Use marker-assisted selection (MAS) and background genome selection to rapidly transfer BLS resistances (Rx-4 and QTL11; on chromosome 11) into well-adapted fresh market tomato breeding lines that already possess strong tolerance to EB (EBT) and resistances and LB (Ph-2 and Ph-3). Outcome: This is the first step to bringing BLS resistance into fungal resistant tomatoes. This Rx-4 and QTL11 “coupling phase” linkage provides broad resistance to BLS, but full control requires Rx-3 (on chromosome 5), which is added in objective 2.
Use marker-assisted selection and background genome selection to rapidly transfer the BSk (Pto), BLS (Rx-3), and SLS (SLS-2) linked resistances on chromosome 5 into the same EB, LB, and BLS resistant tomato being produced in objective 1. Outcome: The combination of Rx-4, QTL-11, and Rx-3 provides the best resistance to all four species of Xanthamonas infecting tomato. The addition of Pto provides a valuable resistance to the primary race of Pseudomonas, race 0, causing BSk. The addition of Pto resistance to bacterial speck and the strongest current resistance to bacterial spot, in lines with genetic control of the major fungal/oomycete diseases of Northeast tomato, would provide an unprecedented level of disease control in fresh market tomato.
The combined results of objectives 1 and 2 will also produce a set of near-isogenic lines (NILs) containing different subsets of bacterial and fungal resistance genes/QTL in adapted fresh market tomato. These are valuable in breaking linkage drag, if present, and to plant pathologists studying interactions among resistance genes and pathogens.
A) Field trial the lines and isolines created by objectives 1 and 2 to determine the acceptability of their horticultural plant/fruit type under low disease control input conditions, permitting the observation of natural infection. B) Also, perform tests of lines with/without BLS resistances (Rx-3 and QTL-11/Rx-4) to determine the degree of control possible in fresh market lines. Outcomes: Field trials will determine the impact of the chromosome 5 and 11 introgressions containing the bacterial resistance genes on the horticultural characteristics of the resulting lines. Characteristics include plant type, fruit size and quality, productivity and maturity. This will determine whether the transfer of either introgression also carries deleterious traits to be eliminated. BLS control will be assessed through naturally and artificially infected replicated trial plots in Cooperation with collaborating breeders and extension/plant pathologists over two years.
Marker-assisted backcross breeding scheme: A Cornell fresh market line, CU151095-146, with strong resistance to late blight, early blight, and Septoria leaf spot was crossed to two donor parents from the Francis OSU breeding program that bore distinct resistance packages. These packages were initially brought into the Cornell genetic background independently but are now being unified in a small number of final lines. A schematic of the breeding scheme pedigree is shown in Figure 1. The first Ohio processing tomato line, OH7536, possesses a coupling phase linkage between the Pto and Rx-3 bacterial resistance genes on chromosome 5. The second, OSU line OH087663, possess a coupling phase linkage between two targeted bacterial spot resistance genes, QTL-11 and Rx-4, on chromosome 11.
Tandem foreground and background marker-assisted selection (MAS) was used to rapidly transfer and combine these resistances into one of our best fresh market tomato backgrounds. Solcap SNPs were adapted into PCR, KASP, and quantitative sequencing-based markers for use in positive foreground selection for targeted regions encoding bacterial resistance genes, and for negative background selection against donor genotypes in the non-target genome to rapidly recover the recurrent parent genetic background. In each segregating generation, MAS was performed in two phases to minimize costs associated with genotyping and greenhouse space and maximize time use-efficiency. Foreground selection was performed on a large population using only a few genetic markers, followed by background selection on the selected subset of the population using genome-wide SNPs placed evenly across a high-density genetic distance map. A small number of selected plants, typically 3-5, were then used to establish the following generation.
Transfer of Bacterial Spot Resistances on Chromosome 11.
BC1F1: After analysis of a large BC1F1 population, 324 plants were identified that possessed the full chromosome 11 region and were heterozygous for processing tomato DNA on 3 – 5 other chromosomes. This degree of progress is what would be expected for two cycles of backcrossing without background selection. The selected BC1F1 plants were backcrossed to the fresh market parent to produce BC2F1 seed.
