PCR-Based InDel Marker Associated with Powdery Mildew-Resistant MR-1

Powdery mildew (PM) is a fungal disease occurring in both field and greenhouse conditions worldwide. It infects many plant species and reduces both the productivity and quality of crops. Melon (Cucumis melo L.) is an economically important crop. In order to develop a molecular marker that can be used more conveniently in the development of PM-resistant melon using MR-1 melon resources, the previously reported cleaved amplified polymorphic sequence (CAPS) marker was improved with a length polymorphism PCR marker. Two cleaved CAPS markers—BSA12-LI3ECORI and BSA12-LI4HINFI—associated with BPm12.1, a major quantitative trait locus (QTL) corresponding to the PM resistance of MR-1, have been reported. In this study, we found that in the BSA12-LI3ECORI CAPS marker specifically, a 41 bp deletion was present in the PCR DNA region of the MR-1 melon genome. A new marker capable of distinguishing polymerase chain reaction (PCR) length polymorphism was produced using insertion-deletion (InDel) information in this region. This PCR-based InDel marker distinguished the genotypes of PM-resistant MR-1, PM-susceptible Top Mark, and their F1 progeny. These results suggest that this InDel marker could be used to develop PM-resistant melon varieties based on MR-1.

Identification of molecular markers associated with PM resistance from PM-resistant melon resources is a prerequisite for the development of PM-resistant melons by molecular breeding [8,9]. Molecular markers used for breeding are primarily aimed at a single gene associated with the target trait [10][11][12][13][14]. Genetic linkage analysis is mainly applied using quantitative trait loci (QTL) derived from between two parents with a contrasting phenotype. With this method, DNA markers for phenotypic data with characteristics of interest are identified based on variations in DNA sequences in segregation generations. In marker-assisted selection (MAS), DNA markers map DNA regions closely related to the target trait in donor lines. Subsequently, the targeted gene region is introduced into an agriculturally superior elite line using crosses, and used as an association marker until the target trait gene is stably inherited in the elite plant [15].
Among the melon resources, MR-1 is derived from PI124111 and exhibits nonspecific tolerance to powdery mildew [25]. However, the resistance gene of MR-1 has never been reported. Recently, Li, et al. [26] conducted next-generation sequencing (NGS)-based bulked segregant analysis (BSA) on 346 F2 progeny individuals derived from crossing MR-1 resistant and susceptible Top Mark to P. xanthii race 1. As a result, a single nucleotide polymorphism (SNP) was analyzed for all genomic sequences in two bulk DNA samples, to find QTL BPm12.1 between BSA12-LI3ECOR1 and BSA12-LI4HIFI, cleaved amplified polymorphic sequence (CAPS) markers associated with PM resistance to MR-1. In this study, we developed a PCR-based InDel marker that can discriminate MR-1 resistance more efficiently by using information from BSA12-LI3ECOR1 and BSA12-LI4HIFI, which are CAPS markers associated with BPm12.1 QTL, associated with MR-1 PM resistance.

Genomic DNA Isolation
Melon genomic DNA was isolated from young plants grown from seeds of thirteen resistant and two susceptible lines. The protocol described in web (https://www.pharm.auth.gr/plant-biotech/ en/protocols.html) was modified and used as follows. Genomic DNA was extracted using a DNA extraction buffer (50 mM Tris-HCl, 20 mM EDTA). One hundred milligrams of sampled leaves were ground with liquid nitrogen. One milliliter of extraction buffer and 66 µL of 10% SDS were added to a sample contained in a 2 mL tube, and then mixed at 65 • C for 30 min. After adding 300 µL of 3M sodium acetate (pH 5.2), the tube containing the mixture was left on ice for 50 min, and centrifuged at 9330× g for 20 min using a high-speed centrifuge. Eight hundred microliters of the supernatant were transferred to a new 2 mL tube and the same volume of isopropanol (IPA) was added. The tube containing the mixture was frozen at −80 • C for 10 min; after centrifugation, the pellet was separated and the supernatant was discarded. The pellet was rinsed twice using 700 µL of 70% EtOH, and, finally, the pellet was dissolved in sterile distilled water to obtain genomic DNA.  Figure 1B) were synthesized using the nucleotide sequence information reported in Li et al. [26]. Genomic DNA-PCR was performed on 100 ng of melon DNA using mixture of 0.75 µM pairs of CAPS markers, 250 µM dNTPs, and 1× reaction buffer with 5-unit NEXpro™ e Taq DNA polymerase (Focus Bioscience, St. Lucia, Australia). PCR was performed under the following conditions: 95 • C for 5 min, 30 cycles of 95 • C for 30 s, 56 • C for 30 s, 72 • C for 1 min, and 72 • C for 10 min. Five microliters of the PCR product was treated with EcoRI or Hinf1 restriction enzyme for 3 h and then subjected to agarose gel electrophoresis to determine whether there was a difference between PM resistance and susceptibility melon lines.

