Original Research published: 03 December 2019 doi: 10.3389/fpls.2019.01583 Mapping Cucumber Vein Yellowing Virus Resistance in Cucumber (Cucumis sativus L.) by Using BSA- seq Analysis Marta Pujol 1,2, Konstantinos G. Alexiou 1,2, Anne-Sophie Fontaine 3, Patricia Mayor 4, Manuel Miras 4, Torben Jahrmann 3, Jordi Garcia-Mas 1,2 and Miguel A. Aranda 4* 1Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Plant and Animal Genomics Program, Barcelona, Spain, 2 Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Genomics and Biotecnology Program, Barcelona, Spain, 3 Semillas Fitó S.A., Biotechnology Department, Barcelona, Spain, 4 Centro de Edafología y Biología Aplicada del Segura (CEBAS)-CSIC, Departamento de Biología del Estrés y Patología Vegetal, Murcia, Spain Cucumber vein yellowing virus (CVYV) causes severe yield losses in cucurbit crops across Edited by: Mediterranean countries. The control of this virus is based on cultural practices to prevent Jaime Prohens, the presence of its vector (Bemisia tabaci) and breeding for natural resistance, which Polytechnic University of Valencia, requires the identification of the loci involved and the development of molecular markers Spain for linkage analysis. In this work, we mapped a monogenic locus for resistance to CVYV Reviewed by: Dirk Janssen, in cucumber by using a Bulked Segregant Analysis (BSA) strategy coupled with whole- IFAPA Centro La Mojonera, Spain genome resequencing. We phenotyped 135 F3 families from a segregating population Zhiyong Liu, Institute of Genetics and between a susceptible pickling cucumber and a resistant Long Dutch type cucumber Developmental Biology, China for CVYV resistance. Phenotypic analysis determined the monogenic and incomplete Xingfang Gu, dominance inheritance of the resistance. We named the locus CsCvy-1. For mapping this Chinese Academy of Agricultural Sciences, China locus, 15 resistant and 15 susceptible homozygous F2 individuals were selected for whole *Correspondence: genome resequencing. By using a customized bioinformatics pipeline, we identified a unique Miguel A. Aranda region in chromosome 5 associated to resistance to CVYV, explaining more than 80% of the m.aranda@cebas.csic.es variability. The resequencing data provided us with additional SNP markers to decrease the Specialty section: interval of CsCvy-1 to 625 kb, containing 24 annotated genes. Markers flanking CsCvy-1 in This article was submitted to a 5.3 cM interval were developed for marker-assisted selection (MAS) in breeding programs Plant Breeding, a section of the journal and will be useful for the identification of the target gene in future studies. Frontiers in Plant Science Keywords: Cucumber vein yellowing virus, cucumber, resistance, mapping, BSA-seq, breeding, Received: 04 July 2019 marker-assisted selection Accepted: 12 November 2019 Published: 03 December 2019 Citation: INTRODUCTION Pujol M, Alexiou KG, Fontaine A-S, Mayor P, Miras M, Jahrmann T, Cucumber vein yellowing virus (CVYV) is an ipomovirus (family Potyviridae) that is transmitted Garcia-Mas J and Aranda MA (2019) in a semi-persistent manner by the whitefly Bemisia tabaci. CVYV was first reported in Eastern Mapping Cucumber Vein Yellowing countries of the Mediterranean basin (Israel, Jordan, Turkey, and Cyprus) later expanding to Western Virus Resistance in Cucumber (Cucumis sativus L.) by Mediterranean countries including Spain, Portugal, France, and Tunisia (reviewed in Navas-Castillo Using BSA-seq Analysis. et al., 2014). CVYV infects cucurbits, causing symptoms of variable intensity. In melon (Cucumis Front. Plant Sci. 10:1583. melo L.) and cucumber (Cucumis sativus L.), it causes a typical severe vein clearing often followed by doi: 10.3389/fpls.2019.01583 generalized chlorosis and necrosis. In cucurbit-producing areas of heavy B. tabaci infestation, it can Frontiers in Plant Science | www.frontiersin.org 1 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq cause epidemics with massive yield losses and dramatic economic MATERIALS AND METHODS consequences. Although significant diversity has been reported for this virus, epidemics in Western Mediterranean countries Plant Material and Phenotyping for seem to be associated to genetically uniform virus populations CVYV Resistance (Desbiez et al., 2019), perhaps as a consequence of single virus Accession CE0754 (hereafter PS), a CVYV-susceptible pickling introductions followed by rapid epidemic expansions (Janssen et cucumber, was crossed with accession CE0749 (hereafter PR), a al., 2007). At the start of the CVYV epidemics, disease control CVYV-resistant Long Dutch type cucumber, to obtain the F1, F2 and relied heavily on early detection (Martínez-García et al., 2004) F2:3 segregating populations used to perform the genetic mapping