Development of novel specific molecular markers for the Sw-5b gene to assist with tomato spotted wilt virus-resistant tomato breeding

Abstract

Tomato spotted wilt virus (TSWV) is a plant pathogen that causes devastating tomato yield losses worldwide. The Sw-5b gene is one of the most effective resistance genes for TSWV control in tomato plants, and has been widely used in resistance breeding. Molecular markers are specific DNA sequences with known locations on the chromosome; they are indispensable tools in marker-assisted selection, which detects the presence of target genes to expedite breeding. We developed gene-specific molecular markers for Sw-5b to facilitate the accurate distinction of resistance (Sw-5b^R) and susceptibility (Sw-5b^S) alleles of Sw-5b. Using these markers, we successfully detected Sw-5b and determined its genotype (homozygous Sw-5b^R, heterozygous Sw-5b^R/S, or homozygous Sw-5b^S) in six tomato varieties. Then we successfully applied these markers to 46 commercial tomato cultivars to detect and determine the genotype of Sw-5b. The results revealed a striking absence of the Sw-5b^R gene and high TSWV susceptibility among most of the analyzed commercial cultivars. With the assistance of the novel Sw-5b-specific molecular markers, we generated a TSWV-resistant and homozygous Sw-5b^R Micro-Tom tomato line, demonstrating the practical application of these markers in plant breeding. In summary, we developed novel gene-specific molecular markers for Sw-5b, and applied them to distinguish Sw-5b alleles for TSWV resistance or susceptibility. This marker set provides a valuable tool for breeding TSWV-resistant tomato varieties.

Background

Tomato spotted wilt virus (TSWV) belongs to the genus Orthotospovirus. It exhibits a remarkably broad host range, with more than 1000 plant species spanning more than 100 families. TSWV causes devastating yield losses in many crops and ornamental plants worldwide, with an estimated annual global economic impact of approximately 1 billion USD (Parrella et al. 2003; Oliver and Whitfield 2016; Turina et al. 2016; Zhu et al. 2019). Consequently, TSWV is considered as the second most important plant virus in the top ten well-studied plant viruses (Scholthof et al. 2011). In nature, thrips, especially the Western flower thrip (Frankliniella occidentalis), transmit TSWV persistently in a circulative-propagative way (Whitfield et al. 2005; Gilbertson et al. 2015; Rotenberg et al. 2015). The TSWV particles harbor three single-stranded genomic RNAs designated L, M, and S RNA. RNA L encodes RNA-dependent RNA polymerase (RdRp) through negative coding strategy (Dehaan et al. 1991; Adkins et al. 1995; van Knippenberg et al. 2002), while the M RNA encodes the movement protein NSm and glycoproteins Gn and Gc (Kormelink et al. 1994; Nagata et al. 2000). The S RNA encodes nonstructural protein NSs and nucleocapsid protein N (Kormelink et al. 1991; Bucher et al. 2003; Snippe et al. 2007; Schnettler et al. 2010).

