance to certain fungicides. The effect of the fungicide sprays in reducing disease severity and thus improving fruit quality has been demonstrated, but under certain conditions the results are inconsistent [5].
Besides peach, several economically important stone fruit crops, including apricot (P. armeniaca), almond (P. dulcis), and plum (P. domestica), can be infected by V. carpophila [1]. Only recently has the taxonomic identity of the pathogens causing scab on stone fruit and other related genera begun to be clarified [6][7][8]. No complete genome sequence of V. carpophila has been reported, although some related species have now been sequenced [9][10][11]. As with these other species, an annotated genome of V. carpophila is a valuable resource for various genomic, genetic, and systematic studies. For example, various genes of interest and importance, such as those related to fungicide resistance, host recognition, or mating type, can be identified for further research to aid in management of the disease. Microsatellites can be developed as informative markers for genetic mapping and diversity studies. Also, the knowledge obtained from the genome can be useful in improving development of resistant cultivars.
In this report, we describe the first high-quality draft genome sequence of V. carpophila and provide a phylogenetic analysis of the fungus and other closely related species. The genome sequence will facilitate further genomic and phylogenetic exploration to understand the pathogen and its relationship with peach.

Classification and features
The sequenced strain of V. carpophila was isolated from a scab-infected peach fruit (cv. Julyprince) from an 8year-old tree in June 2014 at the USDA-ARS-SEFTNRL, Byron, Georgia, USA (Table 1). Asexually produced conidia ( Fig. 1c) of V. carpophila were scraped from a single lesion on the fruit surface using a scalpel, and a dilute spore solution prepared in sterile distilled water. Multiple 0.1 μL aliquots were spread on the surface of replicate water agar plates, amended with lactic acid (0.50 mL/L), streptomycin (0.20 g/L), tetracycline (0.05 g/L) and chloramphenicol (0.05 g/L). Plates were incubated at 27°C for 48 h under fluorescent light on a 12/12 h day/night cycle. A single germinated spore of V. carpophila was excised on an agar plug using a scalpel under a microscope (50×), and was transferred to antibiotic-amended potato dextrose agar, and was grown for 4 weeks under the same conditions described above (Fig. 1d).
The fungus belongs in the Eukaryota, is a member of the Fungal kingdom, phylum Ascomycota, class Dothidiomycetes, and family Venturiaceae (Table 1). Several other economically important plant pathogens are members of the Dothidiomycetes, including apple scab (V. inaequalis), pear scab (V. pyrina), pecan scab (F. effusum), rice scald (Magnaprthe oryzae), and Septoria leaf blotch of wheat (Zymoseptoria tritici syn. Mycosphaerella graminicola). V. carpophila has been classified based on its host range, morphology and some molecular characteristics [1]. The sexual stage (pseudothecia that produce ascospores) of the fungus has been identified and described from Australia [1], but has not been described elsewhere at any time. Its role in the epidemiology of the disease is unknown.
The phylogenetic relationship of V. carpophila to other Ascomycota species based on the 18S rRNA genes shows that it is most closely related to members of the family Venturiaceae, particularly the genera Fusicladium and Venturia (Fig. 2). The 18S rRNA gene was located on scaffold_438 and a 1552 bp sequence aligned with the sequences from the other fungi was used for the analysis. The phylogenetic analysis was performed using the nearest neighbor joining method in CLUSTALX2 [12] with node values based on 1000 replicates. The phylogenetic tree was drawn by TreeView [13]. Members from other genera in the Dothidiomycetes were included as outgroups.

Genome project history
The genome of V. carpophila described here was sequenced in 2015 at the ICBR core facility of the University of Florida, Gainesville, Florida, USA. The genome was assembled and annotated at the USDA-ARS Fruit and Tree Nut Research Laboratory. This Whole Genome Shotgun project has been deposited at DDBJ/ ENA/GenBank under the accession MECS00000000 (Bioproject PRJNA321389). The version described in this paper is version MECS01000000. The project data is summarized in Table 2. The project information is in compliance with MIGS version 2.0 [14].

Growth conditions and genomic DNA preparation
Culture of V. carpophila was on antibiotic-amended potato dextrose agar. The culture was incubated for 4 weeks at 25°C (12 h light/12 h dark), when the DNA was extracted from the sample using a ZymoResearch DNA extraction kit (ZymoResearch, Irvine, CA), following a slightly modified protocol for DNA extraction from fungi [15]. A Qiagen Tissue Lyser (Qiagen, Valencia, CA) was used to lyse the mycelium. Once obtained, the DNA was quantified using a Nanodrop spectrophotometer (Nanodrop Products, Wilmington, DE) and stored in TE buffer at −20°C.

Genome sequencing and assembly
The genome was sequenced using an Illumina paired-end library (a V3 kit, 2 × 300 cycles) and a MiSeq machine, which generated 400,041,052 raw reads consisting of 12,052,356,652 raw nucleotides. The A5-Miseq assembly pipeline was used automatically to check quality, trim adaptors, filter lowquality reads, to correct sequencing errors using The actual genome size is unknown at this stage, but the 263 × genome coverage likely covers more than 95% of the genome. Therefore we can estimate that the genome size of V. carpophila is 38.9 Mb, which is in the typical size range of genomes in the phylum Ascomycota [17].