PCR Analysis for CAPS
In addition, PCR bands were eluted from agarose gel, and DNA sequences were determined by Sanger DNA sequencing.

Design of InDel Marker and PCR Analysis
The InDel  marker  primers  were  constructed  based  on  the  nucleotide  sequences  of  the  PCR  products  generated  by  the  BSA12_LI3ECORI  CAPS marker. The forward primer is 5 -AATCTATCCCAAATCAAAGTC-3 , and the reverse primer is 5 -AAACTTCTAGACCAATATAACTT-3 ( Figure 2B). The genomic DNA PCR reaction mixture consisted of 100 ng of melon DNA with 0.5 µM primers, 250 µM dNTP, 1x reaction buffer and 5-unit NEXpro™ e Taq DNA (Focus Bioscience, St Lucia, Australia). The PCR reaction of this mixture was carried out at 95 • C for 5 min, 30 cycles of 95 • C for 30 s, 49 • C for 30 s, 72 • C for 30 s, and 72 • C for 5 min, and 5 µL of PCR product out of a total of 20 µL of products was subjected to agarose gel electrophoresis to confirm the band.

F1 Generation between MR-1 and Top Mark and Powdery Mildew Disease Test
F1 hybrid seeds were harvested from pollination between MR-1 as the father, and Top Mark as the mother. F1 seeds were germinated and grown under the same conditions as MR-1 and Top Mark. In the summer of 2019, MR-1 and Top Mark parents and F1 seedlings were grown in plastic greenhouses conditioned with 25-30 • C and 60-70% relative humidity to induce spontaneous powdery mildew, and the symptoms of powdery mildew were investigated. The level of infection of powdery mildew was classified on a scale of 1-5 based on previously reported criteria [27]. Class 1 has no symptoms (0%); classes 2, 3, and 4 are low (10%), medium (10-25%), and severe infections (25-50%), respectively; and class 5 is where the entire leaf (50-100%) is infected ( Figure 3C).

PCR Polymorphism Analysis of CAPS Markers BSA12-L13ECOR1 and BSA12-L14HINF1 in Various Melon Resources
Li et al. [26] reported that BPm12.1, the major QTL of PM resistance of MR-1, is located between the CAPS markers BSA12-LI3ECORI and BSA12-LI4HINF1, at 0.02 cM and 0.28 cM intervals, respectively ( Figure 1A). Using the forward and reverse primers of CAPS markers BSA12-LI3ECORI and BSA12-LI4HINF1 ( Figure 1B Figure 1C) were compared for PCR product size ( Figure 1D,E). For PCR products of BSA12-LI3ECORI and BA12-LI4HINF1, length polymorphism was compared using EcoRI and Hinf1 restriction enzyme treatments, respectively ( Figure 1D,E).
Using the BSA12-LI4HINF1 CAPS marker, a PCR band of about 525 bp was amplified in all 15 lines (Figure 1D upper). In Hinf1 treatment, S1 (IranH), R1 (Edisto47), R2 (PMR5), R3 (PMR6), and R5 (TGR1551) were not cut. With the exception of these, 10 lines were cut with Hinf1, which all showed the same 351 bp and 174 bp bands (Figure 1D lower). These results show that the BAS12-LI4HINF1 CAPS marker does not exhibit specific polymorphism between PM resistance and susceptible lines, and against MR-1. Using the BSA12-LI3ECORI CAPS marker, PCR bands corresponding to about 840 bp were amplified in all 15 lines (Figure 1E upper). As a result of cutting the EcoR1 restriction enzyme, IranH was not cut and the remaining 14 lines were cut and divided into a 388 bp band and a 450 bp band. Among the 13 resistive lines, R4 (MR-1) and R7 (2563) lines showed the same size polymorphism band, smaller than 450 bp. Since R7 corresponds to the parent of R4-the MR-1 line-it showed the same pattern (Figure 1E lower). These results suggest that the BSA12-LI3ECORI CAPS marker is specific for MR-1. In addition, since it is close to the BPm12.1 powdery mildew resistance QTL at 0.2 cM [26], it is expected that the BSA12-LI3ECORI CAPS marker itself can be used as a BPm12.1 resistance marker.

