Powdery mildew isolate classification
Leaves from PM-infected melon plants were stained using Coomassie Brilliant Blue, and the conidia therein appeared ovoid in shape. Fibrous bodies were clearly visible in the cytoplasm with a length of 57–76 µm and a width of 38–44 µm, consistent with the characteristics of P. xanthii (Fig. 1). Following inoculation, the susceptibility of 13 different host lines to this isolate was evaluated (Table 1), and resistance responses were consistent with the identity of this isolate as P. xanthii race 1, with no change in the dominant strain having occurred over the course of the year.
Table 1
The PM infection outcomes for 13 differential hosts
Differential hosts
|
Resistance evaluation in 2017
|
Resistance evaluation in 2018
|
IranH
|
S
|
S
|
Top Mark
|
S
|
S
|
Vedrantais
|
S
|
S
|
PMR 45
|
S
|
S
|
PMR 5
|
R
|
R
|
WMR 29
|
R
|
R
|
Edisto 47
|
R
|
R
|
PI 414723
|
R
|
R
|
MR-1
|
R
|
R
|
PI 124111
|
R
|
R
|
PI 124112
|
R
|
R
|
PMR 6
|
R
|
R
|
Nantais Oblong
|
S
|
S
|
Note: R: resistant, S: susceptible |
Genetic Analyses Of Pm-resistant And Pm-susceptible Melon Lines
As shown in Table 2, the stems and leaves of the female parental MR-1 line were disease resistant, whereas those of the male parental M4-7 were susceptible to PM. The leaves of F1 plants were resistant to disease, whereas the stems of these plants were susceptible. F2 plant isolation yielded plants conforming to three phenotypes: leaf and stem resistant (LRSR), leaf resistant stem susceptible (LRSS), and leaf and stem susceptible (LSSS). BC1P1 plants conformed to the LRSR and LRSS phenotypes, whereas BC1P2 plants conformed to the LRSS and LSSS phenotypes.
Table 2
Disease responses for P1, P2, F1, F2, BC1P1, and BC1P2 populations following artificial inoculation with P. xanthii race 1, with Chi-squared verification of the F2, BC1P2 segregation ratios between resistant (R) and susceptible (S) individuals.
Year
|
Generation
|
LRSR
|
LRSS
|
LSSS
|
Excepted Ratio
|
χ2
|
P Value
|
2017
|
P1
|
30
|
-
|
-
|
-
|
-
|
-
|
P2
|
-
|
-
|
30
|
-
|
-
|
-
|
F1
|
|
30
|
-
|
-
|
-
|
-
|
F2
|
224
|
73
|
99
|
9:3:4
|
0.03
|
0.98
|
BC1P1
|
14
|
16
|
-
|
1:1
|
0.13
|
0.72
|
BC1P2
|
-
|
13
|
17
|
1:1
|
0.53
|
0.47
|
2018
|
P1
|
30
|
-
|
-
|
-
|
-
|
-
|
P2
|
-
|
-
|
30
|
-
|
-
|
-
|
F1
|
-
|
30
|
-
|
-
|
-
|
-
|
F2
|
577
|
189
|
256
|
9:3:4
|
0.04
|
0.97
|
A dominant gene (CmPMRl) was found to control leaf resistance to powdery mildew, while a recessive gene (CmPMrs) was found to control resistance to powdery mildew on stems and vines. Analyses of inheritance patterns indicated that CmPMRl had a recessive epistasis effect on CmPMrs. In the F2 population, the ratios of LRSR: LRSS: LSSS were consistent with a 9:3:4 ratio, as determined via χ2 test. In the BC1P1 population, the LRSS:LRSR ratio was consistent with a 1:1 ratio as determined by χ2 test. In the BC1P2 population, the LRSS: LSSS ratio was consistent with a 1:1 ratio as determined by χ2 test.
