Novel Sources of Resistance to Fusarium oxysporum f. sp. lycopersici Race 3 Among Solanum pennellii Accessions

in Journal of the American Society for Horticultural Science
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  • 1 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611
  • | 2 Plant Genetic Resources Unit, U.S. Department of Agriculture, Agricultural Research Service, 630 W. North Street, Geneva, NY 14456
  • | 3 Gulf Coast Research and Education Center, University of Florida, Wimauma, FL 33598; and Plant Pathology Department, University of Florida, Gainesville, FL 32611
  • | 4 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611; Gulf Coast Research and Education Center, University of Florida, Wimauma, FL 33598; and Plant Molecular and Cellular Biology Graduate Program, University of Florida, Gainesville, FL 32611
  • | 5 Horticultural Sciences Department, University of Florida, Gainesville, FL 32611; and Gulf Coast Research and Education Center, University of Florida, Wimauma, FL 33598

Fusarium wilt of tomato (Solanum lycopersicum), caused by fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), is one of the most important diseases in tomato production. Three races of the pathogen are described, and race-specific resistance genes have been applied in commercial tomato cultivars for controlling the disease. Race 3 (Fol3) threatens tomato production in many regions around the world, and novel resistance resources could expand the diversity and durability of Fol resistance. The wild tomato species, Solanum pennellii, is reported to harbor broad resistance to Fol and was the source of two known Fol3 resistance genes. In this study, we evaluated 42 S. pennellii accessions for resistance to each fusarium wilt race. F1 plants, developed from crossing each accession with the Fol3 susceptible line ‘Suncoast’, were evaluated for Fol3 resistance, and BC1F1 plants were screened to determine the likelihood that Fol3 resistance was based on a novel locus (loci). Nearly all accessions showed resistance to Fol3, and many accessions were resistant to all races. Evaluation of F1 plants indicated a dominant resistance effect to Fol3 from most accessions. Genetic analysis indicated 24 accessions are expected to contain one or more novel Fol3 resistance loci other than an allele near the I-3 locus. To investigate genetic structure of the S. pennellii accessions used in this study, we genotyped all 42 accessions using genotyping by sequencing. Approximately 20% of the single nucleotide polymorphism (SNP) loci were heterozygous across accessions, likely due to the outcrossing nature of the species. Genetic structure analysis at 49,120 unique SNP loci across accessions identified small but obvious genetic differentiations.

Abstract

Fusarium wilt of tomato (Solanum lycopersicum), caused by fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), is one of the most important diseases in tomato production. Three races of the pathogen are described, and race-specific resistance genes have been applied in commercial tomato cultivars for controlling the disease. Race 3 (Fol3) threatens tomato production in many regions around the world, and novel resistance resources could expand the diversity and durability of Fol resistance. The wild tomato species, Solanum pennellii, is reported to harbor broad resistance to Fol and was the source of two known Fol3 resistance genes. In this study, we evaluated 42 S. pennellii accessions for resistance to each fusarium wilt race. F1 plants, developed from crossing each accession with the Fol3 susceptible line ‘Suncoast’, were evaluated for Fol3 resistance, and BC1F1 plants were screened to determine the likelihood that Fol3 resistance was based on a novel locus (loci). Nearly all accessions showed resistance to Fol3, and many accessions were resistant to all races. Evaluation of F1 plants indicated a dominant resistance effect to Fol3 from most accessions. Genetic analysis indicated 24 accessions are expected to contain one or more novel Fol3 resistance loci other than an allele near the I-3 locus. To investigate genetic structure of the S. pennellii accessions used in this study, we genotyped all 42 accessions using genotyping by sequencing. Approximately 20% of the single nucleotide polymorphism (SNP) loci were heterozygous across accessions, likely due to the outcrossing nature of the species. Genetic structure analysis at 49,120 unique SNP loci across accessions identified small but obvious genetic differentiations.

Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), is a serious disease of tomato (Solanum lycopersicum) grown in warm production regions. The pathogen penetrates the root and infects the vascular system, blocking the supply of water and nutrition, and results in wilting, stunting, and, ultimately, plant death. The disease can cause devastating crop losses, and heavily infested fields were often abandoned before the availability of resistant cultivars (McGrath et al., 1987; Scott and Jones, 1989). Cultural practices, such as raising the soil pH level, using nitrate-nitrogen, and exercising sanitary practices to reduce spread of inoculum, can contribute to disease management, especially when combined with soil fumigation (Jones and Overman, 1985; Jones and Woltz, 1968, 1983). However, host resistance is the most effective and economical means of controlling Fol and has been the primary strategy for controlling Fol in field-grown tomato production since the 1940s.

There are three races of Fol that infect tomato, and these are distinguished based on their response to three dominant, race-specific resistance genes, each derived from wild tomato species. The I (immunity to fusarium wilt) and I-2 genes confer resistance to races 1 (Fol1) and 2 (Fol2), respectively. Both genes are located on chromosome 11 and were introgressed from accessions of S. pimpinellifolium (Bohn and Tucker, 1939; Paddock, 1950; Simons et al., 1998; Stall and Walter, 1965). I-3, which was introgressed from S. pennellii accession LA716, is located on chromosome 7 and confers resistance to race 3 (Fol3) and is reported to confer resistance to races 1 and 2 as well (Lim et al., 2008; Scott and Jones, 1989). Currently, only the I, I-2, and I-3 genes are used for Fol resistance in commercial cultivars; but besides these, several other resistance genes have been reported. Sela-Buurlage et al. (2001) described I-4, I-5, and I-6 in S. lycopersicum and S. pennellii, each conferring Fol2 resistance. More recently, Gonzalez-Cendales et al. (2016) cloned a second dominant Fol3 resistance gene, I-7, from S. pennellii accession LA1724 (PI 414773). Notably, these authors found that the I-7 transgene conferred resistance against all three races of Fol, suggesting that I-7 may provide broad efficacy. However, expression of this transgene was driven by the CaMV 35S promoter, and the effect of I-7 against other races when under its native promoter has not been clearly demonstrated. Currently, I-7 is not widely used for the development of commercial cultivars, likely because I-3 is already deployed and provides effective Fol3 control, and also because it was only relatively recently that I-7 was distinguished from I-3 (Lim et al., 2006).