and eradication, and whitefly control. Sources of resistance were of the resistance trait. PS, PR, F1, F2, and F2:3 plants were inoculated soon identified in cucumber (e.g. Picó et al., 2003) and indeed, mechanically with CVYV-AILM (Martínez-García et al., 2004) by commercial seed companies are currently selling cucumber rubbing recently-expanded cotyledons with extracts from CVYV- hybrids resistant to CVYV, which represent an excellent solution infected cucumber plants (cv. SMR-58) and were re-inoculated for disease control. Resistant accessions, varieties and hybrids three days after. To measure virus accumulation in PS, PR and seem to share the common characteristic that resistance is partial; F1-inoculated plants, we followed procedures described by Marco symptoms in inoculated plants are mild or absent, and the virus et al. (2003); quantitative dot-blot hybridization was done using can be detected infecting systemically the so-called resistant the probe described by (Martínez-García et al., 2004). Plants were plants, although at reduced levels as compared to susceptible sampled at 9, 16, 23, and 30 days post inoculation (dpi) taking controls (e.g. Galipienso et al., 2013). three leaf discs measuring 8 mm in diameter per leaf sampled. Breeding for CVYV resistance of cucumber varieties requires Symptoms were scored using a 0–3 scale: (0) No symptoms; (1) the development of molecular markers linked to the trait of interest, mild chlorotic mottling in young but fully-expanded leaves in as pathology tests are labor-intensive and time-consuming. For interveinal petiole-proximal leaf areas; (2) similar to (1) plus molecular breeding, several genetic and genomics resources are vein yellowing evident in fully-expanded leaves and incipient in available for cucumber, which have substantially increased after young developing leaves; (3) obvious vein yellowing in all leaves, the release of reference genomes (Huang et al., 2009; Wóycicki including young developing leaves, chlorotic mosaics in fully- et al., 2011; Yang et al., 2012). Cucumber has a relatively small expanded leaves and overall plant growth reduction. A minimum genome (367 Mb, 2n = 2x = 14) with a very narrow genetic of nine plants were used per treatment. Plants were kept in an base (Staub et al., 2008; Wóycicki et al., 2011). The availability insect-proof glasshouse, with temperature control set at 26°C/18°C of high-density consensus maps and whole genome sequencing (day/night) throughout the experiments. has facilitated the identification of NB-LRR resistance genes in the cucumber genome and the map-based cloning of candidate genes (Ren et al., 2009; Yang et al., 2013). These achievements DNA Extraction and NGS Sequencing Young leaves from the parental lines and the F2 population are the basis for efficient marker-assisted selection (MAS). In this were collected, frozen in liquid nitrogen, and stored at −80°C. sense, Bulked Segregant Analysis (BSA) was developed as a rapid DNA was extracted following the CTAB method (Doyle, 1991), method for the detection of molecular markers linked to target adding a purification step using Phenol : Chloroform:Isoamyl traits in mapping populations (Michelmore et al., 1991). The alcohol (25:24:1). The integrity of DNA was evaluated by principle of BSA is the selection of a small group of individuals agarose gel electrophoresis and quantified with the PicoGreen® from a segregating population that belong to phenotypic dsDNA Assay Kit (Life Technologies) according to the contrasting extremes of the target trait. These individuals are manufacturer’s protocol. For NGS sequencing, we pooled then pooled in two bulks, and fingerprinted to obtain genetic equimolar concentrations of DNA from 15 homozygous polymorphisms. When the trait is monogenic, the number of resistant F2 plants (R-Bulk), and from 15 homozygous individuals per bulk can be reduced to 10-20, but in the case of susceptible F2 plants (S-Bulk). Twenty µg aliquots of each quantitative trait loci (QTL) this number should be increased bulked DNA and both parental lines were sent to the National (Sun et al., 2010; Takagi et al., 2013; Zou et al., 2016). With Centre for Genomic Analysis (CNAG-CRG, Barcelona, Spain) the improvement of technologies and the significant reduction for library construction and sequencing. Libraries of 300 bp and of next generation sequencing (NGS) costs, whole-genome 500 bp average insert size for bulks and parents, respectively, resequencing has been coupled to BSA. The combination of BSA were sequenced with Illumina HiSeq 2000 (Illumina, Inc. San with NGS (BSA-seq) has accelerated the identification of tightly Diego, CA, USA), generating 2 × 100 bp paired-end reads for linked markers for important traits, improving the resolution of both