BC2F1: When this strategy was repeated summer of 2017 using 198 BC2F1 plants, 92 plants were identified that were heterozygous for the full chromosome 11 region and were heterozygous for regions on only 1 to 2 additional chromosomes. The selected BC2F1 plants were self-pollinated to produce BC2F2 seed lots.
BC2F2: Screening of two BC2F2 populations in the fall of 2017 with PCR markers identified 10 BC2F2 plants that were homozygous for the chromosome 11 region containing the bacterial spot gene Rx-4 and QTL-11. SNP/marker data to date indicate that these BC2F2 plants are also homozygous for the DNA of the CU fungal resistant parent line for the remaining 11 chromosomes. Additional plants recombinant for the chromosome 11 region were also saved, in case we later discover linkage drag issues associated with the chromosome 11 region. An additional selection was retained that contained an extraneous donor region on the bottom of chromosome 2 that resulted in smaller fruit size but increased overall productivity and the percentage of fruit which were of marketable quality. While not useful for breeding of large-fruited fresh market tomatoes, the lineage derived from this selection is potentially useful for smaller-fruited cherry or plum tomato cultivars.
BC2F3: These BC2F2 selections were self-pollinated in the greenhouse and produced fixed BC2F3 seed which was harvested in March 2018. This seed established the new lines combining the fungal resistances of the CU parental line with the Rx-4 and QTL-11 bacterial speck resistances on chromosome 11.
Additional denser genotyping was performed on the BC2F2 lines during the summer of 2018 to confirm that they were homozygous for fresh market parental DNA throughout the genome and to identify any residual heterozygosity.
Transfer of Bacterial Spot and Speck Resistances on Chromosome 5.
Screening of the BC1F1 populations for the chromosome 5 transfer was completed in June 2017 resulting in a selection of 4 plants that were heterozygous for the full chromosome 5 region carrying prf /pto and Rx-3 and were heterozygous for 3 or 4 additional regions on other chromosomes. These BC1F1 plants were crossed to the recurrent parent to generate BC2F1 seed.
The BC2F1 populations were sown Dec 2017 and were screened with chromosome 5 markers in early Jan 2018, identifying 94 plants that were heterozygous for the entire chromosome 5 region carrying both Prf/Pto and Rx-3, and in which heterozygosity for at least one other chromosome was eliminated. Further SNPs on these plants facilitated the identification of 25 plants which were self-pollinated. Several of these BC2F2 plants revealed novel recombinations within the chromosome 5 region, and so these plants were grown to maturity and seed was collected in case later trials indicated a need to reduce the introgression size/break linkage drag.
Three BC2F2 populations were selected for continuation of the project, two of which had three additional segregating regions besides the CH5 introgression, and one of which had just two. Two distinct populations of 384 plants each were planted in Freeville using two of these seed lots during the summer of 2018, while the third was planted on a grower’s field in the Hudson valley and at the Long Island Agricultural Experiment station, in coordination with Dr. Meg McGrath and Teresa Rusinek of Cornell Cooperative extension. PCR markers run on these populations facilitated the identification of several plants which were homozygous for the chromosome 5 region carrying both Pto/Prf and Rx-3 and homozygous for fresh market tomato DNA at all other regions surveyed. Of these, phenotypic observations on important traits such as maturation period, fruit size, set, and shape, and foliar coverage and growth habit was used to identify 7 superior individuals in Freeville, 2 in the Hudson Valley, and 2 in Long Island. Self-pollinated seed was extracted from the fruit of these individuals to establish BC2F3 fixed near-isogenic lines.
Because pto and SLS-2 are very close in location, but in repulsion, there are two types of chromosome 5 lines: those in which the full chromosome 5 introgression was transferred possess pto/Rx3, in the background of CU151095-146, but no longer have SLS-2 for Septoria resistance. Other lines had a recombination reducing the chromosome 5 introgression, and so have Rx3, but not pto, in the background of CU151095-146, that still includes SLS-2. We will be searching for recombinant individuals that bring pto and SLS-2 together in coupling phase in the summer of 2019 after all the bacterial resistance genes are brought together into a single breeding line (see next section).