BSA and QTL analysis of the dominant resistance gene CmPMRl
MR-1 sequencing produced 134,014,482 reads, of which 95.0% could be mapped to the reference genome, M4-7 sequencing produced 122,001,928 reads, of which 93.8% could be mapped to the reference genome, and a total of 2,761,801 SNPs were identified between MR-1 and M4-7. In addition, 39,710,623 reads were sequenced in LRSR pool, with a reference genome mapped rate of 93.17%, 43,930,213 reads were sequenced in LSSS pool, with a reference genome mapped rate of 91.10%, and 45,151,799 reads were sequenced in LRSS pool, with a reference genome mapped rate of 92.82%.
SNP index values were calculated according to sequencing data from three gene pools with corresponding 95% confidence intervals. The LRSR and LSSS pool results revealed the candidate gene to be localized within the 21.7-23.3Mb interval on chromosome 12 (Fig. 2a). When the LRSS-pool and LSSS-pool were calculated, the location was roughly the same as that calculated using the LRSR-pool and LSSS-pool, extending from 21.6 to 23.2 Mb (Fig. 2b). These results indicated that the dominant CmPMRl gene, which controls disease resistance in leaves and stems, is located in the 21.6–23.3 Mb interval on chromosome 12 (Fig. 2c). Based on the preliminary BSA localization results, 13 polymorphic markers (68.42% polymorphic) were selected within this initial CmPMRl location (Table S2). In total, 93 F2 individuals were selected to construct a validation population, and 13 pairs of primers (PM12-V1 to PM12-V13) were used for F2 genotyping. Phenotypic leaf disease resistance data in the F2 population was then used to localize the main effect locus associated with PM resistance in melon leaves to between PM12-V4 and PM12-V10 (21.59–23.30 Mb) on chromosome 12, with an LOD of 47.2 (Fig. 2d).
BSA and QTL analyses of the recessive resistance gene CmPMrs
Using a marker tightly linked to the CmPMRl gene (PM12R-6), we screened 726 plants with the same genotype and leaf phenotype as the disease-resistant parental MR-1 line. In the F2 population, the stem disease-resistant phenotype separated at a 1:3 ratio, thus eliminating the effect of CmPMRl on CmPMrs (Table S3). Stem resistance was therefore considered to be a trait controlled by a recessive allele. The LRSR-pool and LRSS-pool results revealed the candidate gene to be located within the 0–2.03 Mb interval of chromosome 10 (Fig. 3a, b). Based on preliminary BSA localization results, 12 polymorphic markers were screened in the initial CmPMrs location (63.16% polymorphic) (Table S4), and 93 F3 individuals were selected to construct a validation population, after which 12 primer pairs (PM10-V1 - PM12-V12) were used for F3 genotyping. With respect to stem resistance phenotype data in the F3 population, the main effect loci related to PM resistance was localized between PM10-V1 and PM10-V8 (0–2.04 Mb) on chromosome 10, and the LOD was 31.6 (Fig. 3c).
Finally mapping of the dominant resistance gene CMPMRl and recessive resistance gene CmPMrs
In this study, 1022 F2 plants were genotyped with markers PM12R-1 to PM12R-12 (Table S2) and 20 recombinant plants were identified, further identifying the location of the dominant resistance gene CmPMRl. The CmPMRl was found to exist between markers PM12R-5 and PM12R-7 (22.78–22.91 Mb). Six new CAPS markers (Table S2) were developed and used to genotype the 5 recombinants (F2-395, F2-89, F2-288, F2-715, and F2-1002). Gene recombination occurred between markers PM12R-AD1, PM12R-AD4 and PM12R-AD6, enabling us to determine that the R1 gene was localized between the PM12R-AD1 and PM12R-AD4 markers (Chr12: 22786222–22836405, about 50 Kb) (Fig. 4a, Table S5). No recombination was observed for the PM12R-AD2 and PM12R-AD3 markers, suggesting that this segment is the smallest segment localized to the R1 gene interval in the F2 population.
