Genome-wide melon R-genes and their chromosomal distribution
The latest version (v3.6.1) of the whole-genome sequence of the melon double haploid line DHL90 was constructed using an improved assembly and annotation. This sequence contains 70 R-genes [1, 45]. We retrieved genomic information for these 70 R-genes, including their coding sequences and deduced amino acid sequences, from the cucurbit genome database (http://cucurbitgenomics.org). Detailed genomic information about these R-genes, including their locations on chromosomes, is provided in Table 1. Chromosomal mapping of the 70 R-genes revealed that they are distributed across all 12 melon chromosomes, with 1 to 12 genes per chromosome (Fig. 1; Table 1). Chromosome 9 (Chr09) contains the most R-genes (12), followed by Chr12 and Chr01 (10 and 9 genes, respectively). Chr11 contains the fewest R-genes (2), followed by Chr03 and Chr07 (3 genes each). The genes appear to be clustered, particularly in the telomere regions of chromosomes such as chr09, chr01, and chr04 (Fig. 1).
Gene Structures, Domain Organizations, and Motif Distribution of R-genes in Melon
We analyzed the exon–intron structures of all 70 melon R-genes by comparing their coding sequences with the corresponding genomic sequences using the online tool GSDS2.0 (http://gsds.cbi.pku.edu.cn/). The highest number of exons (22) was in the gene MELO3C013803, followed by 18 in MELO3C007367 (Additional file: Figure S1). Among the 70 R-genes, 21 were mono-exonic, while 12 and 4 genes were bi- and tri-exonic, respectively.
We analyzed the conserved domains of the 70 melon R-genes using the Conserved Domain Database (CDD) at https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi. We detected several disease resistance-related domains encoded by these R-genes, such as NB-ARC (Nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4), LRR (Leucine-rich repeat), TIR (Toll/interleukin-1 receptor), CC (Coiled-coil), and RLK (Receptor-like kinase) domains. The R-genes were grouped into different classes based on the presence of the following conserved domains in their encoded proteins: (i) LRR, (ii) NBS-LRR, (iii) TIR, (iv) TIR-NBS-LRR, (v) NB-ARC, (vi) CC, (vii) RLK, and (viii) DUF (Table 2 and Additional file: Figure S2). Thirty-seven genes encode proteins with only LRR domains, seven encode proteins with NB-ARC domains, two encode proteins with TIR domains, and only one encodes a protein with a CC domain (Table 2). Twelve genes encode three domains (TIR, NBS, and LRR), including MELO3C004288, MELO3C004289, MELO3C004311, MELO3C004313, MELO3C022154, MELO3C022152, MELO3C022146, MELO3C022145, MELO3C022144, MELO3C004309, MELO3C004259, and MELO3C004301. A list of the genes and a description of their domains is provided in Table 2.
Table 1
Information about R-genes throughout the melon genome including chromosomal positions, lengths, and annotated descriptions.
