The parental genotypes MT12 and MT17 were obtained from tomato genetic resources of Multi Tohum (Antalya, Turkey). The tomato line MT12 was used as a resistance source in this study. There is not much information about the genetic background of this genotype. MT17 is a susceptible parent obtained from female (Solanum lycopersicum X S. hirsutum) and male (Solanum lycopersicum X Solanum pimpinellifolium). Tomato cultivars Seval F₁ carrying Mi-1.2 gene and susceptible Tueza F₁ (Multi Tohum, Antalya, Turkey) were used as controls in the experiments. The presence or absence of Mi-1.2 gene in all tomato plants was confirmed using Mi23 primer set (Seah et al., 2007) (Supplementary Figure 1).

Two avirulent isolates of M. incognita, S6 and K7, were used in the experiment to investigate the response of the MT12 at a high temperature (Devran and Sogut, 2009). All isolates used have been continued as pure nematode cultures since 2008 in Devran laboratory. The responses of MT12 to Mi-1.2 natural virulent populations V12 and V21 of M. incognita were also investigated. Only the M. incognita avirulent S6 isolate was used in the mapping and inheritance studies of RRKN1.

Avirulent isolates were multiplied on susceptible tomato cultivar Tueza F₁, while Mi-1.2 natural virulent isolates were multiplied on resistant tomato cultivar Seval F₁ containing the Mi-1.2 gene to maintain their virulence in a growth chamber at 25°C ± 0.5 with a 16:8 hours photoperiod (light: dark) and 65 ± 5% relative humidity. Sixty days after inoculation the plants were removed, and the plant roots were washed with water. Egg masses were collected from the root using a small needle and placed in centrifuge tubes (Özalp and Devran, 2018). They were then stored at 4°C until inoculation.

Tomato seeds were sown in vials including vermiculite, perlite, and peat (v:v:v: 1:1:1) and maintained in a seedling facility. Seedlings with four true leaves were transplanted into 250 ml plastic pots including sterilized sandy soil and were kept in the growth chamber at 25°C for a week to ensure root development. For the tests to be carried out at high temperature, the pots were transferred to a growth chamber at 32°C. Soil temperature was monitored with the probes placed in pots and nematodes were inoculated when the soil temperature reached 32°C. Seedlings were exposed to soil temperatures at 32°C for 7 days.

For nematode inoculation, egg masses were placed in a sieve and fresh second stage juveniles (J2s) from hatched eggs were collected and counted under a light microscope and diluted to 1000 J2s/ml. A total of 1000 J2s were inoculated into two holes (0.5 ml of water per hole) near the stem of each plant. The procedure was performed according to former studies (Öçal et al., 2018; Nas et al., 2023). After seven days, the plants were transferred to a growth chamber at 25°C ± 0.5 with a 16:8 hours photoperiod (light: dark) and 65 ± 5% relative humidity until the end of the experiment.

To observe the response of the MT12 tomato line to nematode isolates at a high temperature, an experiment was conducted under 25°C and 32°C soil temperature conditions. For this purpose, avirulent M. incognita isolates K7 and S6 were used for the inoculation of plants. Besides resistant source MT12, susceptible tomato cultivar Tueza F₁ and resistant tomato cultivar Seval F₁ were used as control plants in the experiment.

To observe the response of the MT12 tomato cultivar to Mi-1.2 natural virulent M. incognita isolates, an experiment was conducted under 25°C soil temperature conditions. For this purpose, Mi-1.2 natural virulent M. incognita isolates V12 and V21 were used for the inoculation of plants.

Both experiments were performed as described above for testing plant lines. The experiments were carried out as five replicates and two repeats according to the completely randomized design.

A cross between the resistant and susceptible parent lines MT12 and MT17, respectively, was generated and the F₁ line was obtained. An F₂ mapping population was generated by selfing the F₁ line. A total of 130 F₂ seeds were obtained from a single F₁ tomato plant and were used in the phenotyping and genotyping experiments.

Plants were uprooted sixty days post inoculation (dpi) and the roots were washed under the tap water. The roots were then stained with Ploxine B, and the galls and egg masses were counted and recorded (Öçal et al., 2018). Egg masses and galls in the roots of the plants were counted under a light microscope.