datasets. maps for gene identification and QTL mapping (Zou et al., 2016). In cucumber, BSA-seq has been successfully applied for mapping traits such as early flowering (Lu et al., 2014), flesh thickness (Xu Conventional Linkage Mapping et al., 2015) and downy mildew resistance (Win et al., 2017). A set of 172 polymorphic cucumber SNP markers, distributed across The aims of this work were to study the inheritance of the the seven chromosomes of the cucumber genome, were selected resistance conferred by the resistant accession CE0749 in a between PR and PS from the resequencing data (Supplementary segregating F2:3 population, to map the CsCvy-1 locus by using Table 1). Kompetitive Allele Specific PCR (KASP, https://www. a BSA-seq approach, and to develop molecular markers that are lgcgroup.com) was used for genotyping a subset of 72 individuals, easily transferable to cucumber breeding programs. with the 172 SNPs converted to KASP markers, following the Frontiers in Plant Science | www.frontiersin.org 2 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq protocol of LGC Genomics. A genetic map was constructed using “runQTLseqAnalysis”. Briefly, SNP-index was calculated for both JoinMap® 5 (Kyazma, B.V.), with 172 SNP markers data and the S- and R-bulk by dividing the number of non-reference alleles phenotypic data used as another marker (CsCvy-1) due to the with the total number of reads in a position. If the SNP-index was monogenic inheritance of the trait. Three F2 individuals and one <0.3 in both bulks, the SNP was discarded. SNP-index values were SNP marker were excluded because of the high amount of missing calculated in sliding windows of 4.7 Mbp with a 10 kb step and the data. Two more SNP markers were excluded for being identical average SNP-index value for each window was recorded along the (similarity value = 1.000) to other nearby markers. Linkage analysis chromosome. In order to avoid regions that generate segregation and marker order were performed with the regression mapping distortion caused by other reasons other than artificial selection algorithm, and genetic distance was calculated using the Kosambi (Takagi et al., 2013), the SNP-index of the S-bulk was subtracted mapping function (Crow, 1990). QTL analysis was performed by the SNP-index of the R-bulk in order to obtain the Δ(SNP- using MapQTL6® (Kyazma B.V.) using both interval mapping and index). If Δ(SNP-index) equaled to 1.0 then the allele originated Kruskal-Wallis (KW) analysis. from the R parent and if Δ(SNP-index) equaled to -1 the allele originated from the S parent. Confidence interval calculations in each SNP position (95% and 99%), considering the null hypothesis Variant Detection and BSA-seq Analysis (Ho: no QTL), was done through a simulation analysis of 10,000 Variant Detection and Functional Effect Annotation replications for two bulks that were randomly generated from the In order to detect variants of the genome linked to the resistance population and calculating, after each iteration, the SNP-index and against CVYV, a BSA-seq strategy was implemented in which the corresponding Δ(SNP-index) in the two simulated bulks. two pools of F2 individuals were chosen depending on their phenotype. 15 homozygous resistant F2 individuals (R-bulk) and 15 homozygous susceptible F2 individuals (S-bulk) were RESULTS selected for pooling and sequencing. Paired-end Illumina sequencing data from parental lines and the two bulks were Phenotyping Analyses trimmed (length ≥35 bp, with a mean sliding window of 4 bp We first analyzed symptom development and virus accumulation phred quality score ≥20) using Trimmomatic (Bolger et al., in PS, PR, and F1 plants in a time course experiment (Figure 1). 2014) and the output was quality checked using FastQC Symptoms started to appear in inoculated PS plants as soon as 6 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). dpi (data not shown) and were conspicuous by 9 dpi, with plants Trimmed data were aligned versus the ChineseLong 9930 v3 showing obvious vein yellowing in all leaves and chlorotic mosaic assembly ftp://cucurbitgenomics.org/pub/cucurbit/genome/ in fully-expanded leaves (Figure 1A). Symptom display was cucumber/Chinese_long/v3/) using the BWA-MEM algorithm delayed, and symptoms were clearly milder in PR-infected plants, (v0.7.16a-r1181; http://bio-bwa.sourceforge.net/bwa.shtml) although by 9 dpi all the plants showed mild interveinal chlorotic with default parameters. After removal of unmapped reads mottling in petiole-proximal leaf areas of fully expanded leaves and marking of PCR duplicates, variant calling was performed (Figure 1A). Symptoms in F1 plants appeared at around 7 dpi, with samtools (v1.5; Li, 2011) using default parameters, except and by 9 dpi they were of intermediate