Development of PCR-based marker for resistance to Fusarium wilt race 2 in lettuce (Lactuca sativa L.)

Resistance to multiple races of Fusarium wilt is a desired trait in lettuce (Lactuca sativa L.) cultivars, as it is directly related to grower profitability. We focused on its resistance to the race 2 pathogen, and analyzed F2 individuals obtained from a cross between ‘VI185’ (resistance to race 2) and ‘ShinanoGreen’ (susceptible to race 2) for genetic mapping of its resistance locus. According to the ddRAD-seq analysis and resistance genotype prediction in each F2 individual by infection assay of derived F3 generation, a single semi-dominant locus on LG1 (qFOL1.2) was found to determine race 2 resistance, and a marker designated as LG1_v8_117.181Mbp showed complete co-segregation with the resistance phenotype. Fine mapping by identified PCR-based markers in this locus allowed to show that qFOL1.2 were located in the position of 116.468–117.974Mbp in LG1. Association of the resistance phenotypes and genotypes of the PCR-based markers for qFOL1.2 in 42 lettuce cultivars showed that genotypes of all the analyzed markers were uniform in all the susceptible cultivars, while their genotypes in the resistant cultivars were not always consistent. Nonetheless, only the genotype of LG1_v8_116.506Mbp was identical across all the 25 resistant cultivars. Thus, we defined that LG1_v8_116.506Mbp was a broadly available marker for selection of race 2-resistance in lettuce cultivars. Our results provide additional breeding tool for resistance to race 2, and can accelerate pyramiding of resistance loci to multiple races of Fusarium wilt in lettuce breeding program.

Abstract Resistance to multiple races of Fusarium wilt is a desired trait in lettuce (Lactuca sativa L.) cultivars, as it is directly related to grower profitability. We focused on its resistance to the race 2 pathogen, and analyzed F 2 individuals obtained from a cross between 'VI185' (resistance to race 2) and 'ShinanoGreen' (susceptible to race 2) for genetic mapping of its resistance locus. According to the ddRAD-seq analysis and resistance genotype prediction in each F 2 individual by infection assay of derived F 3 generation, a single semi-dominant locus on LG1 (qFOL1.2) was found to determine race 2 resistance, and a marker designated as LG1_v8_117.181Mbp showed complete co-segregation with the resistance phenotype. Fine mapping by identified PCR-based markers in this locus allowed to show that qFOL1.2 were located in the position of 116.  in LG1. Association of the resistance phenotypes and genotypes of the PCR-based markers for qFOL1.2 in 42 lettuce cultivars showed that genotypes of all the analyzed markers were uniform in all the susceptible cultivars, while their genotypes in the resistant cultivars were not always consistent. Nonetheless, only the Introduction Fusarium wilt (alias Root Rot Disease) of lettuce caused by Fusarium oxysporum f. sp. lactucae races 1, 2 and 3 presents a serious problem for lettuce production during summer in Japan (Fujinaga et al. 2001(Fujinaga et al. , 2003(Fujinaga et al. , 2005Ogiso et al. 2002). For plant breeding programs, molecular markers are powerful tools in marker-assisted selection (MAS) and gene pyramiding to develop new cultivars with the resistance to multiple races (Ashikari and Matsuoka 2006;Kumar et al. 2011). Race 1 is widespread worldwide, and intensive studies on the resistance to race 1 have been undertaken by various expert groups (Tsuchiya et al. 2004;McCreight et al. 2005;Cabral and Reis 2013;Cabral et al. 2019;Seki et al. 2020a). Race 1-resistance loci were found to be located on LG1, LG2, LG4, LG7, and LG8 (Michelmore 2010(Michelmore , 2013Seki et al. 2020a), and it was succeeded in fine mapping of two QTLs on LG7 and LG8 (Seki et al. 2020a). Resistance to the race 2 was suggested to be determined by a single locus and RAPD marker WF25-42, showing linkage to its locus, was developed (Aruga et al. 2012). However, position of race2resistance locus was undefined and the number of its linked markers was still limited. Our objective, therefore, is to develop a more useful marker tightly linked to resistance gene to race 2 for MAS.
Next generation sequencing (NGS) based genotyping methods are extremely useful for designing molecular marker such as genome wide single nucleotide polymorphisms (SNPs) (Baird et al. 2008;Abe et al. 2012;Matsumura et al. 2014). Double digest restriction site-associated DNA sequencing (ddRADseq), high-throughput analysis of multiple samples, allow for the rapid construction of linkage map. Reference genome sequence of lettuce (Reyes-Chin-Wo et al. 2017) was already provided from Lettuce Genome Resource (LGR; https://lgr.genomecenter. ucdavis.edu), and ddRAD-seq was applied to a F 2 population obtained from a cross between 'VI185' (resistance to race 2) and 'ShinanoGreen' (susceptible to race 2) so far (Seki et al. 2020b). We performed an inoculation test with race 2 pathogen using the F 3 population in this study. In addition, by employing Genomic DNA-seq of parents (Seki et al. 2020b), we tried to develop a reliable PCR-based marker in the genomic vicinity of race 2-resistance locus for MAS breeding.