In response to the widespread devastation caused by TSWV globally, breeders have made great efforts to identify tomato varieties with natural resistance to TSWV. This endeavor has led to the discovery of multiple resistance sources in various tomato lines and cultivars. Among these sources, eight loci designated Sw-1a, Sw-1b, Sw-2 to Sw-7 have been reported to provide TSWV resistance (Turina et al. 2016; Qi et al. 2021). However, the resistance conferred by Sw-1a, Sw-1b, Sw-2, Sw-3, and Sw-4 has been overcome by TSWV and other orthotospoviruses (Stevens et al. 1991; Saidi and Warade 2008). In contrast, Sw-6 and Sw-7 confer resistance to multiple TSWV isolates, but they have yet to be cloned (Rosello et al. 1998; Canady et al. 2001; Rosello et al. 2001; Qi et al. 2021). Sw-5, which was originally found in Solanum peruvianum, is a gene cluster located on the telomeric region of the long arm of chromosome 9, it encompasses six homologous paralogs, which were designated Sw-5a to Sw-5f (Stevens et al. 1991; Stevens et al. 1995b; de Oliveira et al. 2018). However, only Sw-5b is responsible for broad-spectrum resistance against TSWV and other orthotospoviruses (Rosello et al. 1998; Spassova et al. 2001; Peiro et al. 2014). Sw-5b is a classical resistance gene that encodes a nucleotide-binding leucine-rich repeat (NLR) immune receptor. This receptor comprises an extended N-terminal Solanaceae domain (SD), a coiled-coil (CC) domain, a central nucleotide-binding adaptor shared by ApaF-1, resistance proteins, a CED-4 (NB-ARC) domain, and a C-terminal leucine-rich repeat (LRR) domain (Brommonschenkel et al. 2000; Chen et al. 2016; De Oliveira et al. 2016). Sw-5b triggers plant immunity through the recognition of the viral movement protein NSm and provides broad-spectrum resistance to various orthotospoviruses by recognizing a conserved 21-amino acid peptide within NSm (Lopez et al. 2011; Hallwass et al. 2014; Zhao et al. 2016; Leastro et al. 2017; Zhu et al. 2017). Despite the discovery of resistance-breaking (RB) TSWV isolates, Sw-5b has been widely used as a resistance source against orthotospoviruses due to its durability and consistent broad-spectrum resistance (Turina et al. 2016).

The advent of molecular markers has substantially improved the efficiency and precision of trait selection, thereby expediting plant breeding efforts. These markers are closely linked to genes that determine specific traits, marker-assisted selection (MAS) can be applied to determine the presence and absence of a target gene by detecting linked molecular markers. MAS has been employed to assist the selection and identification of TSWV-resistant materials and breeding for commercial tomato cultivars. The development of molecular linkage markers for TSWV resistance genes has primarily focused on Sw-5 and Sw-7 (Qi et al. 2021). Several markers, including the restriction fragment length polymorphism (RFLP) markers CT71 and CT220, have been linked to Sw-5 (Stevens et al. 1995a, 1995b; Garland et al. 2005). Concurrently, gene-specific markers directly targeting the Sw-5b gene have been established. In 2005, a dominant marker representing the Sw-5b gene sequence, combined with an improved cleaved amplified polymorphic sequence marker, were used to form a new marker system for Sw-5 (Garland et al. 2005). Subsequently, Sw-5b-specific single nucleotide polymorphism (SNP) and sequence-characterized amplified regions (SCAR) markers were developed (Shi et al. 2011; Panthee and Ibrahem 2013; Lee et al. 2015). However, only a few of these markers have been found to distinguish homozygous resistant, heterozygous resistant, or homozygous susceptible varieties (Garland et al. 2005).

In this study, we developed novel gene-specific SCAR markers for the tomato Sw-5b gene, which confers broad-spectrum resistance to multiple orthotospoviruses including TSWV. These markers were validated through testing on an array of tomato varieties and cultivars. Our findings may contribute to the development of TSWV-resistant tomato cultivars.