Fol3 was originally discovered in Australia in 1978 and in Florida in 1982, and it now exists in many production regions around the world (Cai et al., 2003; Correll and Jones, 2014; Grattidge and O’Brien, 1982; Volin and Jones, 1982). Cultivars containing I-3 have been commercially available since the early 1990s, but there has been considerable difficulty developing commercially acceptable Fol3-resistant hybrids due to the association of I-3 with several negative traits. Some of the earliest Fol3-resistant parental lines showed a higher incidence of blossom end rot than susceptible lines, and this issue is occasionally observed today (Scott 2004). It was also reported that I-3 has a negative effect on fruit size, resulting in a lower percentage of extra-large fruit—the size for which growers usually receive higher prices (Scott, 1999). Furthermore, the I-3 locus has been associated with greater sensitivity to bacterial spot disease caused by multiple Xanthomonas species, and Hutton et al. (2014) demonstrated that homozygosity for I-3 resulted in up to 20% more disease severity compared with homozygosity for the susceptible allele. Fortunately, the effects on fruit size and bacterial spot are caused by linkage drag, and reducing the size of the I-3 introgression can eliminate these problems while maintaining Fol3 resistance (Chitwood-Brown et al., 2021; Li et al., 2018).

Such negative effects are not associated with the I-7 introgression (Chitwood-Brown et al., 2021) and neither have they been reported for the I or I-2 introgressions, which are common resistance genes in much of the commercial germplasm worldwide. In light of this, we hypothesize that problems such as these may be specific to the I-3 introgression, and that alternative, novel Fol3 resistance genes might be free of such effects. Furthermore, the discovery and deployment of novel resistance genes may play a critical role in preventing the emergence of new Fol races in the future by expanding the diversity of Fol3 resistance mechanisms. There are more than 40 S. pennellii accessions available from the C.M. Rick Tomato Genetics Resource Center (TGRC) at the University of California, Davis (TGRC, 2011). Scott and Jones (1990) tested many of these and reported broad resistance to multiple Fusarium species in all accessions tested. It is possible that Fol3 resistance in some accessions is based on genetics other than I-3 and I-7. Considering this, a genetic structure analysis may provide insight into the relationship among these accessions, allowing research efforts to be focused on a smaller number of accessions that are genetically less similar to one another and possibly to retain diversity in resistance alleles. Likewise, such information may be useful for other research efforts seeking to mine S. pennellii for specific traits or novel alleles.

In this study, we investigate a large collection of S. pennellii accessions as potential sources of novel Fol3 resistance. We characterize 42 accessions for resistance to all three races of fusarium wilt and identify potential sources of broadly effective resistance alleles. We also evaluate interspecific F1 and backcross populations corresponding to each accession to identify those that are likely sources of novel, Fol3 resistance loci. Finally, we use genotyping by sequencing [GBS (Elshire et al., 2011)] to generate a collection of SNPs between S. pennellii accessions and the tomato reference genome for genetic structure analyses.

Materials and Methods

Plant materials.

Seeds of 42 S. pennellii accessions were obtained from TGRC. Each accession was increased by manually sib-mating at least six plants of each accession in a greenhouse located at the University of Florida Gulf Coast Research and Education Center (GCREC) in Wimauma, FL. Information regarding geographic origin for each accession was obtained from the TGRC online database, including latitude, longitude, elevation, and habitat of the collection site, and collection sites were pinpointed on a map (Fig. 1). For each accession, an interspecific F1 was made by bulking pollen from six or more plants of the accession and hybridizing with the Fol3 susceptible cultivar, Suncoast (Scott et al., 1985). In the F1, pollen was likewise bulked from six or more Fol3-resistant plants and then backcrossed to ‘Suncoast’ to create modified BC1F1 populations for each accession.

Fig. 1.
Fig. 1.

Genetic structure of 42 Solanum pennellii accessions used in this study. (A) Genetic structure of 42 S. pennellii accessions primarily corresponds with geographic areas. Symbols (circle, empty triangle, cross) are based on three genetic clusters inferred using discriminant analysis of principal components (DAPC) shown on Supplemental Fig. 1. A single or double asterisk indicates the presence of single or multiple novel Fol3 resistance loci, respectively. (B) Neighbor-joining tree of 42 S. pennellii accessions also reveals an association between accessions’ geographic origins and phylogenetic clustering. Accessions in the tree are represented by different symbols: empty square (accessions collected north of latitude 10°S); half-filled square (between latitudes 10°S and 14°S); and filled square (south of latitude 14°S).

Citation: Journal of the American Society for Horticultural Science 147, 1; 10.21273/JASHS05080-21

Similarly, resistance from accessions LA750, LA1297, LA1299, LA1367, LA1522, LA1734, and LA1926 was advanced into cultivated tomato to the fourth backcross using a modified backcrossing approach, wherein disease assays were again used at each generation to identify resistant plants for backcrossing. Molecular markers linked to I-3 and I-7 (Supplemental Table 1) were used to select away from these known loci and advance novel Fol3 resistance. We selected against the I-3 locus at the BC1F1 generation for most accessions (except for LA1926, for which the I-3 locus was maintained); and we tested to confirm absence of the I-7 locus in BC4F1 plants. Screening for I-7 was not accomplished sooner because these generations were advanced before the discovery of the chromosomal location of I-7 and the development of genetic resources for this locus by Gonzalez-Cendales et al. (2016). Seed was collected from individual BC4F1 plants for each population resulting in segregating BC4F2 populations.