Sl. | Gene IDx | Chr. Number | Position on chromosome | CDS-length (bp) | Peptide length (AA) | Strand | Description |
Start | End |
1 | MELO3C023580.2 | chr01 | 33,386,823 | 33,390,698 | 687 | 288 | - | Disease-resistance protein RGA2-like |
2 | MELO3C023579.2 | chr01 | 33,395,126 | 33,397,789 | 2,664 | 887 | - | Disease-resistance protein RGA2-like isoform X1 |
3 | MELO3C023578.2 | chr01 | 33,410,087 | 33,414,749 | 1,158 | 385 | - | Disease-resistance protein |
4 | MELO3C023577.2 | chr01 | 33,419,963 | 33,423,566 | 2,715 | 904 | - | Disease-resistance protein RGA2-like |
5 | MELO3C023441.2 | chr01 | 34,457,351 | 34,462,055 | 2,766 | 921 | - | Receptor-kinase, putative |
6 | MELO3C023440.2 | chr01 | 34,462,521 | 34,463,915 | 1,338 | 445 | - | LRR receptor-like serine/threonine-protein kinase GSO2 |
7 | MELO3C023439.2 | chr01 | 34,468,416 | 34,473,193 | 3,207 | 1,068 | - | LRR receptor-like serine/threonine-protein kinase GSO2 |
8 | MELO3C023438.2 | chr01 | 34,474,924 | 34,475,353 | 336 | 111 | + | LRR receptor-like serine/threonine-protein kinase GSO2 |
9 | MELO3C023437.2 | chr01 | 34,475,729 | 34,476,367 | 354 | 117 | + | Receptor-kinase, putative |
10 | MELO3C029319.2 | chr02 | 4,111,584 | 4,115,605 | 717 | 238 | + | NBS-LRR type resistance protein |
11 | MELO3C015353.2 | chr02 | 985,162 | 987,242 | 1,737 | 578 | + | Disease-resistance protein RGA2-like |
12 | MELO3C015354.2 | chr02 | 990,582 | 993,823 | 3,240 | 1,080 | + | Disease-resistance protein RGA2-like |
13 | MELO3C029505.2 | chr02 | 7,359,371 | 7,363,388 | 765 | 254 | - | TMV resistance protein N-like |
14 | MELO3C010346.2 | chr02 | 17,481,683 | 17,485,283 | 1,593 | 530 | + | TMV resistance protein N |
15 | MELO3C010827.2 | chr03 | 30,596,169 | 30,600,072 | 3,663 | 1,054 | - | Receptor-kinase, putative |
16 | MELO3C010826.2 | chr03 | 30,600,299 | 30,603,794 | 3,054 | 1,071 | - | Receptor-kinase, putative |
17 | MELO3C010825.2 | chr03 | 30,604,364 | 30,611,770 | 6,069 | 2,022 | - | Receptor-kinase, putative |
18 | MELO3C009695.2 | chr04 | 30,097,463 | 30,100,144 | 2,682 | 893 | + | Disease-resistance protein |
19 | MELO3C009694.2 | chr04 | 30,103,601 | 30,106,071 | 2,391 | 796 | + | Disease-resistance protein |
20 | MELO3C009693.2 | chr04 | 30,110,724 | 30,113,156 | 2,358 | 786 | + | Disease-resistance protein |
21 | MELO3C009179.2 | chr04 | 33,763,652 | 33,766,776 | 3,042 | 1,013 | + | Receptor-kinase, putative |
22 | MELO3C009177.2 | chr04 | 33,766,795 | 33,780,875 | 3,231 | 1,076 | + | Receptor-kinase, putative |
23 | MELO3C004259.2 | chr05 | 25,752,437 | 25,757,292 | 3,951 | 1,316 | + | TMV resistance protein N-like isoform X1 |
24 | MELO3C004288.2 | chr05 | 26,044,574 | 26,052,361 | 3,171 | 1,056 | + | TMV resistance protein N-like |
25 | MELO3C004289.2 | chr05 | 26,065,157 | 26071880 | 3,867 | 1,288 | - | TMV resistance protein N-like |
26 | MELO3C004301.2 | chr05 | 26,231,021 | 26,237,770 | 4,032 | 1,343 | - | TMV resistance protein N-like isoform X1 |
27 | MELO3C004303.2 | chr05 | 26,239,395 | 26,244,020 | 2,052 | 683 | - | TMV resistance protein N-like |
28 | MELO3C004309.2 | chr05 | 26,263,738 | 26,270,641 | 4,134 | 1,377 | + | TMV resistance protein N-like |
29 | MELO3C004311.2 | chr05 | 26,280,801 | 26,299,171 | 3,156 | 1,051 | - | TMV resistance protein N-like |
30 | MELO3C004313.2 | chr05 | 26,311,869 | 26,315,091 | 2,115 | 704 | - | TMV resistance protein N-like |