severity between those for the following: mapping quality ≥10 and base quality ≥20. of PS- and PR-infected plants; fully-expanded leaves showed Variant calling format (VCF) files were filtered by applying the chlorotic mottling but also mild vein yellowing, which was following criteria: genotype quality ≥10, depth ≥10, biallelic starting in young developing leaves (Figure 1A). The uniformity sites, alternative allele frequency ≤ 0.9, no missing data. of symptoms in plants from each accession was remarkable, and Structural variant (SV) analysis between the two parents was thus each accession could be assigned to a symptom severity conducted using DELLY (Rausch et al., 2012) and Pindel (Ye class without uncertainty for each observation time-point. Plants et al., 2009). Both programs were run with default parameters. in this experiment were sampled and virus accumulation was Raw data underwent technical- and visual-based filtering. The measured, differentiating among samples from basal, medium technical filters applied were the following: read depth ≥10 in and apical leaves (Figure 1B). Leaves from PR-infected plants at least one sample, parents variable between them, variant size accumulated significantly less virus than PS-infected leaves larger than 50 bp and smaller than 50,000 bp. Remained variants at all time points, except at 23 dpi in basal leaves, where no were visually inspected in IGV (Robinson et al., 2011) to avoid significant differences were found between both accessions. cases of false positives. For F1-infected plants, differences in virus accumulation in Annotation of the functional effect of the variants was done leaves were less consistent, with an apparent trend suggesting using snpEff (version 4.3t; (Cingolani et al., 2012). intermediate levels of accumulation between those of PR and PS; statistically significant differences (P < 0.027; Kruskal-Wallis BSA-seq Analysis tests) were found among the three accessions at 16 dpi in basal BSA-seq analysis was performed using the R package QTLseqr leaves and at 23 and 30 dpi in intermediate leaves (Figure 1B). (Mansfeld and Grumet, 2018). More specifically, SNPs for the 2 Thus, virus accumulation data was essentially consistent with bulks were first filtered using the function “filterSNPs”, by keeping data on symptom expression, and phenotyping of F1 individuals positions with 30 ≤ total depth ≤ 150, 0.2 ≤ reference allele frequency suggested incomplete dominance of the resistance trait. In any ≤0.8 and genotype quality ≥30. For the QTL detection, the (Takagi case, symptom scoring at 9 dpi appeared to be robust enough to et al., 2013) method was applied, implemented by the function discriminate between susceptible and resistant plants. Frontiers in Plant Science | www.frontiersin.org 3 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq FIGURE 1 | Phenotyping plants of the susceptible (PS) and resistant (PR) parental lines, and their F1, for CVYV susceptibility. Symptoms were scored and virus accumulation was measured at 9, 16, 23 and 30 days post-inoculation (dpi) (A) Symptoms in plants at 9 dpi. Symptoms could be assigned unequivocally to one of the following categories: (0) No symptoms; (1) mild chlorotic mottling in young but fully expanded leaves in interveinal petiole-proximal leaf areas; (2) similar to (1) plus vein yellowing evident in fully expanded leaves and incipient in young developing leaves; (3) obvious vein yellowing in all leaves, including young developing leaves, chlorotic mosaics in fully expanded leaves and overall plant growth reduction. (B) Virus accumulation was measured by quantitative dot-blot hybridization on total plant RNA extracts from basal, intermediate and apical leaves, as indicated for each graph. Three leaf discs (8 mm) were taken per leaf sampled. Virus accumulation in F1 plants was intermediate between the susceptible and resistant parental lines in basal and intermediate leaves at 16, 23 and 30 dpi, respectively; an asterisk marks statistically significant differences in Kruskal-Wallis tests (P < 0.027). A minimum of 9 plants were used per treatment. Symptom category (as in (A)) is indicated for each line on the right side of each graph for each time period after inoculation. To determine the resistance genotype of F2 individuals, 12 2, Supplementary Table 2). The average marker interval was 3.9 individuals from each of 137 F2:3 families were inoculated and cM, with a maximum distance of 23.6 cM. The longest LG was symptom scoring performed at 9 dpi (Supplementary Figure 1). LG4, with 115.1 cM, and the shortest was LG3A with 14 cM. The Out of these, 135 families were unequivocally assigned to CsCvy-1 locus was mapped onto LG05, flanked by two markers, susceptible, resistant or segregating for resistance