Throughout all experiments, seed of accessions, interspecific F1s, and BC1F1 populations were pretreated in a 2.7% sodium hypochlorite solution for 30 min before sowing in spent coal medium (Black Beauty; Harsco Minerals International, Mechanicsburg, PA) in a growth room. Controls used for disease screens included ‘Bonny Best’ (susceptible to all races), ‘Manapal’ [Walter et al., 1961 (contains I; resistant to Fol1 but susceptible to Fol2 and Fol3)], ‘Horizon’ [Scott et al., 1985 (contains I and I-2; resistant to Fol1 and Fol2 but susceptible to Fol3)], and Fla. 7547 [Scott and Jones, 1995 (contains I, I-2, and I-3; resistant to Fol1, Fol2, and Fol3)].

Fusarium wilt disease screening.

Fungal strains of Fol races 1 (GEV2997), 2 (GEV3520), and 3 (GEV1400) were obtained from the University of Florida Plant Pathology laboratory at GCREC. The pathogen was grown on potato dextrose agar media at 28 °C for 1 week before inoculation. Inoculum was prepared by removing the fungal colony with agar from plates and blending into 100-mL deionized water per plate, then adjusting the fungal suspension to a concentration of 107 spores/mL. For each race inoculation, 10- to 14-d-old seedlings were inoculated by dipping the roots into inoculum for 15 s. Inoculated seedlings were immediately transplanted into 128-well trays (38 cm3 cell size; Speedling, Sun City, FL) filled with peat-based soilless media (Speedling) and placed in the greenhouse for disease development. Four to eight plants each of a resistant and a susceptible control were included in each tray; controls included ‘Bonny Best’ and ‘Manapal’ for Fol1 screens, ‘Manapal’ and ‘Horizon’ for Fol2 screens, and ‘Horizon’ and Fla. 7547 for Fol3 screens. Plants were evaluated for disease incidence ≈3 weeks after inoculation according to procedures described previously (Scott et al., 2004). Briefly, plants were rated as diseased if they showed any characteristic external symptoms of fusarium wilt, such as stunting, yellowing of foliage, wilting, enlarged hypocotyl, or death. Stems of plants with questionable symptoms were dissected and rated as diseased if they had vascular browning. Otherwise, plants were scored as healthy and were considered resistant, and numbers of healthy and diseased plants were recorded.

Three separate disease screens were used to evaluate resistance of S. pennellii accessions to Fol1, Fol2, and Fol3. Interspecific F1s were evaluated in two separate Fol3 screens, and both BC1F1 and BC4F2 populations were evaluated in single Fol3 screens. The two screens of interspecific F1s produced similar results, so plant numbers were combined across them. As sufficient plants were available, 32 plants of each accession and each F1, 80 plants of each BC1F1, and 56 plants of each BC4F2 were evaluated in each disease screen.

Genetic analysis of resistance to Fol3.

Each accession and its interspecific F1 were evaluated for Fol3 resistance to assess the presence/absence of dominant Fol3 resistance alleles. BC1F1 families were subsequently evaluated to determine the likelihood that Fol3 resistance is controlled completely or in part by the I-3 locus, or by a locus/loci besides I-3. Three molecular markers, linked to the I-3 locus and distinguishing S. pennellii and S. lycopersicum alleles, were used for determining the presence of an I-3 locus effect. Before disease development, BC1F1 plants were individually screened with one or more of these markers to determine genotype at the I-3 locus. For each BC1F1 backcross population, each plant was assigned to one of the following four groups: 1) healthy and heterozygous for an introgression at the I-3 locus, 2) healthy and lacking this introgression, 3) diseased and heterozygous for this introgression, or 4) diseased and lacking this introgression.

Within each BC1F1 family, a χ2 contingency test was used to determine if the presence of an S. pennellii introgression at the I-3 locus affected Fol3 resistance. The null hypothesis for this test was that an introgression at this locus had no effect on resistance, and there would therefore be no difference in the healthy to diseased ratio for plants that possessed and for plants that lacked an I-3 locus introgression. When the null hypothesis was rejected, it was concluded that resistance in the accession is conferred completely or in part by the I-3 locus. Then, where the null hypothesis was accepted, it was concluded that resistance in the accession is not based on I-3 but is based entirely on alleles other than I-3.

A χ2 test was also used to test for the presence of novel resistance loci. For families of accessions determined to have no effect at the I-3 locus, calculations were based on all BC1F1 plants; and for families of accessions that were determined to harbor a resistance allele at/near the I-3 locus, calculations were based only on plants lacking an S. pennellii introgression in this region. Numbers of healthy and diseased plants were tested for goodness of fit to an expected 1:1 and/or 3:1 ratio to test for the presence of one or two alternative resistance loci, respectively. Where families had an unacceptable fit to the 3:1 ratio due to an excess of healthy plants, it was concluded that resistance may be conferred by more than two alternative resistance loci.

Fol3 resistance was further evaluated in BC4F2 families, and molecular markers linked to I-3 were used as previously described to confirm the presence or absence of this locus. Chi-square analyses of BC4F2 families were used to test healthy to diseased ratios for goodness of fit to an expected 3:1 or 15:1 ratio for the presence of one or two resistance genes, respectively.

Genotyping by sequencing.

Young foliar tissue was collected separately from two individual plants from each S. pennellii accession. Genomic DNA was extracted using the Plant DNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA was normalized to 5 ng·µL−1 based on quantification with a multimode microplate reader (Synergy 2; Biotek Instruments, Winooki, VT) with the Quant-iT PicoGreen (Thermo Fisher Scientific, Waltham, MA) double-stranded DNA. An ApekI-based GBS library was constructed using previously published protocols, barcode-adapters, and polymerase chain reaction primers (Elshire et al., 2011). The final GBS library (total 84 libraries; two libraries per each accession) was analyzed by automated electrophoresis (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA) before sequencing to confirm and examine fragment size distribution and quantities. The GBS library was submitted for 101-cycle single-end sequencing on one lane of an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA) at the Weill Cornell Medical College Genomics Resources Core Facility.