31 | MELO3C006780.2 | chr06 | 5,898,974 | 5,902,420 | 3,447 | 1,148 | - | Disease-resistance protein |
32 | MELO3C006801.2 | chr06 | 6,106,483 | 6,109,133 | 846 | 281 | - | Protein enhanced disease resistance 2-like |
33 | MELO3C016529.2 | chr06 | 27,910,808 | 27,913,125 | 504 | 167 | - | TMV resistance protein N |
34 | MELO3C013803.2 | chr06 | 33,588,343 | 33,599,894 | 2,184 | 727 | + | Protein enhanced disease resistance 2 |
35 | MELO3C017700.2 | chr07 | 26,469,746 | 26,473,637 | 3,141 | 1,046 | - | Disease-resistance protein RGA2-like |
36 | MELO3C017701.2 | chr07 | 26,475,401 | 26,480,759 | 3,192 | 1,063 | + | Disease-resistance protein RGA2-like |
37 | MELO3C017703.2 | chr07 | 26,480,404 | 26,483,226 | 2,823 | 940 | - | Disease-resistance protein RGA2-like |
38 | MELO3C007354.2 | chr08 | 2,332,143 | 2,335,108 | 1,806 | 601 | - | Cysteine-rich receptor-like protein kinase 29 |
39 | MELO3C007358.2 | chr08 | 2,346,707 | 2,353,661 | 4,296 | 1,431 | - | Receptor-like protein kinase |
40 | MELO3C007360.2 | chr08 | 23,53,788 | 2,361,267 | 4,088 | 1,395 | - | Receptor-like protein kinase |
41 | MELO3C007367.2 | chr08 | 2,372,510 | 2,386,472 | 4,656 | 1,551 | - | Receptor-like kinase |
42 | MELO3C022157.2 | chr09 | 665,753 | 668,864 | 2,025 | 674 | - | TMV resistance protein N-like isoform X1 |
43 | MELO3C022154.2 | chr09 | 681,564 | 689,908 | 3,432 | 1,143 | - | TMV resistance protein N-like |
44 | MELO3C022152.2 | chr09 | 700,743 | 713,705 | 4,173 | 1,390 | + | TMV resistance protein N-like |
45 | MELO3C022146.2 | chr09 | 762,107 | 767,613 | 2,274 | 757 | - | TMV resistance protein N-like |
46 | MELO3C022145.2 | chr09 | 768255 | 784,265 | 3,807 | 1,268 | + | TMV resistance protein N-like |
47 | MELO3C022144.2 | chr09 | 784,629 | 792,999 | 4,902 | 1,633 | - | TMV resistance protein N-like |
48 | MELO3C025516.2 | chr09 | 6,632,514 | 6,659,697 | 4,371 | 1,,456 | - | TMV resistance protein N-like |
49 | MELO3C025519.2 | chr09 | 6,674,960 | 6,677,738 | 762 | 253 | - | Disease-resistance protein RGA2-like |
50 | MELO3C025518.2 | chr09 | 6,675,092 | 6,676,395 | 648 | 215 | - | Disease-resistance protein RGA2-like |
51 | MELO3C005450.2 | chr09 | 21,691,401 | 21,694,271 | 2,790 | 929 | - | LRR receptor-like kinase family protein |
52 | MELO3C005451.2 | chr09 | 21,699,468 | 21,702,467 | 3,000 | 999 | - | LRR receptor-like kinase |
53 | MELO3C005452.2 | chr09 | 21,708,265 | 21,711,353 | 28,17 | 938 | - | LRR receptor-like kinase |
54 | MELO3C012268.2 | chr10 | 1,574,521 | 1,579,615 | 1,800 | 599 | + | Leaf rust 10 disease-resistance locus receptor-like protein kinase-like 1.2 isoform X4 |
55 | MELO3C012049.2 | chr10 | 2,989,020 | 2,990,934 | 1,869 | 622 | + | Leaf rust 10 disease-resistance locus receptor-like protein kinase-like 1.5 |
56 | MELO3C012045.2 | chr10 | 3,007,893 | 3,014,091 | 1,503 | 500 | - | Protein enhanced disease resistance 2 |
57 | MELO3C034399.2 | chr10 | 15,627,727 | 15,627,921 | 195 | 64 | + | Disease-resistance protein At4g27190-like |
58 | MELO3C022580.2 | chr10 | 16,222,411 | 16,222,859 | 447 | 148 | - | Disease-resistance protein RGA2-like |
59 | MELO3C022447.2 | chr11 | 33,758,671 | 33,762,610 | 3,030 | 1,009 | - | Receptor-like protein |
60 | MELO3C022449.2 | chr11 | 33,770,307 | 33,772,966 | 2,145 | 714 | - | Receptor-like protein |