phenotypes CVYV_121 and CVYV_122 in an interval of 5.3 cM (Figure 2A). and, therefore, the 135 F2 individuals could be classified as: 37 In order to discard any other minor QTL involved in the homozygous for resistance, 36 homozygous for susceptibility and resistance trait, a QTL analysis was performed. As expected, we 62 heterozygous, which fit well (χ2 value 0.63, P > 0.05) with a obtained a single major QTL on LG05, co-localizing with CsCvy- 1:2:1 segregation ratio. We propose the symbol CsCvy-1 for this 1, with a LOD value of 49 explaining 95.8% of the variance monogenic incompletely-dominant resistance gene. For the BSA- (Figure 2B). No other significant QTLs were observed, in seq analysis, two pools containing 15 F2 individuals homozygous accordance to the monogenic inheritance of the trait. for resistance and susceptibility to CVYV, respectively, were used. Identification of CsCvy-1 Locus by Preliminary F2 Mapping BSA-seq In parallel with the BSA-seq method, we performed F2 mapping Libraries of parental lines (PS and PR), S-bulk and R-bulk were with a subset of the population including the CsCvy-1 locus as a resequenced with the Illumina HiSeq2000 sequencer. In total, phenotypic marker. We used 172 polymorphic SNPs covering the 274,709,874 paired-end clean reads were mapped, after trimming cucumber genome: 24 SNPs in Chr01, 18 SNPs in Chr02, 34 SNPs and adapter removal (Table 1). in Chr03, 36 SNPs in Chr04, 21 SNPs in Chr05, 27 SNPs in Chr06, Small variant calling for the 4 datasets and subsequent variant and 12 SNPs in Chr07 (Supplementary Table 1). We selected filtering (see Material and Methods), generated 186,433; 215,735; 72 F2 individuals (35 homozygous resistant, 34 homozygous 933,846 and 915,524 variants (SNPs and INDELs) for PS, PR, susceptible, 2 heterozygous, and 1 individual without phenotypic S-bulk and R-bulk, respectively, uniformly distributed throughout data) for mapping. The 72 F2 individuals were genotyped, and the genome (Supplementary Table 3). SNP variants from the 2 most of the markers fitted the expected 1:2:1 segregation ratio. bulk datasets were used as input data in the QTLseqr R package However, a group of markers on Chr05 showed a distorted for calculating SNP-index and Δ(SNP-index) for R- and S-bulks, segregation. This distortion was also found for the phenotypic marker based on the Takagi et al., 2013 method. Graphs for SNP-index of CsCvy-1, due to the selection of individuals, with almost all of the R- and S-bulks and the Δ(SNP-index) were plotted (Figure 3). them being homozygous for this trait. The genetic map consisted S-bulk was used as a reference dataset for the calculation of the of eight linkage groups (LGs) spanning 628 cM, with Chr03 split Δ(SNP-index). BSA-seq analysis detected a single genomic region into two linkage groups (LG3A, LG3B) (Supplementary Figure of 2,998,622 bp located in Chr05:5,088,092-8,208,448 bp, at a Frontiers in Plant Science | www.frontiersin.org 4 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq FIGURE 2 | (A) Genetic linkage of SNP markers and the resistance locus CsCvy-1 in Chr05. Genetic distance is expressed as centiMorgans (cM). The CsCvy-1 gene is flanked by two markers in an interval of 5.3 cM. (B) Major QTL detected on Chr05 corresponding to CsCvy-1 locus. TABLE 1 | Results from the resequencing of PS, PR, S-bulk and R-bulk. recombinant informative plants delimited the candidate area of CsCvy-1 to a 626.5 kb interval, between the markers CVYV_181 Samples Clean mapped Mapping rate Median depth Coverage (%)3 reads1 (%)2 and CVYV_122 (Figure 4C). This interval contains 24 annotated genes (Table 2). To further explore the genomic region containing PS 56,694,486 96 20 X 89 the CsCvy-1 locus, we extended our previous small variant analysis PR 60,357,172 96 22 X 89 with the detection of SVs between the parental lines. In total, we S-bulk 77,718,714 95 30 X 90 R-bulk 79,939,502 96 31 X 90 detected 2 SVs of more than 50 bp in length: one 55 bp deletion and a duplication measuring 41,644 bp (Supplementary Table 4). 1Number of reads after trimming and adapter removal. With the purpose of detecting alterations in potential candidate 2Alignment to the Chinese Long 9930 genome assembly v3 (www.cucurbitgenomics.org) 3Coverage (≥ 1 read). genes that could be responsible for the resistance phenotype, we performed an annotation of the effect of small variations in the 24 genes found in the CsCyv-1 region. In total, we annotated 45 SNPs/ confidence interval higher than 0.99, associated with the CsCvy-1 INDELs, of which 17 were highlighted for being within or close locus. The highest Δ(SNP-index) value was -0.528 at position to coding sequences of 10 genes (Supplementary Table 5). The 7,678,525 bp whereas the average Δ(SNP-index) was found to