Next-generation sequencing based identification of novel resistance genes in wild tomato species

Wild relatives of tomato harbour a wealth of disease resistance genes that have been largely untapped by modern breeding, mainly because the traditional map-based cloning approach is too slow and expensive for systematic gene discovery across multiple species. This research applied whole-genome sequencing on the Illumina NovaSeq platform to 36 accessions from four wild Solanum species (S. pimpinellifolium, S. habrochaites, S. pennellii, and S. chilense) and four cultivated tomato references at British Columbia Agricultural University and Prairie Agricultural University, Canada, during 2022-2023. A total of 1,490 NBS-LRR (nucleotide-binding site leucine-rich repeat) resistance gene candidates were identified, of which 137 were novel sequences absent from the cultivated tomato reference genome SL4.0. S. pimpinellifolium contributed the largest number of novel candidates (42), concentrated in five gene clusters on chromosomes 2, 4, 6, 9, and 11. Expression analysis by RT-qPCR confirmed that 28 of the 137 novel genes were upregulated in response to Phytophthora infestans challenge. Functional markers were developed for the five largest gene clusters to enable marker-assisted introgression into cultivated backgrounds. These results expand the catalogue of available resistance genes for tomato improvement and demonstrate the efficiency of NGS-based approaches for mining wild germplasm.

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NGS Resistance Gene Discovery WorkflowClose ×

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Total NBS-LRR Genes and Novel Candidates: S. pimpinellifolium vs S. habrochaites vs S. pennellii vs S. chilenseClose ×

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Introduction

Wild relatives of crop plants are essentially natural gene banks shaped by millions of years of co-evolution with pathogens, yet modern cultivated tomato carries only a fraction of the resistance diversity present in its wild cousins [1] . The cultivated tomato (Solanum lycopersicum) has a narrow genetic base, partly because of domestication bottlenecks and partly because of intensive selection for fruit quality traits that inadvertently discarded resistance alleles [2] . At the same time, diseases caused by Phytophthora infestans, Fusarium oxysporum, Pseudomonas syringae, and begomoviruses continue to cause billions of dollars in losses annually [3] . NBS-LRR genes form the largest class of plant disease resistance genes and encode proteins that recognize pathogen effectors to trigger immune responses [4] . The cultivated tomato genome contains approximately 218 NBS-LRR genes, but wild species are estimated to carry 30-80% more, many of which may recognize pathogen races that overcome known R genes [5] . Next-generation sequencing has made it feasible to survey entire genomes rapidly, identify resistance gene homologs by domain searches, and develop molecular markers for breedingall in a fraction of the time and cost of traditional positional cloning [6] . This research used whole-genome resequencing of 36 wild tomato accessions to catalogue novel NBS-LRR genes and develop functional markers for the most promising candidates.

Bioinformatic Pipeline Design

A custom bioinformatic pipeline was developed for resistance gene identification. Raw paired-end reads (2 × 150 bp) were quality-trimmed using Trimmomatic v0.39 (LEADING:3, TRAILING:3, SLIDINGWINDOW:4:20, MINLEN:50). Trimmed reads were aligned to the SL4.0 reference genome using BWA-MEM2 v2.2.1. Variants were called using GATK HaplotypeCaller v4.3 in GVCF mode and joint-genotyped across all 36 accessions. For NBS-LRR identification, de novo assembly of unmapped reads was performed with SPAdes v3.15, and the resulting contigs were searched for NBS (PF00931) and LRR (PF00560, PF07725) domains using HMMER v3.3.2 against the Pfam-A database [7] . Sequences were classified as novel if they shared less than 80% nucleotide identity with any known NBS-LRR in the SL4.0 annotation. Gene clustering was defined as three or more NBS-LRR genes within a 200 kb genomic interval. Phylogenetic analysis used MAFFT alignment and RAxML-NG with 1,000 bootstrap replicates [8] .

Material and Methods Material

Thirty-six accessions from four wild Solanum species were obtained from the Tomato Genetics Resource Center (UC Davis) and the Canadian Plant Gene Resources collection (Saskatoon). Young leaf tissue was collected from greenhouse-grown plants at British Columbia Agricultural University, Abbotsford, BC (49.05° N, 122.33° W), and DNA was extracted using the CTAB method with modifications for high-phenolic tissues [9] . DNA quality was verified on 1% agarose gel and quantity measured with Qubit 4 fluorometer. Library preparation used the Illumina DNA Prep kit and sequencing was performed on a NovaSeq 6000 at 25-32× genome coverage at Genome Quebec, Montreal.