61 | MELO3C002671.2 | chr12 | 22,199,381 | 22,201,102 | 1,350 | 449 | + | LRR receptor-like kinase |
62 | MELO3C002667.2 | chr12 | 22,209,961 | 22,215,123 | 3,279 | 1,092 | + | LRR receptor-like kinase |
63 | MELO3C002666.2 | chr12 | 22,219,699 | 22,226,478 | 3,114 | 1,037 | + | LRR receptor-like kinase |
64 | MELO3C002506.2 | chr12 | 23,598,469 | 23,607,646 | 2,040 | 679 | - | Receptor-like protein kinase |
65 | MELO3C002504.2 | chr12 | 23611,543 | 23,620,880 | 3,870 | 1,289 | - | Cysteine-rich receptor-like protein kinase 28 |
66 | MELO3C002501.2 | chr12 | 23,633,920 | 23,636,908 | 1,617 | 538 | + | Cysteine-rich receptor-like protein kinase 26 isoform X1 |
67 | MELO3C002394.2 | chr12 | 24,343,418 | 4,346,595 | 2,385 | 794 | - | LRR receptor-like kinase family protein |
68 | MELO3C002393.2 | chr12 | 24,352,898 | 4,355,087 | 2,190 | 729 | - | LRR receptor-like kinase |
69 | MELO3C002392.2 | chr12 | 24,358,807 | 24,361,890 | 3,084 | 1,027 | - | LRR receptor-like serine/threonine-protein kinase GSO1 |
70 | MELO3C002389.2 | chr12 | 24,376,328 | 24,380,811 | 3,786 | 1,261 | + | LRR receptor-like serine/threonine-protein kinase GSO1 |
xGenomic information based on the reference Genome of Melon (DHL92) v3.6.1 retrieved from the Cucurbit Genomics Database (http://cucurbitgenomics.org). |
Table 2
R-genes throughout the melon genome categorized based on functional disease resistance-related domains.
Sl. | Domain | Function | Gene ID |
1 | Leucine-rich repeat (LRR) | Recognition of pathogen and Plant Defense [29, 47] | MELO3C023577.2, MELO3C023579.2, MELO3C015353.2, MELO3C015354.2, MELO3C017700.2, MELO3C017701.2, MELO3C025518.2, MELO3C009695.2, MELO3C006780.2, MELO3C023441.2, MELO3C023437.2, MELO3C023440.2, MELO3C023439.2, MELO3C023438.2, MELO3C004303.2, MELO3C025516.2, MELO3C010346.2, MELO3C005450.2, MELO3C002394.2, MELO3C005451.2, MELO3C005452.2, MELO3C002671.2, MELO3C002667.2, MELO3C022447.2, MELO3C022449.2, MELO3C002392.2, MELO3C002389.2, MELO3C002393.2, MELO3C029505.2, MELO3C034399.2, MELO3C010827.2, MELO3C010826.2, MELO3C010825.2, MELO3C009179.2, MELO3C009177.2, MELO3C007367.2, MELO3C002666.2 |
2 | Nucleotide-binding site leucine-rich repeat (NBS-LRR) | Resistance protein Signaling and Plant Defense [27, 33, 48] | MELO3C029319.2 |
3 | Toll/interleukin-1 receptor homology (TIR) | TMV resistance protein N [34, 47] | MELO3C022157.2, MELO3C016529.2 |
4 | Toll/interleukin-1 receptor homology nucleotide-binding site leucine-rich repeat (TIR-NBS-LRR) | Pathogen specificity and defense [34, 47, 49] | MELO3C004288.2, MELO3C004289.2, MELO3C004311.2, MELO3C004313.2, MELO3C022154.2, MELO3C022152.2, MELO3C022146.2, MELO3C022145.2, MELO3C022144.2, MELO3C004309.2, MELO3C004259.2, MELO3C004301.2, |
5 | Nucleotide-binding adaptor shared by APAF-1, R proteins and CED-4 (NB-ARC) | Molecular switch in activating defenses [28, 31] | MELO3C017703.2, MELO3C025519.2, MELO3C022580.2, MELO3C023578.2, MELO3C009694.2, MELO3C009693.2, MELO3C013803.2 |
6 | Coiled-coil domain (CC) | Pathogen recognition and signaling [31, 32, 50] | MELO3C023580.2 |
7 | Protein kinase (RLK) | Signaling and plant defense [35, 51–53] | MELO3C007354.2, MELO3C007358.2, MELO3C007360.2, MELO3C002506.2, MELO3C012268.2, MELO3C012049.2, MELO3C002504.2, MELO3C002501.2 |
8 | Domain of unknown function (DUF) | Protein enhanced disease resistance 2-like [54, 55] | MELO3C006801.2, MELO3C012045.2 |