Genotyping of 726 F3 plants using markers PM10R-1 to PM10R-12 (Table S4) identified 18 recombinant plants, leading to the location of the recessive resistance gene CmPMrs. The CmPMrs location was thus determined to be between markers PM10R-4 and PM10R-5 (0.47–0.59 Mb). Four new CAPS markers (Table S4) were developed and used to genotype the three recombinants (F3-591, F3-116, and F3-106). Recombination occurred only for labeled PM10R-AD4, with no recombination between labeled PM10R-AD1 and PM10R-AD4. This led us to refine the CmPMrs location between markers PM10R-AD1 and PM10R-AD4 (Chr10:466642–552080, about 86 Kb) (Fig. 4b, Table S6), suggesting that this segment is the smallest that can be used to locate the CmPMrs gene interval.
Candidate Genes Functional Annotation And Sequence Alignment
When the CmPMRl finally-mapping interval was aligned to the DHL92 melon reference genome, we identified nine protein-coding genes (MELO3C002441-MELO3C002449) within this interval, of which six harbored nonsynonymous mutations between the two parental lines. Functional annotation of these genes revealed MELO3C002445 to be unclassified, MELO3C002441 and MELO3C002443 to be involved in signal transduction, MELO3C002442 and MELO3C002444 to be involved in lipid and protein metabolism, MELO3C002446 to play roles in unclear biological processes, MELO3C002447 to be involved in redox reactions, MELO3C002448 to be involved in the control of phosphate hydrolase activity, and MELO3C002449 to be involved in glycolytic enzyme activity (Table 3).
Table 3
List of the predicted candidate genes in the CmPMRl candidate region
Gene ID
|
The number of nsSNPs
|
Description of gene function
|
MELO3C002441
|
1
|
Ankyrin repeat family protein
|
MELO3C002442
|
2
|
Aspartic proteinase
|
MELO3C002443
|
1
|
F-box plant-like protein, putative
|
MELO3C002444
|
0
|
Aminomethyltransferase
|
MELO3C002445
|
2
|
unkown protein
|
MELO3C002446
|
2
|
Glycine-Rich cell wall structural protein
|
MELO3C002447
|
0
|
L-ascorbate oxidase
|
MELO3C002448
|
0
|
phosphatase family protein
|
MELO3C002449
|
1
|
Glucan endo-13-beta-glucosidases
|
Total
|
9
|
|
This same analytical approach led to the identification of 11 candidate genes within the CmPMrs finally-mapping interval, of which seven harbored nonsynonymous mutations between the two parental lines (MELO3C012428-MELO3C012438). Functional annotation of these genes revealed them to be primarily associated with plant physiological and biochemical responses (Table 4), including substance metabolism (MELO3C012428, MELO3C012430, MELO3C0124233, MELO3C012434, MELO3C012435), substance transport (MELO3C012429, MELO3C012436), redox reactions (MELO3C012431), pollen budding and pollen tube elongation (MELO3C012432), ubiquitination (MELO3C012437), and PM resistance (MELO3C012438).
Table 4
List of the predicted candidate genes in the CmPMrs candidate region
Gene ID
|
The number of nsSNPs
|
Description of gene function
|
MELO3C012428
|
0
|
mRNA decapping protein 2
nuclear-transcribed mRNA
|
MELO3C012429
|
0
|
Aquaporin 2
|
MELO3C012430
|
1
|
Alpha/beta hydrolase family protein
|
MELO3C012431
|
9
|
Thioredoxin-like protein
|
MELO3C012432
|
0
|
Pollen-specific leucine-rich repeat extensin-like protein 1
|
MELO3C012433
|
2
|
Patatin
|
MELO3C012434
|
0
|
Anthranilate synthase component I
|
MELO3C012435
|
5
|
phosphatase family protein
|
MELO3C012436
|
1
|
Probable protein kinase
|
MELO3C012437
|
1
|
Ubiquitin-conjugating enzyme, E2
|
MELO3C012438
|
1
|
MLO-like protein
|
Total
|
20
|
|
Assessment Of Candidate Gene Expression Patterns
A qRT-PCR analysis of genes expressed within the CmPMRl locus revealed that MELO3C002441, MELO3C002444, and MELO3C002448 were significantly upregulated in disease-resistant MR-1 leaves following inoculation, whereas MELO3C002446, MELO3C002447, and MELO3C002449 were downregulated, and the expression of the other three genes was unaffected. The expression of MELO3C002441 was also significantly elevated in stem tissues, but was expressed at a lower level therein relative to leaves, consistent with the CmPMRl gene expression pattern (Fig. 5a). Following the inoculation of M4-7 plant materials, relative MELO3C002441 expression was significantly reduced in leaves, whereas it was expressed at an at least three-fold higher level in MR-1 leaves relative to M4-7 leaves, suggesting that this gene is expressed at substantially different levels in PM-resistant and PM-susceptible plants (Fig. 5b). This gene encodes an anchor protein-containing repeat sequence harboring a nonsynonymous SNP that alters the identity of the amino acid in position 587 from a phenylalanine (Phe) in MR-1 plants to a cysteine (Cys) in M4-7 plants. Based on these findings, MELO3C002441 was identified as a CmPMRl candidate gene. For other details regarding expression levels of these genes, see Figures S1-S2.