Genome annotation
The draft genome was annotated using the MAKER pipeline [18]. In summary, repeats were first found and masked using RepeatMasker and the RepBase database [19]; ab initio gene prediction was performed with AUGUSTUS under the parameter Saccharomyces [20]; these predicted genes were annotated by BLAST against the NCBI nonredundant (nr) nucleotide database and also by RPSBLAST (Reverse Position-Specific BLAST) batch search in conserved domain database (CDD v3.14) [21,22]. The CCD is a superset including a total of (See figure on previous page.) Fig. 2 The phylogenetic position of Venturia carpophila in comparison with other related fungal species. The tree was developed based on the 18S rRNA gene of the sequenced isolate of V. carpophila, an accession of the 18S rRNA gene of another isolate of V. carpophila, and accessions of other closely-related members of the family Venturiaceae (genera Fusicladium and Venturia) and with outgroup representatives (Phyllosticta harai, Staniwardia suttonii, Mycosphaerella graminicola, M. verrucosiafricana, Xenomeris raetica and Gibberella moniliformis, all in the class Dothidiomyectes of the phylum Ascomycota). The nearest neighbor joining tree was built by CLUSTALX2 [12] and drawn to scale by TreeView [13] based on 18S rRNA sequence phylogenetic analysis involved in a 1000-replicate bootstrapping process (numbers adjacent to branches are support values from 1000 bootstraps). The branch lengths in the tree were measured as substitutions per site, i.e., 0.1 on the scale bar representing 4 substitutions in 100 bp. The evolutionary history was inferred from 1522 aligned characters. The GenBank accession numbers for each species or strain are shown in parenthesis  a The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome 47,363 position-specific scoring matrix (PSSM) domains curated in the NCBI and imported from Pfam [23], SMART [24], COG [25], PRK [26], and TIGR-FAM [27]. The e-value for BLAST and RPSBLAST search in a database was 1e-50 and 0.01, respectively. In addition, CRISPR regions were identified using the CRISPR Recognition Tool (CRT) [28]; tRNAs were identified by tRNAScan-SE-1.23 [29]; rRNAs were identified by RNAmmer [30]; signal peptides and transmembrane helices were predicted using SignalP [31] and TMHMM [32], respectively. According to BLASTN, 107 of the 657 contigs, accounting for 144,247 bp, only had multiple hits of mitochondrial genome sequences at e-10, suggesting they belong to the organelle genome of the pathogen.

Genome properties
The properties of the V. carpophila genome are summarized in Table 3. The draft genome sequence had 36,917,822 bp in 657 contigs, with a G + C content of 47.36%. The G + C content of the 550 contigs presumable for the nuclear genome is 47.43% and that of the 107 contigs likely belonging to the mitochondrial genome is 30.13%. Of the total 8220 predicted protein-coding genes, 6547 had hits in the nr database. In addition, 4632 had predicted functions, 2694 had Pfam domains, and 3665 were assigned to COGs. The distribution of genes into COG functional categories is presented in Table 4.

Insights from the genome sequence
The genome provides a useful resource for identifying genes of interest in V. carpophila. Furthermore, the phylogenetic analysis presented earlier confirms the relationship of V. carpophila to other members of the Venturiacae and confirms previous observations on the taxonomic relationships among these members of the Ascomycota. Based on the phylogenetic analysis using the sequence of the 18S rRNA gene (Fig. 2), V. carpophila is closely related to other scab-causing fungal pathogens of higher plants, including V. cerasi, causing scab on cherry, and also V. nashicola, cause of scab on Asian pear.

Extended insights
The genome of the peach scab fungus, V. carpophila, will be of great value to research on this organism moving forward. Apart from the opportunity it affords for more detailed analysis of individual genes relating to the secretome, we have identified several genes of interest and all microsatellites for further characterization. First, we have located the full open reading frame for one of the mating type idiomorph genes (MAT1-2) in scaffold_15 (between 199,706-200,916 bp) with two predicted introns (between 199,953-200,006 bp and between 200,318-200,365 bp), which paves the way for a better understanding of the reproduction of this fungus, particularly in the USA [1]. When fully characterized, the mating type idiomorphs provide the basis not only for a more complete understanding of the epidemiology of the pathogen, but also the potential opportunity for management of the disease through control of the sexual stage, as has been demonstrated with the closely related apple scab pathogen [33]. Secondly, we have also identified the cytochrome b gene in scaf-fold_347 (between 2149 and 7821 bp), which contains at least two introns. The accurate boundaries of the introns need further determination by PCR and sequencing of cDNA. This gene is particularly important in plant pathogenic fungi as it plays a critical role in resistance to the quinone outside inhibitor fungicides (also known as strobilurins). Understanding the structure of this gene will form the basis for assessing risk of resistance to the quinone outside inhibitors, as demonstrated in closely related pathogens [34]. Thirdly, we have identified a total of 4021 microsatellites in 322 of the 657 scaffolds. On average, there was about one microsatellite per 10 kb of genome. The tri-type microsatellite was the most abundant. The total length of the largest 101 scaffolds (0 to 100) is 31,436,433 bp, accounting for 85.15% of the total genome size and containing 80.10% of the microsatellites. The count and distribution of microsatellites on scaffold_0 to scaffold-100 are presented (Fig. 3 a and b). Scaffold 9 had the highest count of microsatellites (152), and it was also the longest scaffold. Microsatellites are basic to studying the genetic diversity and population structure of the pathogen, and also have potential to be used for tracking specific genes of interest.

Conclusions
The predicted genes may represent most functional genes in the V. carpophila genome and can be used as a new resource for developing molecular markers for genetic diversity studies, and for other research into the biology, ecology, taxonomy and phylogeny of the pathogen, and for research into host/pathogen coevolution.
the isolate and extracted the DNA. Each author read and approved the final version of the manuscript.