Additional, denser genotyping was also performed on the chromosome 5 lines using semi-random and targeted quantitative sequencing, to determine if they are indeed homozygous for the fresh market parental tomato line throughout the genome, or if any smaller regions of processing tomato DNA remain in the genome that were not previously detected by low density genotyping.
Combining all resistance genes to Bacterial Spot and Speck in fresh market tomato.
Three chromosome 5 lines are currently being crossed to three chromosome 11 lines (in all combinations) to create hybrids to test summer 2019, and to bring together all resistance genes in our fresh market tomato background. F1 seed of this cross is currently being harvested in the greenhouses in Ithaca, NY.
Field trials: During the summer of 2018, the new lines were tested for plant and fruit characteristics in field trials in three locations in NYS, as well as in several inoculated bacterial spot trials at OSU. Plant spacings of 3 feet were used in 6 plant plots on black plastic. Fixed CH11 isolines were replicated four times in each trial in New York state. These were uninoculated and primarily focused on identifying and evaluating the effect of the CH11 introgression on important horticultural traits. Trials in Fremont OH were on 1ft spacing on bare soils and were inoculated with the pathogen as young transplant starts in the greenhouse.
Continuing bacterial breeding work: Several finished chromosome 5 and chromosome 11 lines, along with their F1 hybrids and F2 segregating populations will be planted on research plots and farmer’s fields in Freeville NY, Fremont Ohio, Long Island, and the Hudson valley in cooperation with OSU and Cornell Cooperative Extension in the summer of 2019 to evaluate the horticultural performance of these lines and study the effect of the resistances genes (in homozygous and heterozygous conditions) in resistance to bacterial speck and spot diseases under both inoculated and natural infection conditions. The F2 population will also be screened with genetic markers in the summer of 2018 to select lines that combine, in the homozygous state, both the chromosome 5 and 11 introgressions for the strongest bacterial speck and spot resistances in a fungal-resistant tomato background. These results will complete the breeding and research goals of this work and generate several breeding lines which can be licensed for use in commercial breeding.
Genetic mapping of Early Blight tolerance in cultivated tomato: In order to effectively pyramid resistances to all five major diseases of tomato using only genetic markers, we needed to map QTL that conferred partial early blight resistance within our genetic population. Early blight resistance is the only disease for which these genetic markers did not yet exist. We planted two populations of ~300 individuals, one an F2 derived from the cross of CU151095-146 and OH087663, and the other a triple cross between F1 created for the aforementioned parents and the OH processing tomato line OH7536, at the Terwilliger research farm in Freeville, NY in the summer of 2017. These were inoculated in the second week of July with an Alternaria tomatophila NC isolate No. 10 spore suspension with a concentration of ~30,000 spore/mL using backpack sprayers and inoculum grown in the laboratory. These were planted in 6-plant plots with 4 ft spacing on black plastic and were irrigated by both drip and overhead irrigation to maintain plant health and encourage the spread of disease. Stem and foliar early blight lesions and blights were rated 3+ times throughout the season, and the results were used to identify plants at the extremes of the disease phenotype distribution that would be selectively genotyped using semi-random and targeted quantitative sequencing. QTL mapping was performed with these selected genotypes (using rQTL) in order to identify the large effect QTL underlying partial EB resistance. Genetic markers (KASP and PCR) were created for highly influential SNP genotypes and used to maintain, and in the case of some of the breeding populations, improve the resistance to EB. Two QTL confirmation populations were grown the following year (summer 2018) to evaluate the heritability of these QTL. These populations included ~17 F2:3 families in a replicated trial and a single segregating population generated from the BC1F2 seed of the chromosome 11 bacterial breeding population. These were planted in 8-plant plots with 2.5 ft spacing on black plastic and were irrigated by both drip and overhead irrigation to maintain plant health and encourage the spread of disease. This work supported the project goal of using marker-assisted selection to pyramid resistances to all five major bacterial, fungal, and Oomycete diseases of northeast-grown tomato into our elite fresh-market background.