be vast majority of variations (14 out of 17) were non-synonymous, -0.523 (Figure 4A). This region contained the flanking markers and were annotated as modifiers or with low impact. Among selected in the preliminary mapping. the remaining four variants, three SNPs caused synonymous changes, and one insertion of 2 nucleotides caused a frameshift resulting in a premature stop codon (Supplementary Table 5). Fine Mapping of CsCvy-1 Locus The three missense variants (moderate impact) were located in In order to narrow down the interval of the CsCvy-1 locus, the coding sequence of CsaV3_5G011200, CsaV3_5G011220 fine mapping was performed using the linked SNPs obtained and CsaV3_5G011240 genes, whereas the frameshift variant by BSA-seq on Chr05 to design 19 SNP markers (Figure 4B, (high impact) was located in CsaV3_5G11180 gene. The Supplementary Table 1). A subset of the F2 population was frameshift caused the disruption of the CsaV3_5G11180 gene, genotyped to identify plants with recombination events, by coding for a serine/arginine repetitive matrix protein (SARMP) using the genotyping data of the preliminary mapping. Eleven 2-like, at position 607 (Supplementary Figure 3). A search for Frontiers in Plant Science | www.frontiersin.org 5 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq FIGURE 3 | SNP-index and Δ(SNP-index) distribution (blue line). SNP-index for R-bulk (A) and S-bulk (B) was calculated using sliding windows of 4.7 Mbp in length with a step measuring 10 kb. The corresponding Δ(SNP-index) (C) was calculated as the difference SNP-index R-bulk – SNP-index S-bulk. Regions of statistical significance are detected as those that surpass the threshold of 0.95 (orange line) or 0.99 (green line) confidence intervals. TABLE 2 | List of genes within the interval of CsCVY-1 locus in the Chinese Long conserved motifs detected a Constitutive Photomorphogenic 1 9930 v3 cucumber genome annotation, variants and structural variation analysis (COP1)-interacting protein signature. With regards to structural between PS and PR. variations, we also detected a single duplication event in PR Gene_ID Description Chinese Long v3 Small measuring 41,644 bp in length located in Chr05:7,195,565- variants1 7,237,209 that contained genes CsaV3_5G011170 to CsaV3_5G011220 (Table 2 and Supplementary Figure 4). Genes CsaV3_5G011170* Unknown protein – CsaV3_5G011180* Serine/arginine repetitive matrix protein 2-like INDEL CsaV3_5G011170 and CsaV3_5G011190 encode for unknown isoform X1 proteins, CsaV3_5G11180 encodes the above-mentioned CsaV3_5G011190* Unknown protein – SARMP and genes CsaV3_5G011200 and CsaV3_5G011210 CsaV3_5G011200* RNA-dependent RNA polymerase SNP encode two RNA-dependent RNA polymerases (RDRs) 1a and CsaV3_5G011210* RNA-dependent RNA polymerase SNP CsaV3_5G011220* Endo-1,4-beta-xylanase A-like SNP 1b, respectively. In contrast, the deletion of 55 bp was located in CsaV3_5G011230 Unknown protein SNP an intergenic region. CsaV3_5G011240 SAM dependent carboxyl methyltransferase SNP CsaV3_5G011250 Unknown protein SNP CsaV3_5G011260 SAM dependent carboxyl methyltransferase SNP CsaV3_5G011270 Unknown protein – DISCUSSION CsaV3_5G011280 Integrator complex subunit 9 like SNP CsaV3_5G011290 Cytochrome c – In cucumber, the disease caused by CVYV is a limiting factor CsaV3_5G011300 Serine/threonine protein kinase SNP for production in areas with high pressure from viruliferous CsaV3_5G012300 40S ribosomal protein S6 – whiteflies. In this work, we phenotyped an F2:3 segregating CsaV3_5G012310 Myb family transcription factor – population identifying a single monogenic locus, CsCvy-1, CsaV3_5G012320 Protein kinase, putative – CsaV3_5G012330 E3 ubiquitin-protein ligase ORTHRUS 2-like – that controls resistance to CVYV. The resistance conferred by CsaV3_5G012340 THUMP domain-containing protein 1 – this locus is partial, as PR plants showed viral accumulation in CsaV3_5G012350 Amino acid transporter – systemically infected leaves. However, the progression of the CsaV3_5G012360 Unknown protein – disease was very much reduced as compared to susceptible CsaV3_5G012370 Perakine reductase – CsaV3_5G012380 UBN2_3 domain-containing protein – controls, the growth of the plants was not affected at all, and CsaV3_5G012390 SWI/SNF-related matrix-associated actin- – viral accumulation was significantly reduced as compared to PS dependent regulator of chromatin subfamily and F1 plants. In cucumber, the accession C.sat-10 (Picó et al., A containing DEAD/H box 1 2003) was described as having partial resistance against CVYV, 1Small variants are referred as SNPs or INDELs < 50 bp. and a segregating F2 population was obtained to study the genetic *These genes are inside the duplicated region of 41,644 bp in PR. control of this resistance (Picó et