We analyzed the conserved motifs of these 70 R-genes using the MEME Suite (http://meme-uite.org/tools/meme). A total of 20 conserved motifs were detected in these 70 R-genes, each comprising more than 14 amino acids. The greatest number of motifs was identified in the LRR domain-encoding gene MELO3C002394, whereas the fewest were detected in MELO3C029505, MELO3C023580, and MELO3C006801, which are LRR-, CC-, and DUF-domain-encoding genes, respectively. The distribution of these conserved motifs, along with the motif sequences, are described in Fig. 2.
Microsyntenic relationships of melon R-genes with genes in the watermelon and cucumber genomes
We analyzed the microsyntenic relationships of the 70 R-genes from melon (Cucumis melo) with genes in the watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) genomes using the Circos tool. Most of R-genes from melon share homologous relationships with R-genes from watermelon and cucumber. However, watermelon R-genes on chromosomes 11 and 12 lack homologues in melon (Fig. 3). By contrast, all 70 R-genes in melon have homologues in all chromosomes of cucumber.
Expression profiles of melon R-genes in response to A. citrulli inoculation
We investigated the expression patterns of the 70 melon R-genes in the leaves of resistant and susceptible melon seedlings at 12 h, 1 d, 3 d, and 6 d of inoculation with A. citrulli strain NIHHS15-280 via qRT-PCR. Several genes showed differential expression in the resistant vs. susceptible accession at different time points. Most of the genes were significantly induced within 12 h of infection with A. citrulli in the resistant accession and showed a general increase in expression in this accession. By contrast, in the susceptible accession, the expression of these genes fluctuated, with little or no expression at the 12 h time point. Heatmap analysis of the expression data identified a sub-cluster of six genes (MELO3C023441, MELO3C016529, MELO3C022157, MELO3C022146, MELO3C025518, and MELO3C004303) that showed contrasting trends of expression in the resistant vs. susceptible accession, with progressively increasing expression with increasing days after inoculation with A. citrulli in the resistant but not the susceptible accession (Fig. 4). Extensive analysis of the expression patterns of these six genes indicated that the expression of four genes (MELO3C023441, MELO3C004303, MELO3C022146, and MELO3C025518) increased in the resistant accession and decreased in the susceptible accessions with increasing time after inoculation with A. citrulli (Fig. 4). In the resistant accession, the expression of these four genes (MELO3C023441, MELO3C004303, MELO3C022146, and MELO3C025518) peaked at 6 d after inoculation, with levels approximately 8, 8, 10, and 7-fold those of the control samples, respectively. In the susceptible accession, the expression of two genes did not increase in response to A. citrulli inoculation, whereas the expression of two genes (MELO3C022157 and MELO3C016529) generally increased in response to inoculation, but to a lesser extent than in the resistant accession.
The expression of these two genes increased until 3 d after inoculation (5 fold in MELO3C016529 and 2.5 fold in MELO3C022157), followed by a decrease to their lowest levels at 6 d post inoculation (Fig. 5).