Results

Development of gene-specific molecular markers for Sw-5b

To develop gene-specific molecular markers that can distinguish Sw-5b alleles for TSWV resistance or susceptibility, we analyzed nucleotide sequences of Sw-5b from the resistant tomato cultivar Solanum peruvianum cv. G17-60 (Sw-5b^R), and its susceptibility allele in the cultivar S. lycopersicum cv. Heinz1706 (Sw-5b^S). The sequence alignment result showed that the LRR region exhibited the highest level of polymorphism (Fig. 1a). We designed two primer pairs, Sw-5b-F1/R1 and Sw-5b-F2/R1, to specifically amplify the Sw-5b^R and Sw-5b^S fragments, respectively, based on nucleotide sequence alignment of the LRR regions of Sw-5b^R and Sw-5b^S (Fig. 1b and Table 1). Both primer pairs share a common reverse primer, Sw-5b-R1, which targets both Sw-5b^R and Sw-5b^S. However, the forward primers Sw-5b-F1 and Sw-5b-F2 were designed to match Sw-5b^R and Sw-5b^S, respectively (Fig. 1b, c). The primer pair Sw-5b-F1/R1 was designed to amplify Sw-5b^R exclusively, producing a 660-bp fragment, and Sw-5b-F2/R1 was designed to amplify Sw-5b^S exclusively, resulting in a 459-bp fragment (Fig. 1c). To ascertain the specificity of these primers, genomic DNA was isolated from the homozygous Sw-5b^R tomato cultivar G17-60, heterozygous Sw-5b^R/S hybrid tomato line IVF3545, and homozygous Sw-5b^S tomato cultivar Heinz1706. These DNA samples were used as templates and amplified using the primer pairs Sw-5b-F1/R1 and Sw-5b-F2/R1, respectively. The results showed that Sw-5b-F1/R1 produced a 660-bp product from DNA obtained from homozygous Sw-5b^R and heterozygous Sw-5b^R/S tomato plants, whereas Sw-5b-F2/R1 generated a 459-bp product from the DNA of homozygous Sw-5b^S and heterozygous Sw-5b^R/S tomato plants (Fig. 1d). To confirm the above results, we employed the previously reported Sw-5 SNP marker Sw5-f2/r2 as the control marker. We conducted PCR amplifications by using genomic DNA extracted from tomato cultivars G17-60, IVF3545, and Heinz1706 as templates. The marker successfully amplified Sw-5b^R from G17-60 and IVF3545 (Additional file 1: Figure S1), which validated the results from the two primer pairs. Together, these results indicated that the primer pairs Sw-5b-F1/R1 and Sw-5b-F2/R1 are effective Sw-5b-specific molecular markers for distinguishing the resistance allele Sw-5b^R from the susceptibility allele Sw-5b^S. In addition, combining these two markers, we can identify the genotype of Sw-5b in tomato plants.

Fig. 1

Design of Sw-5b specific molecular markers. a Nucleotide sequence alignment between the resistance allele Sw-5b^R and the susceptibility allele Sw-5b^S. The alignment was used to generate a heatmap using the ggplot2, R package, with black marks indicating positions diverging from the reference Sw-5b^R sequence. Different colors distinguish the SD, CC, NB-ARC, and LRR regions of Sw-5b^R and Sw-5b^S. b Nucleotide sequence alignment between the LRR regions of Sw-5b^R and Sw-5b^S, highlighting the location of the Sw-5b-specific primers Sw-5b-F1, F2, and R1. c Positions of the Sw-5b-specific primer pairs, Sw-5b-F1/R1 and Sw-5b-F2/R1, within the Sw-5b gene. The expected PCR products sizes are indicated. Sw-5b-F1/R1 was designed to amplify Sw-5b^R exclusively, producing a 660-bp fragment, and Sw-5b-F2/R1 to amplify Sw-5b^S exclusively, resulting in a 459-bp fragment. d Validation of the specificity of the Sw-5b-specific molecular markers. Genomic DNA extracted from the tomato cultivar G17-60, IVF3545, and Heinz1706, representing the Sw-5b^R homozygote, Sw-5b^R/S heterozygote, and Sw-5b^S homozygote, respectively, was used as template for PCR amplification. Genomic DNA from Nicotiana benthamiana was used as the negative control

Table 1 Sw-5b-specific primers

Detection of the Sw-5b gene and genotype determination of tomato varieties using Sw-5b-specific molecular markers