Breeding and testing of Bacterial resistances:
Transfer and field testing of Bacterial Spot Resistances on Chromosome 11.
We successfully used tandem foreground and background marker-assisted selection to rapidly introgress the CH11 resistances from the OH parent OH087663 into the background of the CU fresh market tomato line CU151095-146, creating a set of chromosome 11 lines that combine broad spectrum resistances to Xanthamonas sp., narrow-sense resistance to X. perforans (T3), and strong resistances to fungal/oomycete blights including late blight, stem, foliar, and fruit phases of EB, and Septoria leaf spot. We have now performed our first season of performance testing of these lines, with uninoculated trials in New York to evaluate horticultural quality/utility, and in inoculated trials in Ohio (in cooperation with Dr. Francis of OSU) to evaluate the disease resistance of the new lines. Denser genotyping on the CH11 materials, performed by semi-random and targeted quantitative sequencing, revealed a small stretch of heterozygosity on chromosome 1 for some lines that did not appear to be correlated with modifications of important performance-related phenotypes.
Marketable yield of the chromosome 11 lines significantly increased over that of the recurrent parent with the introduction of the QTLs on chromosome 11, going from 14.4 Kg of marketable produce per 6-plant plot to 21.5 Kg (p = 0.03), while there was no significant impact of the introgression on average weight of marketable fruit, the percentage of marketable fruit, or days to maturity (defined as number of days from planting until 50% of the fruit are red-ripe). As expected, there was a significant reduction in fruit size for the subset of chromosome 11 lines that carry an extraneous chromosome 2 donor introgression. In all other traits evaluated, such as foliar coverage and uniformity of set and ripening, the chromosome 11 lines performed at least equivalently to the recurrent parent CU151095-146, indicating the successful introduction of these resistance without negatively impacting quality (linkage drag).
Replicated and inoculated field trials in cooperation with Ohio State University revealed a strong and significant effect (P << 0.001, Type II SS, df = 3,27) of the chromosome 11 introgression from OH7663 at reducing disease symptoms caused by X. perforans (T3). Lines which had functional copies of Rx4 and QTL-11 fared significantly better than the recurrent parent check, susceptible checks, and sister lines that had lost the QTL due to recombination and/or independent assortment. This was as expected, due to the nearly complete control of a true R-gene, characterized by a gene-for-gene interaction between a pathogen effector and a plant pathogen receptor protein. Every line that carried Rx4, and several of the recombinant lines that may have retained Rx4 (The marker density was not high enough to specify where the recombination occurred between the markers flanking the two QTL on chromosome 11 due to a lack of polymorphism between the parents that inhibited the creation of additional descriptive genetic markers), displayed infection severity below the population mean, confirming the successful transfer of an effective bacterial spot resistance gene into the Cornell fresh market tomato background.
Results of the inoculated field trials in Fremont OH were less clear against X. euvesicatoria (T1) and X. gardneri (T2). We have not yet introduced the Rx3 locus (on chromosome 5) into the chromosome 11 lines, so resistance to these pathogens would have been derived from the partial resistance conferred by QTL-11 and any marginal effect of the Rx4gene against non-target pathogen races. Materials that were homozygous for QTL-11 were in fact significantly more resistant (P<0.05, Type II SS, df = 3,27) to X. euvesicatoria than the Cornell recurrent parent, but curiously were not significantly different from two sister lines that had apparently lost the QTL-11 during recombination and/or independent assortment. This could be due to the small sample size (there were only two of these lines entered into the replicated trial). Alternatively, this could occur from the not-unlikely scenario that the QTL was present in these lines despite the genotypes indicated by the flanking markers. Due to our poor knowledge regarding the exact location of this centromeric QTL, and to the absence of nearby and tightly-linked flanking markers because of low polymorphism between the OH and CU parents on chromosome 11, we cannot rule out this possibility. Indeed, these are the challenges of working within elite, cultivated materials that already have high levels of quantitative resistance and that lack genetic polymorphism due to the significant genetic bottleneck that occurred during domestication.