One multiplexed sequencing run generated a total of 140,179,147 reads with barcodes from 42 S. pennellii accessions. The raw sequencing data were processed using the TASSEL 5 GBS v2 pipeline (Bradbury et al., 2007). Bowtie 2 with the –very-sensitive option (Langmead and Salzberg, 2012) was used to align the reads to the tomato reference genome (Tomato SL2.50 ITAG2.4; Sol Genomics Network, 2019). Only 80–base pair kmers (reads) present at least five times in the dataset were considered. Minimum quality score within the barcode and read length was set for 20. SNPs were not filtered by minor allele frequency. For each SNP, only sequence polymorphism common to both individual plant samples of each accession were considered. SNPs with >95% missing data were discarded.

Genetic structure analyses.

We explored genetic differentiation among 42 S. pennellii accessions genotyped using the GBS method. We used K-means clustering, which clusters objects to maximize between group variation, for K = 1 through 8 implemented in the adegenet package (Jombart, 2008) for the R software (version 3.0.1; R Foundation, Vienna, Austria). We used the Bayesian information criterion to select the number of clusters that best describes those data. Using the multivariate method [discriminant analysis of principal components (DAPC) (Jombart et al., 2010)], accessions were grouped into three clusters that were genetically diverse from one another. In addition, we built a phylogenetic tree for these accessions using the neighbor-joining method implemented in TASSEL 5 and the same genotype dataset that was used for DAPC.

Results

Fol resistance among S. pennellii accessions.

Disease screens of 42 accessions confirmed high levels of resistance to Fol in the S. pennellii species (Table 1). Overall, these accessions were less resistant to Fol1 than to Fol2 or Fol3. Whereas 27 accessions demonstrated some level of Fol1 resistance (≥50% healthy plants), only five of these exceeded 90% healthy plants, whereas 14 accessions had less than 50% healthy plants. Among the latter, accessions LA1926 (6% healthy) and LA2657 (15% healthy) were considered susceptible to Fol1. Most accessions were resistant to Fol2, and the percentage of Fol2-resistant plants ranged from 50% to 90% among these. LA1926 was the only accession clearly susceptible to Fol2, having 7% healthy plants. Resistance was highest and most consistent for Fol3, for which 31 accessions had more than 90% healthy plants, and 39 accessions had more than 70% healthy plants. LA1733 and LA1912 had less than 70% healthy plants.

Table 1.

Resistance among Solanum pennellii accessions to Fusarium oxysporum f. sp. lycopersici (Fol) races 1, 2, and 3, and resistance of corresponding Solanum lycopersicum × S. pennellii interspecific hybrids to race 3.

Table 1.
Table 1.

Many of the interspecific F1s were resistant to Fol3 and displayed similar levels of resistance as the parental accession, suggesting dominant gene action and/or homogeneity of resistance alleles within the accession. Some accessions (LA1724, LA1732, LA1809, LA1911, LA1912, LA1926) displayed lower levels of resistance in the interspecific F1, suggesting a lack of dominance and/or segregation of resistance. The possibility of heterogeneity for resistance was suggested for LA1912, as this parental accession LA1912 had only 63% healthy plants in the Fol3 assay and 27% healthy plants in the interspecific F1 (Table 1).

To further characterize Fol3 resistance in these materials, BC1F1 families corresponding to 40 of the accessions were evaluated in Fol3 disease screens, and molecular markers were used to detect the presence of an introgression at the I-3 locus (Table 2). Because only two crosses to susceptible cultivated tomato had been made, segregation for an introgression at the I-3 locus was expected for all families. However, this locus may or may not confer Fol3 resistance. To identify potential sources of novel resistance loci, we sought first to determine whether the I-3 introgression contributed to Fol3 resistance; and second, whether additional resistance loci are likely to exist.

Table 2.

Genetic analysis of Solanum lycopersicum × Solanum pennellii BC1F1 populations for identifying potential sources of novel resistance to fusarium wilt race 3.

Table 2.

Segregation for Fol3 resistance was observed for each BC1F1 family. An introgression near the I-3 locus was detected in all populations except for that derived from LA751; here, the linked markers were not polymorphic, and we were therefore unable to test for an I-3 locus effect. Chi-square analysis was used to determine an effect of the I-3 locus by comparing the total numbers of healthy and diseased plants with and without an introgression at I-3, which indicated the presence of a resistance allele at or near this locus in 18 of the populations (Table 2). For these populations, the presence of alternative resistance loci was determined with χ2 tests using the plants without an introgression at I-3. As expected, an effect at I-3 was detected in LA716, the source of I-3 in commercial germplasm, but analysis also indicated the presence of an alternative resistance locus besides I-3 in this accession. The possibility of one additional resistance locus was also indicated for LA750, LA1272, LA1297, LA1299, LA1340, LA1515, LA1522, LA1649, and LA1942. In LA1273, LA1303, LA1735, LA1911, LA1920, LA1926, and LA1946, segregation ratios among plants lacking an introgression at the I-3 locus did not support the presence of additional resistance alleles, and these accessions may thus serve little utility toward the identification of novel Fol3 resistance alleles. In LA1302, segregation ratios suggested the possibility of two resistance loci other than I-3.

For the 21 populations demonstrating no effect at the I-3 locus, the potential number of novel resistance loci were predicted with χ2 tests using the total number of plants screened (Table 2). Based on these analyses, resistance in 10 of the BC1F1 families may be conferred by a single locus, while two resistance loci likely confer Fol3 resistance in accessions LA1282, LA1656, and LA1674.

Besides those mentioned previously, eight BC1F1 families fit none of the segregation ratios tested, primarily because of a deficiency of resistant plants (Table 2). LA1912 was among these and demonstrated the lowest levels of resistance of all BC1F1 families. LA1367 was also among these, but in the case of this accession, there was no deficiency of resistant plants. Rather, its 51:29 ratio of healthy:diseased plants was slightly skewed such that it fit neither a 1:1 nor a 3:1 segregation ratio. Thus, although χ2 analyses were not conclusive, there is evidence that LA1367 contains at least one alternative Fol3 resistance locus.

