Abstract
Cucumber (Cucumis sativus) production is negatively affected by Zucchini yellow mosaic virus (ZYMV). Three sources of ZYMV resistance have been commercially deployed and all three resistances are conditioned by a single recessive gene. A vacuolar protein sorting–associated protein 4-like (VPS4-like) gene has been proposed as a candidate for ZYMV resistance from cucumber line A192-18. We analyzed the genomic region across the VPS4-like gene for three independent sources of ZYMV resistance in cucumber (A192-18, Dina-1, and TMG-1) and identified three haplotypes across the coding region and considerable variation in the introns. However, the haplotypes in the coding regions of the VPS4-like gene of A192-18, Dina-1, and TMG-1 encode the same protein sequence, revealing the genetic uniformity for ZYMV resistance from diverse germplasm sources.
The potyviruses (Potyviridae) are especially destructive for cucumber (Cucumis sativus L.) and production is often negatively affected by Watermelon mosaic virus (WMV), the watermelon strain of the Papaya ringspot virus (PRSV-W), and ZYMV. ZYMV is one of the most devastating and economically important virus diseases of cucumber worldwide (Desbiez and Lecoq, 1997; Lecoq et al., 1983; Provvidenti et al., 1984a), and several strains have been differentiated by serological and molecular approaches (Amano et al., 2013; Mahgoub et al., 1997; Nováková et al., 2014; Provvidenti et al., 1984a, 1984b). Resistance to ZYMV has been reported in cucumber. Provvidenti (1987) and Provvidenti et al. (1984a) identified a single recessive gene (zymTMG-1) in a selection (TMG-1) from the Chinese cucumber ‘Taichung Mou Gua’, which has been widely used because it conditioned resistance to all of 173 ZYMV isolates (Lecoq et al., 2002) as well as other potyviruses such as PRSV-W, WMV (Kabelka and Grumet, 1997; Provvidenti, 1985), and Zucchini yellow fleck virus (Gilbert-Albertini et al., 1995). The commercial cucumber cultivar Dina-1 (Abul Hayja and Al-Shahwan, 1991; Al-Shahwan et al., 1995) possesses ZYMV resistance that is recessively inherited (zymDina-1) and is allelic or closely linked to zymTMG-1 (Kabelka et al., 1997). A Japanese slicing cucumber from Saitama Gensyu Ikuseikai Seed Company (A192-18) derived its ZYMV resistance from line G22 and is also inherited as a single recessive gene zymA192-18 (Amano et al., 2013; Svoboda et al., 2013). Subtle phenotypic differences have been observed for these sources of ZYMV resistance in cucumber. Resistance conditioned by the zymTMG-1 and zymDina-1 alleles constrains systemic movement of the virus through the plant (Ullah and Grumet, 2002). Al-Shahwan et al. (1995) and Provvidenti (1987) reported that ZYMV resistances from TMG-1 and Dina-1 occasionally developed a few chlorotic veins on the lower leaves. Kabelka et al. (1997) found that inoculating cotyledons of Dina-1 resulted in interveinal chlorosis on lower leaves; whereas TMG-1 had no symptoms regardless of inoculant placement. Svoboda et al. (2013) reported A192-18, G22, and TMG-1 as immune to ZYMV-H because no virus was detected by using enzyme-linked immunosorbent assay and electron microscopy.
Amano et al. (2013) proposed a VPS4-like gene as the candidate for zymA192-18, which may affect virus movement throughout the plant and it is the first example of association between a vacuolar sorting protein and a recessively inherited virus resistance in plants. In this research, we analyzed sequences of the VPS4-like gene from three sources of ZYMV resistance and two susceptible cucumbers, and revealed that in spite of different haplotypes, all three sources of ZYMV resistance encode the same protein sequence.
Materials and Methods
DNA was isolated from cucumber accessions ZYMV-resistant TMG-1 (Park et al., 2000; Provvidenti, 1987; Provvidenti et al., 1984a) and Dina-1 (Abul Hayja and Al-Shahwan, 1991), ZYMV-susceptible ‘Straight 8’ (St 8), and 49 F6 recombinant inbred lines (RILs) from a cross of TMG-1 and St 8 (Park et al., 2000) from bulked leaf tissue from at least five plants using the DNeasy® Plant Maxi Kit protocol (Qiagen, Valencia, CA). We genotyped the candidate gene for zym in St 8, TMG-1, and the 49 RILs using the cleaved amplified polymorphic (dCAPS-G99A) marker developed by Amano et al. (2013). Digested amplicons were visualized after running through 8% polyacrylamide gels and silver stained. Then, we compared the mean disease severity index scores of RILs (Park et al., 2000) carrying the resistant or susceptible genotype at the CAPS marker using a linear model in analysis of variance in RStudio (2016) version 1.0.136.