Nucleotide Sequence Analysis of PCR Product of BSA12-L13ECOR1 CAPS Marker
DNA isolated from two PM-susceptible and 13 -resistant melon lines was amplified via PCR, using the BSA12_LI3ECORI CAPS primers. Resultantly, the nucleotide sequence information corresponding to 680bp of the PCR product was determined (Figure 2A, Data S1). The nucleotide sequence of this region is an AT-rich region that is highly conserved on melon DNA and is a noncoding sequence without an open reading frame (ORF) coding region. In the R4 (MR-1) and the R7 (parent of MR-1), a total of 36-37 bp was deleted in three areas. Eight nucleotides of "AATAATTT" from 116 bp to 124 bp, 20 nucleotides of "GT(C/T)AATATTT(C/T)AAGTCCATA" from 238 bp to 257 bp, and 8 or 9 nucleotides of " (A/-)CTTGATTA" from 504 bp to 512 bp were deleted. The EcoRI recognition sequence "GAATTC" was present at 384 bp to 390 bp in all lines of PCR products except for the S1 (IranH) line ( Figure 2A). Again, with the exception of the IranH line, the BSA12_LI3ECORI CAPS primer PCR product appeared in two bands at the EcoRI site ( Figure 1E lower). Based on EcoRI, the band corresponding to the upstream part was indicated as 362 bp by a 28 bp deletion in MR-1, and a 390 bp band, in which 28 bp existed in the remaining lines except IranH. In conclusion, we found that the MR-1 BSA12_LI3ECORI CAPS marker region has specific deletions that are not found in other PM-susceptible and -resistant lines.
The phylogenetic tree constructed by the nucleotide sequences of the BSA12_LI3ECORI CAPS marker region divided 15 melon resources into three clades ( Figure S1). Clade I included PM-sensitive lines (S1 (IranH Among the 13 PM-resistant melon resources in the phylogenetic tree, PMR45, Edisto47, PMR5, PI12412, and MR-1 are used for powdery mildew race identification [27][28][29]. These melons show differences in resistance and susceptibility for eight Podosphaera xanthii races: 1, N1, N2, 5 A, S, O, and N5, respectively [29]. PMR45 is susceptible to races N2, 5, S, O, and N5. Edisto47 is susceptible to races 5, O, and N5. PMR5 and PI122412 are only susceptible to race O out of eight races. MR-1 is resistant to these all eight races, but only moderately resistant to races S and O (Table S1). Taken together, lineage differentiation by the nucleotide sequence of the BSA12-LI3ECOR1 CAPS marker region shows a similar tendency to specific resistance to PM races.

Redesign of BSA12-L13ECOR1-Based InDel PCR Marker and Its Validation
Instead of the BSA12-L13ECOR1 CAPS marker associated with PM resistance of MR-1, an InDel PCR marker was developed that can more easily discriminate polymorphisms through differences in PCR product size. In the case of the MR-1 line, a total of 28 bp deletions exist specifically in the upstream region based on the EcoR1 site; so, a PCR primer pair capable of distinguishing the size of this part was prepared ( Figure 2B). When PCR was performed on a total of 14 melon lines using this PCR primer pair, only the MR-1 line specifically identified a band with a small size of 303 bp, while the rest of the powdery mildew sensitivity and resistance lines showed a larger 330 bp PCR band ( Figure 2C). These results suggest that the newly designed InDel PCR primer pair can replace the BSA12-L13ECOR1 CAPS marker. Indeed, the InDel PCR markers showed a 303 bp band in PM-resistant MR-1, and a 330 bp band in PM-sensitive Top Mark. F1 obtained by crossing the two showed two bands of 330 and 303 bp ( Figure 3A). As a result of the test using naturally occurring PM, MR-1 was Class 1 (resistant to PM), and Top Mark was Class 5 (sensitive to PM). However, F1 showed moderate resistance to PM, Classes 2-4 ( Figure 3B). Moderate resistance in F1 suggests the possibility that race S or O occurred simultaneously with other races in spontaneous occurrence. These results suggest that the InDel PCR marker is associated with MR-1 PM resistance and that the BPm12.1 PM resistance gene is present near the marker.
The InDel PCR marker developed in this study is 0.2 cM to the left from the BPm12.1 position, a PM-resistant QTL locus. There are 78 kb between the BSA12-LI3ECOR1 and BSA12-LI4HINFI CAPS marker regions, and 12 genes are present in Cucumis melo L. cv. DHL92, the melon reference genome [30] (Figure S2). In the future, it remains to find the BPm12.1 locus among 12 candidate genes in the MR-1 melon.

Conclusions
It was found that there was an MR-1-specific sequence deletion in the BSA12-L13ECOR1 CAPS marker region, 0.2 cM away from BPm12.1, a major QTL related to PM resistance of MR-1 melon. InDel PCR markers that are more convenient to use than CAPS markers were constructed using the MR-1 deletion polymorphism. Correlations were confirmed between PCR product polymorphism and PM-resistance in MR-1, PM-susceptible Top Mark, and F1 progeny. The newly designed InDel PCR marker used in this study can be used to discriminately introduce a MR-1 PM resistance gene for the development of PM-resistant melon varieties.