We continued to advance resistance from some accessions into cultivated tomato by selecting resistant plants from disease assays for backcrossing. Resistance to Fol3 in several BC4F2 families is shown in Table 3. Chi-square tests of segregation ratios were used to substantiate predictions made in the BC1F1 families. We also used molecular markers linked to I-3 to confirm absence of an introgression (or in the case of LA1926, genotype for each BC4F2 plant) at this locus in each family. LA750, LA1297, LA1299, and LA1522 were previously determined to have an effect at the I-3 locus in addition to one alternative resistance locus. Here, I-3–linked markers confirmed the absence of an I-3 introgression in these BC4F2 families, and segregation ratios further supported the presence of one alternative locus conferring resistance in each family. As mentioned previously, the presence of at least one alternative Fol3 resistance locus was evidenced, although segregation ratios in its BC1F1 family did not fit either a 1:1 or 3:1 ratio. However, χ2 analyses of the BC4F2 population for LA1367 indicated two alternative genes conferring resistance (Table 3). LA1734 was formerly determined to have no effect at the I-3 locus and to contain one alternative resistance locus; analyses confirmed the absence of any introgression at the I-3 locus and supported the presence of a single novel locus conferring resistance. Resistance from LA1926, which was previously determined to be conferred solely by the I-3 locus, was also advanced, and the segregating BC4F2 population was subjected to disease and marker screens, as previously described. Chi-square analyses confirmed segregation of a single resistance locus, and further testing verified that the I-3 locus in fact cosegregated with resistance in this line (P < 0.0001).

Table 3.

Analysis of advanced resistance to fusarium wilt race 3 in Solanum lycopersicum BC4F2 populations.

Table 3.

Genetic structure analyses for S. pennellii accessions.

In this study, all accessions originated from Peru (Fig. 1A). Their geographic collection sites are distributed along the entire west coast, with the greatest distance between collection sites being 1537 km between accessions LA1089 and LA2963. The smallest distance between collection sites was less than 1 km, and this was observed between LA751 and LA1282, between LA1911 and LA1912, and between LA1942 and LA1943.

Analysis of data from GBS identified 1,378,871 SNPs across all accessions (Supplemental Tables 2 and 3). The number of monoallelic SNPs per accession ranged from 24,157 to 33,735, and the number of heterozygous SNPs per accession ranged from 3005 to 7205. The proportion of heterozygous SNPs was high, ranging from 10% to 27% across accessions and averaging 20%. This was likely due to the outcrossing nature of the species, and for this reason, heterozygous SNPs were not excluded from further analyses. Upon comparing all SNP loci among the accessions, a final set of 49,120 unique SNP loci were determined, and these were used for subsequent analyses.

K-means clustering indicated that different patterns of genetic structure could be used to describe the genotype data of all 42 accessions (Supplemental Fig. 1). The mode of K = 3 found significant structuring between accessions collected from areas above an approximate latitude 14°S (two clusters with 29 accessions) and accessions collected from below this latitude (a single cluster with 13 accessions) (Fig. 1A). A third cluster with three accessions (LA751, LA1732, LA1303) showed no clear geographic differentiation from the rest of the accessions collected from areas above an approximate latitude 14°S, but was reassigned to a different genetic structure.

We also investigated genetic structure using a phylogenetic analysis, and this likewise revealed an association between geographic origins and the occurrence of phylogenetic clustering (Fig. 1B). Importantly, we observed that the accessions with novel Fol3 resistance alleles are not confined to a single clade but are present in various clades. Among all accessions, the smallest pairwise genetic distance was between accessions LA716 and LA1943, and the largest genetic distance was between LA1943 and LA2560.

Discussion

Much of the disease resistance used in hybrid tomato breeding is conferred by dominant resistance genes. The present study investigated a collection of 42 S. pennellii accessions for their potential to harbor novel Fol3 resistance loci. The use of screenings at the F1, BC1F1, and subsequent BCF1 generations has likely biased our results toward detection of dominant resistance alleles that confer a high level of resistance. We used χ2 analyses to test segregation ratios in backcross families to determine which accessions are likely to contain novel Fol3 resistance loci, thereby providing some direction for programs that may wish to pursue research in this area. Although these estimates can be useful, they may not be completely accurate in all cases, because segregation ratios in wide crosses such as ours can deviate from normal (Rick, 1969). The estimates were also based on the assumption that all detectable resistance alleles were homogeneously present in the interspecific F1. Although we used only Fol3-resistant F1 plants to generate BC1F1 seed, we recognize that S. pennellii is primarily an outcrossing species, and that this assumption may have been violated in some instances if multiple resistance alleles were present and segregating within an accession.

Our screening confirmed a very high level of Fol3 resistance in most S. pennellii accessions. Resistance to Fol3 appeared to be dominant in most accessions, as evidenced by the high survival rates among interspecific F1s. This is of particular practical importance, given the utility of dominant resistance genes for hybrid breeding. In general, survival rates in the F1s were slightly lower relative to the donor accessions, which might indicate some level of incomplete dominance. But another likely explanation is that this resulted from heterogeneity of resistance, which may also explain why Fol3 resistance deviated from 100% for several of the accessions. F1 survival rates for some of the accessions (i.e., LA1724, LA1732, LA1911, and LA1912) were markedly low, possibly due to heterogeneity and/or heterozygosity of resistance within the accessions, incomplete dominance of resistance, or segregation distortion for the resistance alleles. In the analysis of BC1F1 populations, both LA1724 and LA1732 were predicted to contain one resistance locus. LA1724 is the reported source of Fol3 resistance gene I-7, and it is likely that the resistance in this population is due to this gene. Furthermore, Catanzariti et al. (2015) demonstrated that I-7 confers resistance to Fol1 and Fol2, and we also observed LA1724 to have resistance to Fol1 (78% healthy) and Fol2 (83% healthy) (Table 1). Therefore, for the purpose of identifying novel sources of Fol resistance, this accession may be of minimal interest.