al., 2008); the segregation fitted Frontiers in Plant Science | www.frontiersin.org 6 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq FIGURE 4 | Scheme of fine mapping of the CsCvy-1 locus. (A) DeltaSNP graph on Chr05 with the statistical confidence interval of 0.95 (orange line) or 0.99 (green line) (B) Physical map of Chr05 with all the markers used for mapping the CsCvy-1 locus: preliminary mapping (black), flanking markers (magenta), fine mapping markers (blue). Distance is expressed in Mb on the bottom of the bar. (C) Genotyping result of recombinant individuals with the markers covering CsCvy-1 locus. Alleles are represented in three colors: black for homozygous PR allele, dark grey for heterozygous PR/PS, and light grey for homozygous PS allele. a monogenic control with dominance. These features are very tolerance in HSD 2458, and Cvy-3, showing dominance for similar to what we have described here; however, in the case severe mosaic symptoms in Ouzbèque 2. of C.sat-10, no mapping studies were performed to determine Breeding for resistance requires the availability of highly linked the localization of the locus in the cucumber genome. Thus, markers that can be utilized for performing rapid and specific although our resistance data fit well with those from Picó et al. introgressions of the desired trait. For marker development, (2003; 2008), we cannot rule out that both resistances to CVYV conventional gene mapping is based on the phenotyping and characterized in cucumber are independent. By comparing with genotyping of a large number of individuals in a population, and closely related species such as melon, Pitrat et al. (2012) evaluated it is time-consuming, costly and limiting in terms of the size of the a collection of 1,188 accessions for resistance against CVYV, and population (Takagi et al., 2013). One way to improve conventional studied the inheritance in F1, F2 and BC progenies. Three loci mapping is to use BSA-seq, in which the number of individuals to were detected in their work: Cvy-1, controlling resistance in PI be analyzed can be narrowed down to two representative bulks, 164323 and necrosis in HSD 93-20-A; cvy-2, showing recessive and at the same time as the mapping, the resequencing data offers Frontiers in Plant Science | www.frontiersin.org 7 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq the possibility of obtaining a high number of markers linked to enhanced antiviral activity in the PR line as a consequence of the trait (Yang et al., 2013). BSA-seq has been successfully applied enhanced RDR1a and/or 1b expression; this, in turn, would for the mapping of important agronomical traits in many crops be the consequence of the described gene duplication. This is such as rice (Abe et al., 2012; Takagi et al., 2013; Yang et al., 2013; an attractive hypothesis that awaits further testing, although at Sun et al., 2018), lettuce (Huo et al., 2016), potato (Kaminski least a good alternative candidate gene (i.e., the gene encoding a et al., 2016), soybean (Song et al., 2017), broccoli (Shu et al., 2018; SARMP2-like protein) exists. From the point of view of resistance Branham and Farnham, 2019) or sorghum (Han et al., 2015). stability when confronted to different CVYV strains (Desbiez Moreover, in cucurbits BSA-seq has enabled the identification et al., 2019), the potential implication of RDR1a/b represents of candidate genes for dwarfism (Dong et al., 2018), yellow skin an optimistic perspective, given its broad spectrum of action (Dou et al., 2018) and light rind color (Oren et al., 2019) in (Leibman et al., 2018). watermelon; mapping flavor traits in melon (Zhang et al., 2016); or In conclusion, in this work we identified the monogenic locus the identification of candidate genes for flesh thickness (Xu et al., CsCvy-1, inherited under incomplete dominance, in a short 2015), aphid resistance (Liang et al., 2016), early flowering QTL interval of 5.3 cM containing 24 genes. This is the first report (Lu et al., 2014), two major QTLs for downy mildew resistance where a CVYV resistance locus has been mapped in cucumber, (Win et al., 2017), and three major QTLs conferring subgynoecy and valuable molecular markers for MAS breeding programs (Win et al., 2019) in cucumber. In the present study, through the have been developed. Moreover, our findings will be the basis use of a BSA-seq strategy, the CsCvy-1 locus has been successfully for further map-based cloning and functional validation of the mapped to a region of 2.9 Mb in chromosome 5, whereas no other resistance gene. regions of the genome exhibited significant association with the resistance. These results confirmed previous analysis performed with the conventional mapping approach and were later used for DATA AVAILABILITY STATEMENT