Next, we used the Sw-5b-specific molecular markers to detect the Sw-5b gene in six distinct tomato varieties (Table 2). The cultivars G17-60, IVF3545, and Heinz1706 were used as controls representing the Sw-5b^R homozygote, Sw-5b^R/S heterozygote, and Sw-5b^S homozygote, respectively. Polymerase chain reaction (PCR) analysis detected a 660-bp product from DNA extracted from LA3667, Paronset, and G17-60; a 459-bp product form DNA extracted from D17-6, D17-18, 365, Heinz1706, and Micro-Tom; and 660-bp and 459-bp products from DNA extracted from IVF3545 (Fig. 2a). These results indicated that, similar to G17-60, the varieties LA3667 and Paronset were homozygous for Sw-5b^R. By contrast, D17-6, D17-18, 365, and Micro-Tom were homozygous for Sw-5b^S, mirroring Heinz1706. To determine whether these plants were able to confer resistance against TSWV, each tomato variety was mechanically inoculated with TSWV-infected crude leaf extracts. A dot-enzyme-linked immunosorbent assay (Dot-ELISA) and reverse transcription (RT)-PCR assay showed that LA3667, Paronset, IVF3545, and G17-60 were not infected by TSWV, whereas the remaining tomato plants were infected (Fig. 2b, c and Table 2). These results suggest that the Sw-5b^R homozygote varieties LA3667, Paronset, and G17-60 and the Sw-5b^R/S heterozygote variety IVF3545 exhibit resistance to TSWV, whereas the Sw-5b^S homozygote varieties D17-6, D17-18, 365, Heinz1706, and Micro-Tom are susceptible to TSWV. Thus, the Sw-5b-specific molecular markers were applied successfully to determine whether tomato varieties carry Sw-5b^R, Sw-5b^S, or both.

Table 2 Evaluation of TSWV resistance in six tomato varieties through the Sw-5b-specific molecular markers and virus inoculation experiments

Fig. 2

Detection of the Sw-5b gene and genotype determination of tomato varieties using Sw-5b-specific molecular markers. a Detection and genotyping of Sw-5b gene in six tomato varieties using PCR with Sw-5b-specific molecular markers. Genomic DNA from Nicotiana benthamiana was used as the negative control. b Dot-ELISA to detect TSWV infection in tomato plants. Tomato plants were inoculated with TSWV and uninoculated upper leaves were collected and subjected to TSWV infection detection at 14 dpi. c RT-PCR to confirm TSWV infection in tomato plants. Uninoculated upper leaves of TSWV-infected tomato plants were subjected to RT-PCR analysis at 14 dpi. TSWV-infected tomato leaves were used as positive control (+), while leaves from uninfected healthy tomato plants were used as negative control (−)

Application of Sw-5b-specific molecular makers to detect Sw-5b in commercial tomato cultivars

The use of resistance genes in crop breeding is one of the most efficient approaches to mitigating viral diseases. To determine whether commercial hybrid tomato cultivars contain the Sw-5b gene and/or confer TSWV resistance, we collected samples from 46 cultivars (Additional file 2: Table S1) and applied the Sw-5b-specific molecular markers for gene detection. Only 1 of the 46 cultivars (ANX) harbored the Sw-5b^R gene, which is the Sw-5b^R/S heterozygotes. The vast majority of the commercial cultivars were identified as Sw-5b^S homozygotes, and the remaining three cultivars (GQ6, JP1, and ZZ202) did not carry the Sw-5b gene (Fig. 3). To exclude the possibility of DNA sample quality issues from the three cultivars, we evaluated the DNA quality by amplifying the tomato internal gene Actin using the DNA from these cultivars as templates. The DNA extracted from cultivars G17-60, IVF3545, and Heinz1706, representing the Sw-5b^R homozygote, Sw-5b^R/S heterozygote, and Sw-5b^S homozygote, respectively, were used as controls. The PCR results confirmed the successful amplification of the Actin gene from all DNA samples (Additional file 1: Figure S2a), affirming the quality of DNA extracted from these cultivars. Next, we inoculated these cultivars with TSWV and tested the plants for virus infection at 14 days post inoculation (dpi). The Dot-ELISA results showed that, only 1 of the 46 cultivars (ANX) was not infected by TSWV (Additional file 1: Figure S2b, c). These findings were summarized in Table 3, which reveals a noticeable lack of the Sw-5b^R gene and high susceptibility to TSWV among the 46 tested cultivars.