Single-end, 100 base pair (bp) sequencing runs for St 8 and TMG-1 were performed on the HiSeq2000 sequencer, version 1, SBS chemistry (Illumina, San Diego, CA). Raw reads were filtered for base quality ≥28 and length ≥41 nucleotides. Reads were mapped to scaffold03904 on chromosome 6 of the Gy 14 reference (Cavagnaro et al., 2010) with Bowtie2, version 2.1.0, (Langmead and Salzberg, 2012) and converted to sorted bam format with Samtools version 0.1.18 (Li, 2011).
Polymorphisms relative to the reference were visualized with Integrative Genome Viewer, version 2.3 (Robinson et al., 2011; Thorvaldsdottir et al., 2013). Sequences of St 8 [Sequence Read Archive (SRA) SRX1091587], TMG-1 (SRA SRX1091638), A192-18 (AB819729), and Dina-1 (KY775581) were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/) and translated into amino acid (AA) sequences. Alignments of the coding and AA sequences of Gy 14 (Cavagnaro et al., 2010), St 8, TMG-1, A192-18, and Dina-1 were performed using CLUSTAL O, version 1.2.4 (Li et al., 2015).
The polymerase chain reaction (PCR) protocol used to amplify the ≈5-kb genomic region from Dina-1 carrying the candidate gene for zym consisted of two separately prepared solutions, one which had the template DNA and the other with Taq polymerase. For a single reaction, solution 1 contained 4 µL of 2.5 mm dNTPs, 2 µL of combined forward and reverse primers (5′-TTCTTTGCTTGATTTGAGATAGTCAAAG and 5′-CAACTCTGATGGGTTGTATGCA) (20 µm/L), 17 µL of double-distilled (dd) H2O, and 2 µL of 20 ng/µL template DNA. Solution 2 had 19.25 µL of dd H2O, 5 µL of 10× Expand High Fidelity Buffer with 15 mm MgCl2, and 0.75 µL of Expand High Fidelity Enzyme Mix (Roche, Madison, WI). The two mixes were combined into a single tube with PCR conditions of 94 °C for 2 min, 30 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 4.2 min, and 72 °C for 7 min. Amplicons were visualized in 0.6% agarose gels and remaining reaction was purified to a concentration of 50 ng/µL using the NucleoSpin® PCR clean-up kit (Macherey-Nagel, Düren, Germany). Amplicons were Sanger sequenced using forward primers (Supplemental Table 1) and the reverse primer listed previously by Functional Biosciences (Madison, WI), and reads were analyzed using SnapGene software version 3.3.3 (GSL Biotech, Chicago, IL).
Results and Discussion
The dCAPS-G99A marker (Amano et al., 2013) was significantly (P = 2.2 × 10−16) associated with reactions to ZYMV across the RILs from St 8 × TMG-1 (Park et al., 2000), indicating that zymTMG-1 and zymA192-18 are alleles at the same locus or tightly linked loci. Kabelka et al. (1997) previously reported that zymDina-1 and zymTMG-1 are also allelic or very closely linked. Together these results indicate that these three independent sources of ZYMV resistance may be one in the same.
Amano et al. (2013) reported that the candidate VPS4-like gene has eight exons and seven introns. We conducted a BLAST search using the Cucurbit Genomics Database (http://www.icugi.org/) of both the genomic and coding regions of the candidate VPS4-like gene in cucumbers 9930 and Gy 14, and only one highly similar sequence was identified and no evidence of a duplicated gene was found. However, other gene(s) with minor effects on ZYMV resistance could exist. Further evaluation of the sequences revealed substantial variation in the VPS4-like gene among the cucumber accessions Gy 14, Dina-1, St 8, and TMG-1, with most polymorphisms in the intronic regions. Across the eight exons, six single-nucleotide polymorphisms (SNPs) define four haplotypes corresponding to Gy 14; St 8; TMG-1 and Dina-1; and A192-18 (Table 1). Across the seven introns, we identified 29 SNPs and four indels (insertions or deletions). SNPs were found in all introns (one, twelve, three, four, two, one, and six SNPs in introns one through seven, respectively). In the first intron, Dina-1 has a 37-bp duplication. In the third intron, there are four copies of a seven-bp region in Dina-1 vs. three copies in the other accessions. There are also deleted regions in intron 3, a deletion of 63-bp in Dina-1 and a 33-bp deletion at the same position in TMG-1. Intron seven carries a 3-bp deletion in Dina-1, St 8, and TMG-1 relative to Gy 14.