Plant materials and pathogen
All plant material was grown at the Nagano Vegetable and Ornamental Crops Experiment Station (Shiojiri City, Nagano prefecture, Japan; 36°10 0 N, 137°93 0 E). The crisphead types 'VI185', 'Shi-nanoGreen' are cultivars from the Nagano Vegetable and Ornamental Crops Experiment Station lettuce breeding program. The F 2 population was derived from a cross between 'VI185' and 'Shi-nanoGreen'. Ninety-six individuals of the F 2 progeny from the 'VI185' by 'ShinanoGreen' cross were used for linkage analysis and to produce selfed F 3 populations, which were used to evaluate genotypes of disease resistance in the F 2 individuals. For the infection assay of Fusarium oxysporum f. sp. lactucae race 2, the Japanese isolate F-9501 (Fujinaga et al. 2001) was employed.
Infection assay for disease resistance Infection assay for disease resistance was performed at greenhouse of the Nagano Vegetable and Ornamental Crops Experiment Station. Infested soils using the isolate F-9501 (race 2) were prepared by mixing bran culture medium and sterile soil at a ratio of 1:19 by volume (Tsuchiya et al. 2004;Seki et al. 2020a). Seeds were sown directly into artificially inoculated soil, and seedlings were grown in the greenhouse conditions at a temperature between 15°C and 35°C under natural day length. Approximately 1 month after germination, the respective seedlings were examined and scored using the disease index (0, no symptoms; 1, partial necrosis of leaf only; 2, leaf necrosis, stunting, and wilting; 3, severe wilting and death). The disease severity was calculated as described by Aruga et al. (2012). Twenty individuals of 'VI185 (resistance to race 2)', 'ShinanoGreen (susceptible to race 2)', and each of 96 F 3 populations derived from a cross 'VI185' and 'ShinanoGreen' were evaluated one repetition.
Linkage map by ddRAD-seq and whole genome sequencing by Genomic DNA-seq Previously constructed linkage map (Seki et al. 2020b) was used for QTL mapping of race 2-resistance locus. Previously analyzed whole genome sequencing data (Seki et al. 2020b) was used for genetic polymorphism analysis and designing PCR-based markers. Using the annotations of reference genome sequence, genetic polymorphism analysis was performed using the IGV software between 'VI185' and 'ShinanoGreen' about coding regions of the gene models.

Designing PCR-based markers and their amplification
Polymorphisms between parental lines around the QTL, including insertion, deletion and SNP, were surveyed to identify the marker sites using the IGV software. Primers for amplifying the markers were designed using the Primer3 website (http://bioinfo.ut. ee/primer3-0.4.0/), and their IDs (names) were defined as (Linkage group) _ (genome version) _ (genome position). PCR was performed using 0.5 lL DNA template, 0.4 lL of each primer (50 lM), 2 lL dNTP (2 mM), 5 lL 2 9 PCR Buffer, 0.2 lL KOD FX (1 U/lL, TOYOBO, Japan), and distilled water (dH 2 O) to a final volume of 10 lL. PCR conditions were as follows: 94°C for 5 min, 30 cycles of 94°C for 30 s, and 61°C for 30 s, followed by one cycle at 72°C for 4 min. The amplification products were electrophoresed using capillary electrophoresis apparatus, MCE202 MultiNA (Shimadzu, Kyoto, Japan) and visualized by control and MultiNA Viewer software.