Fig. 3

Detection and genotyping of the Sw-5b gene in commercial tomato cultivars. Detection of the Sw-5b gene in 46 commercial tomato cultivars through PCR amplification using Sw-5b-specific molecular markers. Genomic DNA obtained from tomato cultivars G17-60, IVF3545, and Heinz1706 was used as template for PCR amplification, representing the Sw-5b^R homozygote, Sw-5b^R/S heterozygote, and Sw-5b^S homozygote, respectively

Table 3 Evaluation of TSWV resistance in 46 commercial tomato cultivars through the Sw-5b-specific molecular markers and virus inoculation experiments

Breeding of TSWV-resistant tomato lines with the assistance of Sw-5b-specific molecular markers

In marker-assisted selection, molecular markers are applied directly to screen for plants carrying a target resistance gene in every generation during the breeding process, as well as to determine its genotype (Qi et al. 2021). The tomato variety Micro-Tom is characterized by its smaller size, shorter lifecycle, and amenability to efficient transformation. These traits have led to the widespread use of Micro-Tom in tomato functional genomic research (Okabe et al. 2013; Shikata and Ezura 2016). Using the Sw-5b-specific molecular markers, we identified Micro-Tom as an Sw-5b^S homozygote (Fig. 2a) and cultivated an Sw-5b^R homozygous Micro-Tom line (Fig. 4). Using G17-60 as the paternal line and Micro-Tom as the maternal line, we developed the F₁ generation. In examining the resultant F₁ generation, we discovered that all 12 grown plants exhibited faster growth than G17-60. Genomic DNA extractions followed by Sw-5b-specific molecular markers assays showed that all these plants carried Sw-5b^R. Next, we repeatedly backcrossed the F₁ generation with Micro-Tom as the recurrent parent. We used the Sw-5b-specific molecular markers to determine the genotypes of the resulting plants of each round of backcrossing. Tomato plants carrying Sw-5b^R and that were similar in size to Micro-Tom were selected for subsequent backcrossing. Following five rounds of backcrossing, the resultant BC₅ plants were uniformly small, with rapid growth similar to Micro-Tom. Detection using the Sw-5b-specific molecular markers showed that 33% of these plants carried the Sw-5b^R gene, and these plants were used for subsequent self-crossing. The obtained BC₅F₁ plants were tested with Sw-5b-specific molecular markers; the results showed that only BC₅F₁-3 was an Sw-5b^R homozygote. Next, the BC₅F₁-3 plant was used for self-crossing, and generated BC₅F₂. All BC₅F₂ plants showed the small, rapid-growth phenotype; and PCR using Sw-5b-specific molecular markers confirmed that their genotype was Sw-5b^R homozygous. Next, we inoculated these plants with TSWV and found that they were all resistant to viral infection (Additional file 1: Figure S3). The proportions of plants carrying Sw-5b^R and size phenotypes produced throughout the breeding process are listed in Table 4. These results demonstrate the successful generation of an Sw-5b^R homozygous Micro-Tom line with the assistance of the Sw-5b-specific molecular markers.