Finally, there was no significant difference among the genotype classes at the 5% Type I error rate for resistance to X. garneri (P = 0.463, Type II SS, df = 3,27). Reasons for this result may include the relatively high level of quantitative resistance already present in the Cornell materials, the lower trait heritability due to the QTL, or the relatively low disease pressure, and hence smaller phenotypic range, in the X. garneri trial.
Transfer of Bacterial Spot and Speck Resistances on Chromosome 5.
We successfully transferred the chromosome 5 resistances from OH7536 into the elite fresh-market tomato line CU151095-146. This creating a set of chromosome 5 lines that combine X. euvesicatoria (T1) bacterial spot resistance from the Rx-3 locus, pto-derived resistance to Pseudomonas syringae p.v. tomato, the causative agent of bacterial speck of tomato, and the previously outlined fungal and oomycete resistances in CU151095-146. There are two types of chromosome 5 lines: those with the full chromosome 5 introgression and therefore have Rx3 and pto but lack SLS-2, and other with recombinant introgressions so that they maintained SLS-2 and now have Rx3, but not Pto. These lineages are one generation behind the transfer of the chromosome 11 bacterial resistance genes to the Cornell blight resistant parental line, and so have not yet been extensively field tested for horticultural and disease resistance performance. These field tests will occur summer 2019. Denser genotyping on the CH5 materials did not indicate any residual heterozygosity not identified by low-density genotyping throughout the breeding process, indicating the usefulness of the marker-assisted backcross breeding approach used in this work to more rapidly generate near-isogenic lines. We perceived time-saving gains of ~3 backcross generations with the implementation of this tandem MAS approach, compared to MAS backcross breeding with only foreground markers about the target QTL, and traditional MAS breeding based on phenotypic selection alone.
Mapping and validation of Early Blight Resistances.
Large effect EB resistance QTL, for both stem and foliar EB infection phases, were identified in our population and mapped to three chromosomes. Several additional minor effect QTL were also identified, but these were not prioritized for future work. The impacts of these QTL were confirmed in the 2018 confirmation populations. These QTL are now in the process of being fine-mapped. The details regarding these QTL will be published in an upcoming academic journal publication (Spring 2019) and further outlined in the final SARE report and VBI reports
This section will be completed in the final report.
Education & Outreach Activities and Participation Summary
- A farm demonstration / field day presentation in August 2018 at the Thompson research farm in Freeville, NY showcasing the isogenic bacterial speck and spot resistant breeding lines in progress.
- A farm demonstration / field day presentation in August 2018 at the Terwilliger research farm in Freeville, NY showcasing the effects of early blight resistance QTL which were identified in the course of completing this project.
- One industry-focused VBI report covering work completed to date and ongoing projects, as well as information and listings on tomato breeding lines which are currently, or will shortly be available for licensing.
- A presentation to the Tomato Breeder’s Round Table about this funded research.
- A presentation to the Cornell Plant Breeding community about this funded research.
- A Jan 2019 presentation to the Empire State Agricultural Expo in Syracuse NY (please note that while I was planning on giving this talk in person I have to fly to California for a funeral during this time and so have prepared the slides, but asked a cooperator, Dr. Meg McGrath, to fill in for me as the presenter).
- Two additional industry-focused VBI reports (Summer 2019 and Summer 2020) covering ongoing work and breeding materials which will become available for licensing over the next two years.
- An additional Tomato Breeder’s Round Table presentation in late 2019.
- 2+ additional Vegetable Breeding institute field day presentations in 2019 and possibly 2020.
- An academic journal article publication concerning this work (Late 2019 / Early 2020).
- Several upcoming presentations (including an exit seminar) to the Cornell community which are recorded and uploaded onto the College of Agricultural Science’s YouTube page (Through late 2020).
- Additional lines are to be developed and marketed to growers and seed companies to help disseminate the results of this funded research. Advertisement avenues include those listed above (Cornell Cooperative extension, the Vegetable Breeding Institute, the Empire State Agricultural Expo, and the Tomato Breeder’s Round-table).
This will be included in the Final report.
This will be included in the Final report.