QTL mapping of disease resistance
The disease severity of the F 3 population derived from ninety-six individuals of the F 2 progeny were evaluated according to the results of infection assay. Genetic mapping of the F. oxysporum f. sp. lactucae race 2 resistance locus in 'VI185' was performed using previously analyzed ddRAD-seq data from an F 2 population derived from a cross between 'VI185' and 'ShinanoGreen'. Composite interval mapping (CIM) was conducted using the Haley-Knott regression in R using R/qtl package (Broman et al. 2003). The genome wide LOD threshold at 1% significance level was determined using a 10,000 permutation test. The proportion of phenotypic variance was calculated from the value at the peak indicated by CIM. A detailed script is described in CIM_script.R (https:// github.com/KousukeSEKI/RAD-seq_scripts).
The resistance phenotype was visualized graphically with the plotPXG function (Broman et al. 2003). MapChart ver. 2.3 was used to depict the linkage map and QTL (Voorrips 2002).

Results
Inheritance of resistance to F. oxysporum f. sp. lactucae race 2 Disease-resistance genotypes of the F 2 individuals from a cross between resistant 'VI185' and susceptible 'ShinanoGreen' were determined by the infection assay of the subsequent F 3 generation by F. oxysporum f. sp. lactucae race 2. Since the correlation between their disease severity of shoot and root has already been reported by Aruga et al. (2012) and Seki et al. (2020a), we focused on disease resistance in shoot tissue. Resistance to race 2 derived from 'VI185' was suggested to be controlled by a single semi-dominant gene, according to segregation of putative genotype of resistance gene showing 1:2:1 ratio in the F 2 population (Table 1). This result coincides to the previous studies (Shimazu et al. 2009;Aruga et al. 2012).
ddRAD-seq analysis and fine mapping of resistance to race 2 Genetic mapping of the F. oxysporum f. sp. lactucae race 2 resistance derived from 'VI185' was performed using previously analyzed RAD-seq data and linkage map of F 2 population derived from a cross between 'VI185' and 'ShinanoGreen' (Seki et al. 2020b). CIM analysis was performed using the disease severity scores determined by race 2 pathogen infection assay and marker genotype data from the ddRAD-seq in 96 F 2 individuals. From the CIM analysis, a single locus for the resistance to the race 2 was mapped in LG1 between two markers (LG1_v8_113.602Mbp and LG1_v8_130.852Mbp) at a 9.55 cM interval, and designated as qFOL1.2. Genotype of the LG1_v8_117.181Mbp marker, located in the qFOL1.2 locus, showed complete co-segregation with resistance to race 2 in the F 2 population (Figs. 1, 2), and qFOL1.2 accounted for approximately 96.47% of the phenotypic variance. The additive effect was -48.18, and the dominant effect was -6.64. According to the genotypes of biallelic RAD tags around qFOL1.2 and segregation of the deduced resistant locus genotype deduced in F 2 plants, qFOL1.2 was located between LG1_v8_116.506Mbp and phenotypes (disease severity to Fusarium wilt). Twenty individuals of each of F 2:3 plants derived from a cross 'VI185 (resistant)' and 'ShinanoGreen (susceptible)' were applied to genotyping and phenotype scoring. In genotype, ''A'' or ''B'' allele was derived from 'VI185' or 'ShinanoGreen', respectively 116.225 to 118.058 Mbp in LG1 (Fig. 2). Furtherly, its fine mapping using PCR-based markers (Table 2) demonstrated that location of qFOL1.2 was narrowed down between LG1_v8_116.468Mbp and LG1_v8_117.974Mbp markers in LG1 (Table 2, Fig. 2). Genetic distance between these two marker loci was 1.028 cM, corresponding to 1.505Mbp as physical distance. To develop practical DNA markers for qFOL1.2 applicable to the marker assisted selection (MAS) in the breeding program, polymorphisms between parental lines were surveyed in the genome sequences within the range between 116.468-117.974Mbp in LG1. The eight PCR-based markers were identified by employing the selected polymorphisms. The marker types of the eight PCRbased markers were one ARMS marker designed using SNPs, two SSR markers, and five INDEL markers (Table 2). These selected eight PCR-based markers were applied to genotyping analysis of 25 resistant and 17 susceptible lettuce cultivars to race 2 (Tables 2, 3, Fig. S1). In all the analyzed cultivars, all the marker genotypes represented homozygous genotypes. Genotypes of these markers demonstrated the susceptible cultivars uniformly showed the same genotype in this region, while inconsistent genotypes of most analyzed markers were observed in the resistant cultivars (Table  3). Of the 8 markers, only LG1_v8_116.506Mbp showed the consistent genotype in all the analyzed resistant cultivars. Therefore, LG1_v8_116.506Mbp was the widely available marker for MAS of race 2 resistant plants.
Genetic polymorphism analysis of the gene models located in the qFOL1.2 According to the annotation of 'Salinas' genome sequence, 23 open reading frames (ORFs) are located in the qFOL2.1, but there are no disease resistance gene candidates. Comparing genomic sequences of those 23 ORFs between 'VI185' and 'ShinanoGreen', 6 ORFs have identical genomic sequences. Three ORFs have nucleotide substitutions, and 12 ORFs have amino acid substitutions. Three ORFs have deletion mutations in 'VI185' (Table 4).