fine mapping. The BSA-seq analysis provided enough SNPs to fine map the trait within a narrow interval of 625 kb, containing only The raw data supporting the conclusions of this manuscript will 24 annotated genes. be made available by the authors, without undue reservation, In order to identify candidate genes for CsCvy-1, we to any qualified researcher. Variation data were deposited to performed an analysis of small variants and structural variation European Variation Archive (EVA: https://www.ebi.ac.uk/eva) around this locus. Most of the variations had a small, if any, with the project accession number PRJEB34274. predicted impact, except for the insertion of 2 nucleotides in the gene encoding a SARMP2-like, which causes a frameshift mutation in the coding sequence with the subsequent truncation AUTHOR CONTRIBUTIONS of the protein. A functional analysis of the SARMP-like protein MA, JG-M, TJ, and MP conceived and designed the research. sequence detected a COP1-interacting protein signature. PM, MM and MA provided the plant material and performed Interestingly, COP1 has been associated with plant pathogen the tests with CVYV. A-SF conducted the conventional mapping. resistance (Lim et al., 2018) and the Arabidopsis thaliana COP1- KA performed the bioinformatics analysis of the BSA-seq. MP interacting protein is a positive regulator of ABA response (Ren conducted marker development, mapping analysis, and wrote et al., 2016), which is an essential regulator of plant immunity the manuscript with important contributions from MA and KA. (Berens et al., 2017). Nevertheless, perhaps the most appealing All authors read and approved the final manuscript. modification within the CsCvy-1 region is the duplication of the 41 Kb fragment containing the genes encoding RDRs 1a and 1b (CsaV3_5G011200 and CsaV3_5G011210). RDRs are critical ACKNOWLEDGMENTS players in RNA silencing pathways; they are the key enzymes in the process of amplification of double-stranded RNAs that The authors would like to thank Vanessa Alfaro and Daniel activate gene silencing after nuclease processing. A role for RDRs Alonso for their technical assistance. This work was supported by in antiviral immunity has long been acknowledged, in particular Semillas Fitó S.A., and by the CERCA Programme/Generalitat de for members of the RDR1 and RDR6 clades (Willmann et al., Catalunya. The CRAG and CEBAS acknowledge financial support 2012). For instance, the absence of a functional RDR1 in from the Spanish Ministry of Economy and Competitiveness, Nicotiana benthamiana can explain enhanced susceptibility to through the “Severo Ochoa Programme for Centres of Excellence many viruses in this species (Yang et al., 2004). In relation to virus in R&D” 2016–2019 (SEV-2015-0533)” and grant AGL2015- resistance in crop species, it has been recently demonstrated that 72804-EXP, respectively. the tomato genes Ty-1 and Ty-3 for resistance to tomato yellow leaf curl virus are alleles of an RDR gene (Verlaan et al., 2013). In cucumber, a recent report shows that the RDR1a and 1b genes SUPPLEMENTARY MATERIAL have enhanced expression in natural or engineered lines showing broad virus resistance; importantly, one of the viruses tested The Supplementary Material for this article can be found online was CVYV (Leibman et al., 2018). Taking these data together, at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01583/ a mechanistic explanation for our observations may consist of full#supplementary-material Frontiers in Plant Science | www.frontiersin.org 8 December 2019 | Volume 10 | Article 1583 Pujol et al. Mapping CVYV Resistance by BSA-seq REFERENCES resistance via double-stranded RNA binding proteins. 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A natural variant of a host RNA-dependent RNA polymerase is associated with Copyright © 2019 Pujol, Alexiou, Fontaine, Mayor, Miras, Jahrmann, increased susceptibility to viruses by Nicotiana benthamiana. Proc. Natl. Acad. Garcia-Mas and Aranda. This is an open-access article distributed under the Sci. 101, 6297–6302. doi: 10.1073/pnas.0304346101 terms of the Creative Commons Attribution License (CC BY). The use, distribution Yang, L., Koo, D. H., Li, Y., Zhang, X., Luan, F., Havey, M. J., et al. (2012). or reproduction in other forums is permitted, provided the original author(s) Chromosome rearrangements during domestication of cucumber as revealed and the copyright owner(s) are credited and that the original publication in by high-density genetic mapping and draft genome assembly. Plant J. 71, 895– this journal is cited, in accordance with accepted academic practice. No use, 906. doi: 10.1111/j.1365-313X.2012.05017.x distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 10 December 2019 | Volume 10 | Article 1583