Four haplotypes across the eight exons of the proposed candidate gene vacuolar protein sorting–associated protein 4-like for the Zucchini yellow mosaic locus relative to the reference sequence of Gy 14.
The four haplotypes in the coding region for the VPS4-like gene (Table 1; Supplemental Fig. 1) encode three protein variants (Table 2; Fig. 1). The two key SNPs (T86C and G99A), which translate into two AA changes (F29S and M33I) in zymA192-18 allele (Amano et al., 2013), are also present in the zymTMG-1 and zymDina-1 alleles (Table 2). ZYMV-susceptible Gy 14 and St 8 do not possess these two key SNPs, and St 8 has a unique SNP (G136T) that results in a premature stop codon (E46-*) (Table 2). Although A192-18, Dina-1, and TMG-1 possess different haplotypes at the VPS4-like candidate gene for zym and do not trace back to the same source population, all three haplotypes encode the same protein sequence. The most parsimonious explanation would be that the two key SNPs (T86C and G99A) occurred in a common progenitor of the original sources of ZYMV resistance introduced into Dina-1, G22, and TMG-1. Later polymorphisms may have accumulated in the coding and/or intronic regions of the gene to produce the haplotype diversity that we uncovered. Our research documents that independently identified and introgressed sources of ZYMV resistance in cucumber possess the same genetic basis and undesirable genetic uniformity exists in cucumber for potyvirus resistance that could potentially be overcome by the viruses.
Three amino acid variants across the eight exons of the proposed candidate gene vacuolar protein sorting–associated protein 4-like for the Zucchini yellow mosaic locus relative to the reference sequence of Gy 14.
CLUSTAL O (version 1.2.4) alignment of predicted amino-acid sequences of five cucumber accessions. TMG-1, Dina-1, and A192-18 are resistant to Zucchini yellow mosaic virus, and Gy 14 and St 8 are susceptible. Polymorphisms are shown with bold text.
Citation: HortScience 52, 8; 10.21273/HORTSCI11951-17
CLUSTAL O (version 1.2.4) alignment of predicted amino-acid sequences of five cucumber accessions. TMG-1, Dina-1, and A192-18 are resistant to Zucchini yellow mosaic virus, and Gy 14 and St 8 are susceptible. Polymorphisms are shown with bold text.
Citation: HortScience 52, 8; 10.21273/HORTSCI11951-17
CLUSTAL O (version 1.2.4) alignment of predicted amino-acid sequences of five cucumber accessions. TMG-1, Dina-1, and A192-18 are resistant to Zucchini yellow mosaic virus, and Gy 14 and St 8 are susceptible. Polymorphisms are shown with bold text.
Citation: HortScience 52, 8; 10.21273/HORTSCI11951-17
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CLUSTAL O (version 1.2.4) alignment of nucleotide coding sequences of five cucumber accessions. TMG-1, Dina-1, and A192-18 are resistant to Zucchini yellow mosaic virus, and Gy 14 and St 8 are susceptible. Polymorphisms are shown with bold text.
Citation: HortScience 52, 8; 10.21273/HORTSCI11951-17
CLUSTAL O (version 1.2.4) alignment of nucleotide coding sequences of five cucumber accessions. TMG-1, Dina-1, and A192-18 are resistant to Zucchini yellow mosaic virus, and Gy 14 and St 8 are susceptible. Polymorphisms are shown with bold text.
Citation: HortScience 52, 8; 10.21273/HORTSCI11951-17
CLUSTAL O (version 1.2.4) alignment of nucleotide coding sequences of five cucumber accessions. TMG-1, Dina-1, and A192-18 are resistant to Zucchini yellow mosaic virus, and Gy 14 and St 8 are susceptible. Polymorphisms are shown with bold text.
Citation: HortScience 52, 8; 10.21273/HORTSCI11951-17
List of primers used for sequencing of the vacuolar protein sorting–associated protein 4-like gene.