Discussion
Following the previous QTL mapping study for the resistance to Fusarium wilt race 1 (Seki et al. 2020a), we succeeded in rapid genetic mapping of resistance to race 2 in lettuce, since F 2 population derived from a cross between 'VI185' and 'ShinanoGreen' with their genome-wide marker genotypes (Seki et al. 2020b) were already available for the QTL analysis. Consequently, a single major QTL, qFOL1.2, derived from 'VI185' was efficiently identified in LG1. According to the genetic mapping using these ddRAD-seq and PCR-based markers, resistance to race 2 was probably determined by a single locus, and located between 116.468Mbp and 117.974Mbp in LG1 of the reference genome (Tables 1, 3, Fig. 2). Previously, we developed a marker WF25-42 linked to the resistance locus to race 2 by RAPD and AFLP analysis (Aruga et al. 2012) and it also located at the position of 117.9Mbp in LG1 (data not shown), supporting that qFOL1.2 was the locus for the resistance to race 2.

974Mbp
NaganoVenus 10  LG1_v8_116.506Mbp, can discriminate between resistant and susceptible to race 2 perfectly, suggesting that it is widely applicable to the selection of qFOL1.2 in the breeding program and suggested to be closed to the causal gene for the resistance. Notably, no disease resistance gene candidate was found in the annotation database for 'Salinas' genome even though this locus, between 116.468Mbp and 117.974Mbp in LG1, was located in a major resistance clusters (MRCs) ( Table 4) (Christopoulou et al. 2015). Because 'Salinas' is a susceptible cultivar to race 2, it is possible that it may lack its disease resistance gene. Therefore, further studies, including genome resequencing analysis of the resistant cultivar, are necessary to determine the causal gene located in qFOL1.2.
Fusarium wilt frequently occurs under high-temperature environment (Tsuchiya et al. 2004), like summer season in Japan. Therefore, global warming advance was supposed to promote Fusarium wilt occurrence in other countries. Three races of Fusarium wilt (race 1,2, and 3) were already found in Japan (Fujinaga et al. 2001(Fujinaga et al. , 2003. Furthermore, its race 4  (Gilardi et al. 2017(Gilardi et al. , 2019Claerbout et al. 2018). Considering difficulty of soil sterilization in the field, development of resistance cultivars is the most efficient strategy for avoiding disease. Because there were the many cultivars with race 1-resistance already reported (Tsuchiya et al. 2004;Michelmore 2010Michelmore , 2013Cabral et al. 2019;Seki et al. 2020a), MAS to introduce race 2-resistance to those cultivars with race 1-resistance allow for the cultivars with more durable resistance to Fusarium wilt. The findings of this study will contribute to the pyramiding approach for disease resistance loci to multiple races of Fusarium wilt.