Fig. 4

Breeding TSWV-resistant tomato lines with the assistance of Sw-5b-specific molecular markers. a Flow chart illustrating the cultivation of Sw-5b^R homozygote Micro-Tom lines. b Phenotypes and genotypes of the plants obtained in each round of the breeding process of Sw-5b^R homozygous Micro-Tom lines. In each round, plant phenotypes were observed, and the genotypes were determined using the Sw-5b-specific molecular markers

Table 4 The proportions of plants carrying Sw-5b^R and size phenotypes produced in the breeding process of Sw-5b^R homozygous Micro-Tom lines

Discussion

The Sw-5b gene is the most widely applied and effective resistance gene against TSWV in tomato (Turina et al. 2016). Therefore, breeders are striving to cultivate tomato variants that both carry the Sw-5b gene and harbor excellent horticultural traits. MAS has played a significant role in hastening the breeding process, and the development of precise and highly specific molecular markers, particularly the gene-specific markers, has been critical to breeding success. Among the reported molecular markers for the Sw-5b gene, the SNP markers Sw-5b-SNP and Sw5-f2/r2 and the SCAR marker NCSW-012 have demonstrated the ability to effectively distinguish Sw-5b^R and Sw-5b^S in numerous tomato populations (Shi et al. 2011; Panthee and Ibrahem 2013; Lee et al. 2015). However, these markers can only detect the presence of Sw-5b^R, without providing information about its genotype, i.e., whether it is an Sw-5b^R homozygote or Sw-5b^R/S heterozygote. Some researchers have combined the Sw-5b-specific molecular marker Sw-5b-LRR with the Sw-5 locus molecular marker CT220 to create a marker system that can discern Sw-5b^R homozygotes from Sw-5b^R/S heterozygotes. However, this marker system employs an RFLP marker CT220, which requires an enzyme digestion reaction following PCR (Garland et al. 2005). Previous studies revealed that the LRR domain of Sw-5b is responsible for recognizing the viral effector NSm. A comparison of the amino acid sequences of Sw-5b^R and Sw-5b^S showed that the LRR domain exhibited the most polymorphic sites (Zhu et al. 2017; Li et al. 2019). Among these polymorphic sites, four polymorphic ones, polymorphic sites 3, 4, 5, and 6 within the Sw-5b LRR domain, are critical for NSm binding and recognition, which subsequently determines the resistance or susceptibility of tomatoes to TSWV (Zhu et al. 2017). Furthermore, polymorphic sites 2 and 5 displayed the most significant polymorphisms at the nucleic acid level. Therefore, we designed the Sw-5b-F1, which corresponds to the region of polymorphic site 2 and specifically matches Sw-5b^R, as well as Sw-5b-F2, which corresponds to the region of polymorphic site 5 and exclusively matches Sw-5b^S. Both forward primers were utilized in combining with a shared reverse primer, Sw-5b-R1, targeting both Sw-5b^R and Sw-5b^S. These primer pairs were designed to amplify specific bands of different sizes. Testing across a range of tomato varieties and cultivars revealed that these primers could accurately differentiate Sw-5b^R from Sw-5b^S. Next, we combined these primers to develop a specific marker system for the Sw-5b gene that would accurately distinguish Sw-5b^R from Sw-5b^S and precisely identify the genotype of tomato plants carrying Sw-5b. This Sw-5b-specific marker system improves the detection and selection of tomato germplasm resources carrying the Sw-5b gene, thereby facilitating the process of breeding for TSWV-resistant tomatoes.

China is a preeminent producer and exporter of tomato products, with a crop area of more than 1.1 million ha and the highest total tomato yield worldwide, producing more than 66.2 million tons in 2022. However, viral diseases pose a significant threat to tomato yield and quality (Qi et al. 2021; Tettey et al. 2023), among which TSWV is particularly destructive. The most effective control strategy is the use of disease-resistant genes in cultivar breeding (Turina et al. 2016; Liu and Tang 2023). To date, the Sw-5b is the most effective resistance gene against TSWV in tomato. In this study, we examined a broad range of commercial tomato cultivars from China for their resistance to TSWV, using our novel Sw-5b-specific molecular markers, followed by TSWV inoculation. Among the tested 46 cultivars, only one carried the Sw-5b^R gene, which was Sw-5b^R/S heterozygotes. After TSWV inoculation, this cultivar was found to be resistant to the virus. This suggests that very few commercial tomato cultivars carry the Sw-5b^R gene and possess TSWV resistance. Therefore, more effort is needed to breed and popularize TSWV-resistant tomato plants. Notably, the resistance spectrum of Sw-5b was recently been expanded via stepwise artificial evolution, thereby enabling it to defend against RB TSWV isolates. Ongoing advancements in genome editing technology are anticipated to lead to the emergence of innovative methods surpassing traditional disease-resistant breeding to develop new TSWV-resistant materials.