Several accessions displayed lower resistance or were susceptible to Fol1 and/or Fol2. These accessions may therefore be less desirable as potential sources of novel alleles, if the goal is to identify sources of broad resistance toward improved durability. Bournival et al. (1990) suggested that the I-3 introgression confers resistance to Fol3 as well as Fol1 and Fol2, but it was unclear from this work whether resistances to races 1 and 2 were conferred by I-3 or by linked genes. Although Scott et al. (2004) and Sarfatti et al. (1991) reported that Fol1 resistance in LA716 is conferred by the I-1 gene located near I-3, findings of Do et al. (2016) rather suggest that I-3 alone confers at least partial resistance to Fol1 in addition to resistance to races 2 and 3. Despite the lack of continuity among these studies, the evidence that I-3 provides at least partial resistance to multiple races supports that I-3 should be pyramided with, rather than replaced by, any novel Fol3 resistance alleles that may later be discovered, especially if these do not provide comparable disease control to Fol races 1 to 3 as I-3. Our findings demonstrate that resistance to all three Fol races is present in multiple accessions, but further work will be needed to determine if a specific novel Fol3 resistance locus is likewise effective against races 1 and 2.

Whereas Fol3 resistance cosegregated with the I-3 locus in some backcross populations, 23 accessions are expected to contain one or more alternative resistance loci. Seven of these (i.e., LA750, LA1282, LA1340, LA1376, LA1522, LA1649, and LA1693) had questionable Fol1 and/or Fol2 resistance and thus may not be first choices in pursuit of novel, durable Fol3 resistance genes. The remaining 16 accessions (i.e., LA716, LA1272, LA1275, LA1277, LA1297, LA1299, LA1302, LA1356, LA1515, LA1656, LA1674, LA1732, LA1734, LA1941, LA1942, and LA1943) are all strong candidate sources for novel Fol3 resistance genes and also demonstrated high levels of Fol1 and Fol2 resistance. For each of these accessions, Fol3 resistant BC1F1 plants that lacked an I-3 locus introgression were used for further backcrossing, and resistance was advanced to the fourth backcross. Segregation in BC4F2 families for several of these sources also supported the alternative locus predictions in the BC1F1. One alternative resistance locus likely confers resistance in BC4F2 families of LA750, LA1297, LA1299, LA1522, and LA1734; while LA1367 could contribute two novel resistance genes.

Because of our intentional selection efforts against known Fol3 resistance loci, it is not expected that any of the advanced backcross populations generated through this work will contain I-3, I-7, or alternative alleles at these loci. However, it is possible that other reported Fol resistance loci may be responsible for the resistance responses that were observed. Sela-Buurlage et al. (2001) identified I-5 and I-6 from S. pennellii accession LA716, each of which conferred resistance to Fol2. The authors also identified regions on chromosome 11 that were associated with resistances to races 1 and 2, concluding that these regions may contain S. pennellii alleles of the I and/or I-2 genes from S. pimpinellifolium. Further research is under way to determine the locations of alternative resistance loci in several accessions and to verify whether resistance is conferred by novel or by previously reported loci. In their study, Sela-Buurlage et al. (2001) reported the locus syntenic with the I gene to confer almost complete resistance to Fol2. This suggests the possibility that some alternative alleles may confer resistance to multiple races of fusarium wilt, increasing their utility toward developing more durable resistance to this pathogen.

Data from GBS was successfully used to identify SNPs and to genotype all 42 S. pennellii accessions used in this study. This information may provide a valuable resource for researchers wishing to use the S. pennellii species in their programs; for example, sequence polymorphisms identified in diverse sources could be used to capture existing genetic diversity and develop molecular markers linked to resistance in each specific accession or to obtain marker saturation near the target gene(s) region in mapping populations.

In general, the genetic structure of S. pennellii elucidated from GBS data evidences the divergence of the southern accessions from the northern accessions, corresponding with geographic areas that span ≈10° latitude. However, no clear geographic pattern was evident with regard to presence of alternative Fol3 resistance loci. In addition, genetic structure did not primarily correspond to the observed alternative loci. The range in genetic distance among all accessions was relatively small, which resulted from the fact that all accessions belong to the same species. Nonetheless, the distinguishable genetic structure among accessions means that the GBS data can potentially be used to classify accessions based on their similarity with one another. Thus, whether investigating novel Fol3 resistance loci or other S. pennellii traits, researchers may choose to work with one or more accessions from each of the two major genetic clusters (viz. the circle and the cross symbols in Fig. 1) to maximize the genetic diversity represented in their populations. Likewise, the highly significant correlation between genetic distance and location of origin that was observed suggests that one could accomplish the same goal by arbitrarily choosing accessions from the northern and southern geographical collection sites. With regard to Fol3 resistance, our hypothesis is that accessions that are least related to one another may be less likely to contain the same alternative resistance gene to Fol3, and this also could be investigated with further research.

Despite the adoption of cultural practices that improve management of fusarium wilt in tomato, the utilization of Fol-resistant cultivars has been the primary strategy for disease control since 1939. Cultivar resistance is primarily based on three single, dominant resistance genes: I, I-2, and I-3. Considering the history of R genes in many crops, together with the fact that both I and I-2 were overcome with time, it is almost certain that I-3 will eventually be overcome by the emergence of a new Fol strain(s). However, the introduction of additional Fol resistance genes for pyramiding with those currently being used is likely to improve the durability of resistance. As an example, the I-3 and I-7 genes belong to different gene families and also differ in their mechanisms of resistance to Fol3, and these genes are already being pyramided in some breeding programs (Catanzariti et al., 2015; Gonzalez-Cendales et al., 2016; Lim et al., 2006). Similarly, sources of novel Fol3 resistance loci identified through the present study and currently being pursued may help to expand the Fol resistance gene resources that are available toward this end.