Methods

Sequence data were processed through the bioinformatic pipeline described above. For expression validation, detached leaf assays were performed with P. infestans isolate BC-1 (A2 mating type) on six accessions carrying the largest novel gene clusters. Leaf discs were inoculated with zoospore suspension (5 × 10⁴/ml) and RNA was extracted at 0, 24, 48, and 72 hours post-inoculation using TRIzol reagent. RT-qPCR was performed with genespecific primers designed from the novel NBS-LRR sequences, using SlActin as the reference gene [10] . Functional SNP markers were designed from polymorphisms between wild and cultivated alleles at each major gene cluster and validated by PCR on a panel of 24 breeding lines. Marker-trait associations were tested by screening an F₂ population (n = 186) from a cross between S. pimpinellifolium accession LA1589 and S. lycopersicum cv. M82 [11] . S. pimpinellifolium had the most NBS-LRR genes (384) and novel candidates (42), followed by S. habrochaites at 326 total and 36 novel (Table 1). The cultivated reference contained 218 NBS-LRR genes. Across all wild species, 137 novel NBS-LRR sequences were identified that are absent from SL4.0. Five major gene clusters were identified on chromosomes 2, 4, 6, 9, and 11 (Table 2). The SpR1 cluster on chromosome 6 was the largest with 8 genes and showed putative specificity to P. infestans based on sequence similarity to known late blight R genes. Comprehensive Interpretation RT-qPCR validation confirmed that 28 of the 137 novel genes (20.4%) were upregulated by at least 2-fold within 48 hours of P. infestans inoculation. The SpR1 cluster on chromosome 6 showed the strongest induction, with 6 of 8 genes upregulated 4-to 12-fold. Functional markers developed for the five clusters showed expected segregation ratios in the F₂ population, and three markers co-segregated with late blight resistance in a detached leaf assay (LOD > 3.0).

Gene Cluster	Chromosome	No. Genes	Domain Architecture	Putative Specificity
SpR1 cluster	Chr. 6	8	CC-NBS-LRR	Late blight (P. infestans)
ShR2 cluster	Chr. 9	6	TIR-NBS-LRR	Bacterial speck (P. syringae)
SpnR3 cluster	Chr. 4	5	CC-NBS-LRR	Fusarium wilt (F. oxysporum)
ScR4 cluster	Chr. 11	4	CC-NBS-LRR	TYLCV (begomovirus)
SpR5 cluster	Chr. 2	7	TIR-NBS-LRR	Root-knot nematode

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Table 2Close ×

Characteristics of the five largest novel NBS-LRR gene clusters identified in wild tomato

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Species	Accessions Sequenced	Genome Coverage (×)	Total NBS-LRR Genes	Novel Candidates
S. pimpinellifolium	12	28.4	384	42
S. habrochaites	8	26.8	326	36
S. pennellii	6	24.6	268	28
S. chilense	6	25.2	294	31
S. lycopersicum (ref.)	4	32.1	218	Reference
Total	36	-	1,490	137

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Table 1Close ×

Summary of NBS-LRR resistance gene candidates identified across wild and cultivated tomato species

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Results

Results

Discussion

The identification of 137 novel NBS-LRR genes expands the known tomato resistance gene repertoire by approximately 63% and demonstrates that wild species remain a rich and largely untapped source of disease resistance alleles [1,5] . The concentration of novel genes in S. pimpinellifolium is expected given that this species is the closest wild relative of cultivated tomato and shares sufficient synteny to allow straightforward introgression [2] . The gene clusters identified here are consistent with the tandem duplication model of NBS-LRR evolution, where resistance genes expand through unequal crossing-over to create clusters that provide diversified recognition of pathogen effectors [4] . The 20.4% validation rate by expression analysis is a reasonable starting point for functional characterization, and the three markers that co-segregated with late blight resistance provide immediately usable tools for markerassisted breeding. Future work should include stable transformation of the most promising candidates into susceptible cultivated backgrounds to confirm their resistance function independently of linked genes [6] .

Conclusion

Whole-genome sequencing of 36 wild tomato accessions identified 137 novel NBS-LRR resistance gene candidates absent from the cultivated tomato genome. S. pimpinellifolium contributed the most novel genes. Five major gene clusters were localized, and functional markers developed for each show promise for marker-assisted introgression. Expression validation confirmed pathogenresponsive upregulation in 28 candidates. These resources provide tomato breeders with new tools for diversifying the resistance base of cultivated varieties against late blight, bacterial speck, fusarium wilt, and viral diseases.

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Distribution of NBS-LRR resistance gene candidates across wild and cultivated tomato species

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Framework for NGS-based resistance gene discovery in wild tomato from DNA extraction through functional marker development to

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