Plants in mapping population were classified as resistant if the individual root system had less than 25 egg masses, or susceptible if the individual root system had 25 or more egg masses (Veremis et al., 1999; Ammiraju et al., 2003; Wang et al., 2013).

Data were analyzed by analysis of variance (ANOVA). Differences among means were examined using the Tukey multiple comparison test. Chi-square tests for specific proportions for goodness of fit were carried out using the PROC FREQ function. All statistical analysis was performed using with the SAS statistical program (v. 9.0 for Windows; SAS Institute Inc., Cary, NC, USA).

Genomic DNA was extracted from young leaves collected from parental lines and F₂ plants using the Wizard Magnetic Kit (Promega) following the manufacturer’s instructions. DNA was then run on an agarose gel and checked for degradation or their high molecular weight. The quality control (QC) of the DNA was determined by Novogene UK (Cambridge, United Kingdom) before proceeding to the sequencing.

The sequencing has been carried out by Novogene UK (Cambridge, United Kingdom), generating 150 bp paired-end read data for each parent line (resistant and susceptible) with NovaSeq 6000.

As previously described (Kahveci et al., 2021), we took the NGS analysis approach and trimmed the raw reads using BBDuk (filterk=27, trimk=27; https://sourceforge.net/projects/bbmap/) to remove Illumina adapters and to quality-trim both ends to Q12. Trimmed sequences from parental lines were then mapped onto chromosomes 1 – 12 of the SL3.1 version of the available reference tomato genome (S. lycopersicum cultivar Heinz 1706, GenBank: GCA_000188115.4) using BBMap (https://sourceforge.net/projects/bbmap/) and the alignment data were converted to the BAM format (Li et al., 2009). The variant detection was performed using BCFtools (Li et al., 2009) and SNPsFromPileups (https://github.com/davidjstudholme/SNPsFromPileups) as previously described (Devran et al., 2018; Kahveci et al., 2021). Integrative Genomics Viewer (IGV) was used to visualize the alignment results (Robinson et al., 2011). We used the Solanaceae Genomics Network web portal (Fernandez-Pozo et al., 2015) to browse and visualize the tomato genomic interval and identify the genes contained therein.

At the beginning of the study, there was no information on the location of the RRKN1 gene. Therefore, we used three different strategies to determine the RRKN1 locus.

Genomic sequences flanking and including SNPs were determined using Geneious Prime 2019 (Biomatters) and sent to LGC Biosearch Technologies (https://www.biosearchtech.com) to develop KASP primers. The KASP reactions were performed according to previous studies (Devran and Kahveci, 2019; Kahveci et al., 2021) and carried out using the Hydrocycler (LGC Biosearch Technologies, UK) with a 61-55°C touchdown protocol. The fluorescent readings were performed for 2 min at 25°C using a FluOstar Omega Microplate Reader (BMG LABTECH Ortenberg, Germany).

The raw sequence reads of parent lines have been deposited in the Sequence Read Archive (SRA) under accession numbers SRR25056976 and SRR25056975 and are accessible via BioProject accession PRJNA988534.

In this experiment, we investigated changes in the resistance status of tomato plants inoculated with M. incognita K7 and S6 isolates when exposed to soil temperatures of 25°C and 32°C. MT12 (mi-1.2/mi1.2) and Seval (Mi-1.2/mi-1.2) F₁ plants were resistant to both isolates at 25°C soil temperature, whilst the Tueza (mi1.2/mi-1.2) F₁ cultivar was susceptible, exhibiting a large number of galls and egg masses, as expected (see Supplementary Figure 1 for genotypes of plant lines used). No significant differences were observed in the gall and egg mass numbers of both isolates on MT12 and Seval F₁ cultivar when exposed to 25°C soil temperature (p ≤ 0.05) (Table 1). Among the plants exposed to 32°C soil temperature, the highest egg mass and gall number was counted in Tueza F₁. Resistance was broken at 32°C in Seval F₁ carrying the Mi-1.2 gene; however, the gall and egg mass numbers were about half of those observed in the susceptible Tueza F1. Conversely, MT12 showed a resistant response against K7 and S6 isolates despite being exposed to 32°C soil temperature for one week (Table 1).