Literature Cited

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    • Search Google Scholar
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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Jones, J.P. & Woltz, S.S. 1983 Cultural control of fusarium wilt race 3 of tomato Proc. Florida State Hort. Soc. 96 82 83

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    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Sarfatti, M., Abu-Abied, M., Katan, J. & Zamir, D. 1991 RFLP mapping of I1, a new locus in tomato conferring resistance against Fusarium oxysporum f. sp. lycopersici race 1 Theor. Appl. Genet. 82 22 26 https://doi.org/10.1007/BF00231273

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Supplemental Fig. 1.
Supplemental Fig. 1.

Genetic clusters inferred using discriminant analysis of principal components (DAPC). Clusters are shown by different colors and inertia ellipses, and dots represent individual Solanum pennellii accessions. The red, blue, and yellow groups represent the circle, empty triangle, and cross symbols in Fig. 1A, respectively. The inset indicates the eigenvalues of the analysis, which retains two principal components.

Citation: Journal of the American Society for Horticultural Science 147, 1; 10.21273/JASHS05080-21

Supplemental Table 1.

Molecular markers used to detect the Solanum pennellii introgressions containing either I-3 or I-7.

Supplemental Table 1.
Supplemental Table 2.

Sequence polymorphisms identified in 42 Solanum pennellii accessions using the genotyping by sequencing method.

Supplemental Table 2.

Contributor Notes

S.F.H. is the corresponding author. E-mail: sfhutton@ufl.edu.

Supplementary Materials

  • View in gallery

    Genetic structure of 42 Solanum pennellii accessions used in this study. (A) Genetic structure of 42 S. pennellii accessions primarily corresponds with geographic areas. Symbols (circle, empty triangle, cross) are based on three genetic clusters inferred using discriminant analysis of principal components (DAPC) shown on Supplemental Fig. 1. A single or double asterisk indicates the presence of single or multiple novel Fol3 resistance loci, respectively. (B) Neighbor-joining tree of 42 S. pennellii accessions also reveals an association between accessions’ geographic origins and phylogenetic clustering. Accessions in the tree are represented by different symbols: empty square (accessions collected north of latitude 10°S); half-filled square (between latitudes 10°S and 14°S); and filled square (south of latitude 14°S).

  • View in gallery

    Genetic clusters inferred using discriminant analysis of principal components (DAPC). Clusters are shown by different colors and inertia ellipses, and dots represent individual Solanum pennellii accessions. The red, blue, and yellow groups represent the circle, empty triangle, and cross symbols in Fig. 1A, respectively. The inset indicates the eigenvalues of the analysis, which retains two principal components.

  • Bradbury, P.J., Zhang, Z., Kroon, D.E., Casstevens, T.M., Ramdoss, Y. & Buckler, E.S. 2007 TASSEL: Software for association mapping of complex traits in diverse samples Bioinformatics 23 2633 2635 https://doi.org/10.1093/bioinformatics/btm308

    • Search Google Scholar
    • Export Citation
  • Bohn, G.W. & Tucker, C.M. 1939 Immunity to fusarium wilt in the tomato Science 89 603 604

  • Bournival, B.L., Vallejos, C.E. & Scott, J.W. 1990 Genetic analysis of resistances to races 1 and 2 of Fusarium oxysporum f. sp. lycopersici from the wild tomato Lycopersicon pennellii Theor. Appl. Genet. 79 641 645 https://doi.org/10.1007/BF00226877

    • Search Google Scholar
    • Export Citation
  • Cai, G., Gale, L.R., Schneider, R.W., Kistler, H.C., Davis, R.M., Elias, K.S. & Miyao, E.M. 2003 Origin of race 3 of Fusarium oxysporum f. sp. lycopersici at a single site in California Phytopathology 93 1014 1022 https://doi.org/10.1094/PHYTO.2003.93.8.1014

    • Search Google Scholar
    • Export Citation
  • Catanzariti, A.M., Lim, G.T. & Jones, D.A. 2015 The tomato I-3 gene: A novel gene for resistance to fusarium wilt disease New Phytol. 207 106 118 https://doi.org/10.1111/nph.13348

    • Search Google Scholar
    • Export Citation
  • Chitwood-Brown, J., Vallad, G.E., Lee, T.G. & Hutton, S.F. 2021 Characterization and elimination of linkage-drag associated with fusarium wilt race 3 resistance genes Theor. Appl. Genet. 134 2129 2140 https://doi.org/10.1007/s00122-021-03810-5

    • Search Google Scholar
    • Export Citation
  • Correll, J. & Jones, J.P. 2014 Fusarium wilt 28 29 Jones, J.B., Zitter, T.A., Momol, T.M. & Miller, S.A. Compendium of tomato diseases and pests 2nd ed. Amer. Phytopathol. Soc. St. Paul, MN

    • Search Google Scholar
    • Export Citation
  • Do, T.T.H., Catanzariti, A.M., Lim, G.T.T. & Jones, D.A. 2016 Evidence against the existence of genes for resistance to Fusarium oxysporum f. sp. lycopersici races 1 and 2 on Solanum pennellii chromosome 7 additional to I-3 Acta Hort. 1207 19 26 https://doi.org/10.17660/ActaHortic.2018.1207.2

    • Search Google Scholar
    • Export Citation
  • Elshire, R.J., Glaubitz, J.C., Sun, Q., Poland, J.A., Kawamoto, K., Buckler, E.S. & Mitchell, S.E. 2011 A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species PLoS One 6 e19379 https://doi.org/10.1371/journal.pone.0019379