Micro-Tom is a model tomato variety used in functional genomics studies for its small size, short lifecycle, and adaptability to greenhouses (Shikata and Ezura 2016). Our previous studies have revealed the mechanisms underlying the broad-spectrum resistance conferred by Sw-5b against orthotospoviruses, and illustrated how Sw-5b recognizes NSm to trigger plant immunity (Chen et al. 2016; Zhu et al. 2017; Li et al. 2019). To gain deeper insights into the signaling pathways activated by Sw-5b post-activation, we cultivated an Sw-5b^R homozygous Micro-Tom line. Throughout this breeding process, we applied our novel Sw-5b-specific markers to test the plants obtained in each generation. This procedure allowed the rapid identification of plants carrying Sw-5b^R during the seedling stage, which could then be used in the next round of backcrossing or crossbreeding. The selection of resistant plants using Sw-5b-specific markers requires only basic PCR to detect and genotype the Sw-5b gene, bypassing the need for TSWV inoculation in resistance determination. This approach reduces the costs associated with breeding and accelerates the breeding process. The Sw-5b^R homozygous Micro-Tom line generated in this study provides an invaluable resource for scientific research and can be used as a TSWV-resistant tomato germplasm resource.

Conclusions

We developed gene-specific molecular markers for the tomato resistance gene Sw-5b that were able to distinguish its resistance (Sw-5b^R) and susceptibility (Sw-5b^S) alleles. We successfully detected the gene and determined its genotype in six tomato varieties. Our screening of 46 commercial tomato cultivars using these markers revealed a striking absence of the Sw-5b^R gene and high TSWV susceptibility among the majority of the analyzed cultivars. With the assistance of the Sw-5b-specific molecular markers, we generated a TSWV-resistant, Sw-5b^R homozygous Micro-Tom tomato line. The Sw-5b-specific markers will be valuable tools for breeding TSWV-resistant tomato varieties and mitigating viral diseases.

Methods

Plant materials and growth conditions

Tomato seeds were subjected to surface sterilization and then germinated in Petri dishes in an incubator under controlled conditions (25 °C, 60% humidity, and a 16 h light/8 h dark photoperiod). Once the seedlings produced two true leaves, they were transplanted into soil and transferred to a greenhouse with a 16 h light/8 h dark photoperiod.

Sequence alignment

The nucleotide sequences of the resistance (Sw-5b^R) and susceptibility (Sw-5b^S) alleles of the Sw-5b gene were aligned to generate a difference matrix consisting of two rows, the first representing Sw-5b^R and the second representing Sw-5b^S. A heatmap was generated using the ggplot2 package in the software R (R Core Team, Vienna, Austria).

Extraction of tomato genomic DNA

For genomic DNA extraction, 0.1 g 5-week-old tomato leaves was ground to a fine powder using liquid nitrogen. The powder was mixed with 300 µL extraction buffer (1.5 M Tris-HCI pH 8.0, 5 M NaCl, 0.5 M EDTA, 2% CTAB) and incubated at 65 °C for 10 min. After cooling, 300 µL chloroform was added, and the mixture was vigorously shaken and centrifuged at 13,200 × g for 5 min. Then the supernatant was mixed with 300 µL isopropanol, followed by centrifugation at 13,200 × g for 5 min. The supernatant was discarded, and the precipitate was washed with 500 µL 70% ethanol. After centrifugation at 13,200 × g for 3 min, the precipitate was dried at room temperature and suspended in 100 µL water.