We also tested MT12 with nematode isolates that are naturally Mi-1.2-virulent at 25˚C soil temperature. The Mi-1.2 naturally virulent isolates V12 and V21 multiplied very well and produced egg masses on the roots of MT12, indicating that MT12 did not provide resistance to Mi-1.2 naturally virulent isolates. Also, Seval F₁ lines bearing the Mi-1.2 gene were overcome by the Mi-1.2-virulent isolates as expected. The isolates produced egg masses on roots of susceptible Tueza F₁ and caused galls. No statistically significant differences were observed in the numbers of galls between the two virulent isolates in the three tested plant lines (p ≤ 0.05) (Table 2).

We tested the parents and F₁ plants derived from the cross between the susceptible and resistant tomato lines for resistance to M. incognita at 32°C soil temperature. The F₁ individuals were resistant to M. incognita. The F₁ plant was selfed to obtain F₂ population. Of 130 F₂ individuals subjected to M. incognita, screening showed 91 resistant and 39 susceptible plants, giving a segregation ratio of 3:1 resistant:susceptible (χ2 = 1,73, p=0.18). This indicates that the resistance is controlled by a single dominant gene designated R esistant to R oot-K not N ematode 1 (RRKN1) at 32°C soil temperature.

Initially 55 SNPs on chromosome 6 of the tomato genome from SolCap were selected and converted to KASP markers. We first screened parents with these markers for polymorphism and 9 of them were found to be polymorphic (Supplementary Table 1). Then, F₂ populations were screened with these polymorphic markers. Phenotype and genotype data indicated that RRKN1 may be located on chromosome 6.

We also took a global genome mapping approach. We developed KASP markers for all chromosomes, except for 8 and 9, for which no SNPs were identified. Markers (Supplementary Tables 2, 3) were first used to screen parents to confirm polymorphism and the F₂ mapping population was screened. Results showed that only those KASP markers developed from chromosome 6 were linked to the pathological data for RRKN1. These findings supported our initial results where we used 9 KASP markers developed from SolCap. Therefore, we focused on chromosome 6 to confirm the location of RRKN1 gene and developed further new 17 KASP markers from chromosome 6 (Supplementary Table 2) to fine map the gene. Two flanking markers, KASP-6-2649872 and KASP-6-2919895, were identified placing the gene(s) in a 270 kb interval on chromosome 6 (Table 3; Figure 1).

We identified genes within the interval for RRKN1, using the ITAG annotation of tomato reference genomes SL.3 and SL.4 (Hosmani et al, 2019). There are 28 predicted genes within the RRKN1 interval according to ITAG 3.2 (Table 4) and 24 predicted genes according to ITAG version 4.0. The numbers of genes differ as genes Solyc06g008770.2 (Mi-1E, CNL4), Solyc06g008790.3 (Mi-1F, Mi-1.1) and Solyc06g008800.2 (Mi-1G, CNL6) in ITAG 3.2 disappeared in ITAG 4.0 and two genes encoding transport inhibitor response 1 protein are also missing from ITAG 3.2.

The genes Mi-1E, Mi-1F and Mi-1G within the interval fall within a clade of nematode-specific R genes, as sequence similarity searches using BLASTP against the NCBI’s Non-Redundant Proteins database revealed highest degree of similarity with R-genes previously implicated in resistance to nematodes in tomato and pepper. As tomato accession MT12 does not carry a functional allele of Mi-1.2, the most likely candidates for RRKN1 are Mi-1E, Mi-F and Mi-1G. Amino acid sequence alignments of these three putative R-proteins along with Mi-1.2 indicated these proteins are highly similar to each other but not identical (Figure 2).

As the mapping population was generated by crossing two F₁ parents, the genomic vicinity of the resistance gene is expected to be heterozygous. Therefore, we developed five new KASP markers were developed within the interval (Supplementary Table 2) and the markers were then used these to screen parents and the mapping population. The results confirmed the region to be heterozygous (Supplementary Table 4).