Progress report for LNE21-427R
The long-term goal of our research program is to use natural genetic variation in tomato to increase sustainability of crop management systems by providing breeding tools for resistance to key pests and diseases of tomato. Septoria leaf spot (SLS) of tomato is becoming an increasingly important fungal disease in the Northeast and many neighboring states in the eastern seaboard of the US where hot and humid weather prevails during the growing season in most years. This aggressive foliar disease can devastate tomato crops, to which varieties with effective tolerance are not available. A recently released variety (‘Iron Lady’) was claimed to be tolerant to SLS, but our trials showed that it does not offer sufficient protection against the predominant pathogen strain in mid-Appalachia. There is a critical need of deconvoluting the genetics behind this trait and to develop resistant varieties via introgression of robust SLS resistance from wild species into commercial varieties of tomato. This research project, therefore, aims to respond to our farmers’ needs by identifying strong sources of resistance against SLS in the tomato germplasm, introgressing it into a cultivated variety, and creating breeding tools. The specific aims of this project are: 1) to introgress the novel trait ‘SLS resistance’ from wild accessions into a popular heirloom tomato variety (‘Cherokee’) by creating interspecific hybrids via embryo rescue; 2) to identify the genetic locus responsible for SLS resistance (through a mapping-by-sequencing genomic approach); and 3) to develop bona fide CAPS markers for breeding. Backcrossing will be carried out in greenhouse conditions. Selection will occur in both, greenhouse as well as yearly in the field for performance evaluation at the WVU Organic Farm with the most advanced inbred line available. As preliminary work, we have circumvented the major bottlenecks of this project (thus, making it a low-risk, high-reward investment) by identifying several sources of genetic tolerance (including complete resistance) to Septoria lycopersici in wild tomato accessions, producing 10 viable hybrids, and we have already started the introgression work into the cultivated tomato by recurrent backcrossing and selection. An advisory panel has been formed with crop specialists and farmers, who will meet virtually with the research team once a year for updates and discussion of the directions and priorities of the project. We have pre-built connections with farmers in WV and neighboring Appalachian regions, who will be actively involved in beta-testing and selecting the finalist genotypes prior to final selection for variety release. The main end-product generated from this research will be a long-term, science-based sustainable solution for management of an increasingly important foliar disease to growers who will adopt this variety. The plant material and molecular markers developed in this project will be made available to farmers, breeders, and researchers.
As a direct response to farmers’ needs, the research objective is to develop a tomato cultivar that is resistant to Septoria Leaf Spot (SLS) by introgressing a novel natural resistance source we identified in wild tomatoes. No effective resistance against SLS is available in any tomato cultivar. We produced several F1 hybrids via embryo rescue. We will also map the resistance locus, identify the gene, and develop molecular markers for breeding. Our research will deliver a breeding toolkit to tomato breeders interested in incorporating SLS resistance in their cultivars, and the resistant materials will be available to farmers and breeders.
Research Goals: 1) Identify and introgress genetic resistance to Septoria leaf spot from wild tomato accessions into a cultivated variety, and make the genotype available to growers and seed companies; 2) Map the genetic resistance on the tomato genome and develop a molecular marker for selection (a bonus will be the identification of the culprit gene involved in the resistance mechanism).
- Backcrossing of F1 hybrids with cultivated tomato (cultivar 'WV-63') to clean up the genome from non-domesticated alleles and introgress the resistance trait into Solanum lycopersicum. In later introgression stages, we will use the more conventional 'Cherokee' cultivar (or another variety chosen by the farmers, according to suggestions from the Project Advisory Committee) and will select not only for Septoria resistance but also for the other pathogens which the cv. 'WV-63' is uniquely known for being resistant to (Phytophthora infestans, Fusarium oxysporum, and Verticillium albo-atrum).
- Generation of segregating F2 populations to characterize the inheritance of the resistance (dominant/recessive, mono- or polygenic) and enable genetic mapping;
- Development of CAPS markers to map the resistance and, eventually, be used for marker-assisted selection (MAS) in breeding programs.