    • Search Google Scholar
    • Export Citation
  • Gonzalez-Cendales, Y., Catanzariti, A.M., Baker, B., McGrath, D.J. & Jones, D.A. 2016 Identification of I-7 expands the repertoire of genes for resistance to fusarium wilt in tomato to three resistance gene classes Mol. Plant Pathol. 17 448 463 https://doi.org/10.1111/mpp.12294

    • Search Google Scholar
    • Export Citation
  • Grattidge, R. & O’Brien, R.G. 1982 Occurrence of a third race of fusarium wilt of tomatoes in Queensland Plant Dis. 66 165 166 https://doi.org/10.1094/PD-66-165

    • Search Google Scholar
    • Export Citation
  • Hutton, S.F., Scott, J.W. & Vallad, G.E. 2014 Association of fusarium wilt race 3 resistance gene, I-3, on chromosome 7 with increased susceptibility to bacterial spot race T4 in tomato J. Amer. Soc. Hort. Sci. 139 282 289 https://doi.org/10.21273/JASHS.139.3.282

    • Search Google Scholar
    • Export Citation
  • Jombart, T. 2008 Adegenet: A R package for the multivariate analysis of genetic markers Bioinformatics 24 1403 1405 https://doi.org/10.1093/bioinformatics/btn129

    • Search Google Scholar
    • Export Citation
  • Jombart, T., Devillard, S. & Balloux, F. 2010 Discriminant analysis of principal components: A new method for the analysis of genetically structured populations BMC Genet. 11 94 https://doi.org/10.1186/1471-2156-11-94

    • Search Google Scholar
    • Export Citation
  • Jones, J.P. & Overman, A.J. 1985 Management of fusarium wilt, fusarium crown rot, verticillium wilt (race 2), southern blight, and root-knot of tomato on fine sandy soils Proc. Florida State Hort. Soc. 98 225 231

    • Search Google Scholar
    • Export Citation
  • Jones, J.P. & Woltz, S.S. 1968 Field control of fusarium wilt (race 2) of tomato by liming and stake disinfestation Proc. Florida State Hort. Soc. 81 187 191

    • Search Google Scholar
    • Export Citation
  • Jones, J.P. & Woltz, S.S. 1983 Cultural control of fusarium wilt race 3 of tomato Proc. Florida State Hort. Soc. 96 82 83

  • Langmead, B. & Salzberg, S.L. 2012 Fast gapped-read alignment with Bowtie 2 Nat. Methods 9 357 359 https://doi.org/10.1038/nmeth.1923

  • Li, J., Chitwood, J., Menda, N., Mueller, L. & Hutton, S.F. 2018 Linkage between the I-3 gene for resistance to fusarium wilt race 3 and increased sensitivity to bacterial spot in tomato Theor. Appl. Genet. 131 145 155 https://doi.org/10.1007/s00122-017-2991-4

    • Search Google Scholar
    • Export Citation
  • Lim, G.T.T., Wang, G.P., Hemming, M.N., Basuki, S., McGrath, D.J., Carroll, B.J. & Jones, D.A. 2006 Mapping the I-3 gene for resistance to fusarium wilt in tomato: Application of an I-3 marker in tomato improvement and progress towards the cloning of I-3 Australas. Plant Pathol. 35 671 680 https://doi.org/10.1071/AP06073

    • Search Google Scholar
    • Export Citation
  • Lim, G.T.T., Wang, G.P., Hemming, M.N., McGrath, D.J. & Jones, D.A. 2008 High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplication on Arabidopsis chromosomes 1, 2 and 3 Theor. Appl. Genet. 118 57 75 https://doi.org/10.1007/s00122-008-0876-2

    • Search Google Scholar
    • Export Citation
  • McGrath, D.J., Gillespie, D. & Vawdrey, L. 1987 Inheritance of resistance to Fusarium oxysporum f. sp. lycopersici races 2 and 3 in Lycopersicon pennellii Aust. J. Agr. Res. 38 729 733 https://doi.org/10.1071/AR9870729

    • Search Google Scholar
    • Export Citation
  • Paddock, E.F. 1950 A tentative assignment of fusarium-immunity locus to linkage group 5 in tomato Genetics 35 683 684

  • Rick, C.M. 1969 Controlled introgression of chromosomes of Solanum pennellii into Lycopersicon esculentum: Segregation and recombination Genetics 62 753 768

    • Search Google Scholar
    • Export Citation
  • Sarfatti, M., Abu-Abied, M., Katan, J. & Zamir, D. 1991 RFLP mapping of I1, a new locus in tomato conferring resistance against Fusarium oxysporum f. sp. lycopersici race 1 Theor. Appl. Genet. 82 22 26 https://doi.org/10.1007/BF00231273

    • Search Google Scholar
    • Export Citation
  • Scott, J.W. 1999 Tomato plants heterozygous for fusarium wilt race 3 resistance develop larger fruit than homozygous resistant plants Proc. Florida State Hort. Soc. 112 305 307

    • Search Google Scholar
    • Export Citation
  • Scott, J.W. 2004 Fla. 7946 tomato breeding line resistant to Fusarium oxysporum f.sp. lycopersici races 1, 2, and 3 HortScience 39 440 441 https://doi.org/10.21273/HORTSCI.39.2.440

    • Search Google Scholar
    • Export Citation
  • Scott, J.W. & Jones, J.P. 1989 Monogenic resistance in tomato to Fusarium oxysporum f.sp. lycopersici race 3 Euphytica 40 49 53 https://doi.org/10.1007/BF00023296

    • Search Google Scholar
    • Export Citation
  • Scott, J.W. & Jones, J.P. 1990 Soil-borne fungal resistance in Lycopersicon pennellii accessions HortScience 25 1068 (abstr.)

  • Scott, J.W. & Jones, J.P. 1995 Fla. 7547 and Fla. 7481 tomato breeding lines resistant to Fusarium oxysporum f. sp. lycopersici races 1, 2 and 3 HortScience 30 645 646 https://doi.org/10.21273/HORTSCI.30.3.645

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