PCR-based detection and genotyping of Sw-5b

To detect Sw-5b and determine its genotype in different tomato varieties, two pairs of primers were designed separately. The primer pair Sw-5b-F1/R1 was designed to amplify Sw-5b^R exclusively, producing a 660-bp fragment, and Sw-5b-F2/R1 was designed to amplify Sw-5b^S specifically, generating a 459-bp fragment (Table 1). The optimum annealing temperature for PCR amplification with the Sw-5b-F1/R1 primer pair is 52 °C, whereas for the Sw-5b-F2/R1 primer pair, it is 56 °C. Genomic DNA extracted from tomato samples was used as template and subjected to PCR amplification using the primer pairs Sw-5b-F1/R1 and Sw-5b-F2/R1, respectively, with Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China). The resulting PCR products were visualized through agarose gel electrophoresis.

TSWV inoculation

Virus inoculation assays were performed as described previously (Zhu et al. 2017). TSWV was isolated from local diseased plants in Yunnan Province, China, and propagated on Nicotiana benthamiana plants. For TSWV inoculation, leaf tissues infected with TSWV were collected and ground in 1 × phosphate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄, pH 7.4). The resulting crude leaf extracts were gently rubbed onto the leaves of 6-week-old tomato plants. After inoculation, the plants were grown in a growth chamber with 16 h light /8 h dark photoperiod.

Dot-ELISA

Dot-ELISA was performed to detect TSWV in tomato plants. At 14 dpi, uninoculated systemic leaves were collected and placed in a centrifuge tube. Then, 100 µL of encapsulating solution (0.5 M carbonate buffer) was added to the tube, and the leaf tissues were ground with a grinding rod. The resulting mixture was centrifuged at 1467 × g for 4 min. From the supernatant, 3 µL was pipetted onto nitrocellulose membranes. The resulting blots were probed using an anti-TSWV N (1:5000; produced in our laboratory) and detected using alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:10,000; no. A3687, Sigma-Aldrich, St. Louis, MO, USA), followed by staining with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (Sangon Biotech, Shanghai, China).

RT-PCR to detect TSWV infection

To validate the Dot-ELISA results, RT-PCR was conducted as described previously (Wu et al. 2023). Total RNA was isolated from tomato leaves using the RNA Simple Rapid Isolation Kit (Tiangen, Beijing, China). Then, first-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China) was used to perform PCR reactions to detect the N gene in tomato plants. The tomato Actin gene was used as an internal reference gene.

Breeding Sw-5b ^R homozygous Micro-Tom lines

To cultivate Sw-5b^R homozygous Micro-Tom lines, the Sw-5b^R homozygous tomato cultivar G17-60 was used as the paternal parent and the Sw-5b^S homozygous tomato cultivar Micro-Tom was used as the maternal parent. A cross was made between these two parents to generate the F₁ generation. The F₁ plants were identified as Sw-5b^R/S heterozygous using the Sw-5b-specific molecular markers. To continue the breeding process, the F₁ generation was repeatedly backcrossed with Micro-Tom as the recurrent parent. The resulting progeny, designated BC₁, were backcrossed again with Micro-Tom and the genotype of the resulting plants were determined using the Sw-5b-specific molecular markers. Then BC₁ plants carrying Sw-5b^R were backcrossed with Micro-Tom to obtain BC₂. After five rounds of backcrossing, the obtained BC₅ plants were found to exhibit the same rapid-growth phenotype as Micro-Tom, with Sw-5b^R/S heterozygous genotype. BC₅F₁ plants were obtained from the BC₅ population through self-crossing. These plants were subsequently screened for the Sw-5b^R homozygous genotype using Sw-5b-specific molecular markers. The selected BC₅F₁ plants were further self-crossed and the resulting BC₅F₂ plants were all Sw-5b^R homozygous, conferred resistance to TSWV, and exhibited the same growth phenotype as Micro-Tom.