A qRT-PCR analysis of genes in the CmPMrs locus revealed MELO3C012429 and MELO3C012438 to be significantly upregulated in stem tissues after susceptible M4-7 plant material inoculation, whereas MELO3C012430, MELO3C012432, MELO3C012433, and MELO3C012436 were significantly downregulated, and the expression of the other five genes was unaffected. Of the two upregulated genes, MELO3C012429 was significantly upregulated in stem tissues but did not exhibit altered expression levels in leaves. MELO3C012438 expression rose significantly in the stem on day 4, with a > 2-fold increase in relative expression, whereas its expression levels in leaves were reduced such that this gene was expressed at significantly lower levels in leaf tissues relative to stem tissues at specific time points following inoculation (Fig. 5c). MELO3C012429 was expressed at significantly higher levels in the leaves of disease-resistant MR-1 plants relative to stems, and such expression was not altered following inoculation. In contrast, MELO3C012438 expression rose significantly in both leaf and stem tissues following inoculation, with significantly higher expression in stems relative to leaves at specific time points (Fig. 5d). MELO3C012438 encodes an MLO-like protein harboring a nonsynonymous SNP that alters the identity of the amino acid in position 191 from an isoleucine (Ile) in MR-1 plants to a threonine (Thr) in M4-7 plants. Based on these results, we identified MELO3C012438 as a candidate for the CmPMrs gene. For details regarding the expression levels of other genes, see Figure S3-S4.
Candidate Gene Subcellular Localization
We next evaluated the localization of MELO3C002441 and MELO3C012438 within cells (Fig. 6), with chloroplast autofluorescent signal being used to guide localization efforts. This analysis revealed that the MELO3C002441-GFP fusion protein primarily localized to the cell membrane (Fig. 6a-d). MELO3C012438 exhibited localization largely identical to that of MELO3C002441, localizing to the cell membrane in a manner consistent with transmembrane domain predictions (Fig. 6e-h). The subcellular localization of these proteins may be linked to their functional role in the context of PM resistance, as they control melon epidermal cell resistance to PM infection.
Phylogenetic Analysis Of Candidate Genes
Phylogenetic analyses indicated that the MELO3C002441 protein clustered with soybean GmNPR4 and GmNRP4-like proteins, and was genetically distant from NPR proteins in other crops, suggesting that this gene is an anchor protein repeat sequence-containing gene that is functionally similar to the soybean NPR4 gene (Ankyrin repeat protein). Conservative structural domain analysis revealed that this gene harbored an N-terminal ANK-2 type anchor protein repeat sequence and a C-terminal PPG structural domain (Figure S5a, S6a).
The MELO3C012438 protein clustered with the cucumber CsaV35G036400 and watermelon Cla020573 proteins, which in turn clustered with the Arabidopsis AtMLO2, AtMLO6, and AtMLO12 proteins. These three proteins were genetically distant from the other MLO proteins, suggesting that this gene is an MLO family gene (Mildew Resistance Locus O) with functional similarity to Cucumber CsMLO8 and Arabidopsis AtMLO2, AtMLO6, and AtMLO12. Conserved structural domain analysis indicated that the gene encodes a conserved MLO structural domain (Figure S5b, S6b).