In year 1 of the project, we focused on producing genetic resources to enable our research:
Backcrossing: BC1 has been completed for five F1 hybrids with cv. 'WV'63'. We are conducting a selection for Septoria resistance in order to identify the best materials (highest resistance levels) to move forward. Once the backcrossing/selection pipeline is well established and the graduate student is well trained, since we conduct the backcrossing in the greenhouse, we expect to advance ~2.5 generations per year. We will switch to more common variety (to be determined upon discussion with farmers in 2022) and select for three more disease resistances from the BC3 generation on;
- Four F1 hybrids were evaluated in field conditions (WVU Organic Farm) in summer 2021. Last year was quite humid and hot, thus creating ideal conditions for the aggressive development of Septoria leaf spot. Indeed, the accessions of cultivated tomato cultivated in our organic plot (intentionally without any disease control) were devastated
- Generation of an F2 segregating population: As expected, some F1 hybrids were self-incompatible and could not be selfed to generate F2 plants. however, we were able to produce pseudo-F2 populations by crossing different hybrids from the same genotypes (e.g., H1 and H3, both derived from crossings between cv. 'WV-63' and LA1984 (Solanum arcanum). We were also lucky to identify a hybrid that is self-fertile (H4: 'WV-17B' x LA1984). H4 was not only self-fertile but also the line that produced most seeds and showed the highest resistance level. We have decided to move forward with segregation analyses with H4 for these reasons. Later, the other pseudo-F2 populations will be used to assess whether the genetic resistance behaves similarly when coming from other wild accessions. Characterization of genetic resistance in the H4-F2 population is currently underway.
- Development of CAPS markers to map the genetic resistance to Septoria leaf spot: 24 markers were developed (one per chromosome arm of the tomato genome - this is possible due to the high degree of synteny in the genus Solanum). Markers have been designed to distinguish the domesticated chromosome arm and any wild tomato species under study (S. peruvianum, S. corneliomulleri, and S. arcanum) based on the genome sequence information available to us. The markers have been tested and optimized, and are ready to be employed on the H4-F2 population to identify in which chromosome arm(s) the resistance is mapped. Later, we will develop markers to zoom in/refine the genetic locus with further development of markers on the identified arm in order to narrow down the locus further and further. We also used these CAPS markers to successfully confirm the hybrid nature of the lines we are using (and identified hybrid H5 as an escape, which only had the genome from the maternal progenitor, cv. 'WV-63').
Education & Outreach Activities and Participation Summary
The current pandemic certainly creates hurdles for in-person interactions and outreach activities. Nevertheless, we made efforts to have a presence with the farmers through participating in the WVU Organic Farm Field Day (a yearly event at WVU, which occurred on August 28, 2021) by displaying a plot with our hybrids alongside other tomato cultivars. This year, the Septoria leaf spot disease was very aggressive on tomato cultivars (see picture 1), but impressively the five H1 hybrids we tested were mostly unharmed by them (see picture 2 - commercial varieties on the left and our F1 hybrids on the right). The plot was visited by around 30 local organic farmer families (a much lower number than previous years, due to heavy rain on that Saturday and the pandemic) and counted with 10 agricultural educators and extension specialists.
In future years, after the pandemic is over, we aim to attend local and national in-person conferences, but for now, we will limit our participation in open-field and online to guarantee the safety of our researchers.
A Masters thesis by Estefania Tavares Flores (WVU Genetics and Developmental Biology Program, title: "Traditional and modern breeding strategies towards developing resilient crops: Two case studies in tomato"; advisor: Prof. Vagner Benedito) was defended in July 2021. Chapter II (Introgressing Septoria leaf spot resistance from wild tomato accessions into West Virginia cultivars using in vitro techniques and genetic markers: Developing alternatives for organic farmers) is a product of this research.
In our WVU Organic Farm Field Day, which occurred on Aug 28, 2021, we were able to interact with 30 farmer families and 10 agricultural specialists, who were educated about Septoria leaf spot and other tomato diseases and could witness the strong disease resistance in our hybrid. Since this is year 1 of our project, we have not actively employed farmer participation but intend to do so for testing variety performance in later stages, when we have more advanced introgressions.