Progress 12/01/16 to 09/30/17
Outputs
Target Audience:I provided the training of extension agents and industry workers through formal programs, personal farm visits, field days, workshops, and newsletter articles. To deliver knowledge of molecular-assisted breeding in strawberry, I especially provided workshops and presentations during national and regional meetings, such as strawberry field days, AgriTech, and AgExpo. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Phtophthora crown rot resistance To better characterize the 1.5 Mb FaRPc2 region, more DNA markers has been developed markers for the genomic region. Using the SNP whole genome genotyping and sequencing data, a total of 24 SNP-based high resolution melting (HRM) markers were developed and further 63 SSR markers were selected for the FaRPc2 region. All the markers have been testing on a set of 24 strawberry varieties and breeding selections that inherited putative functional allele combinations of Pc2pc2 (heterozygous resistant) or pc2pc2 (susceptible) based on SNP haplotypes from the QTL analysis. The seven HRM and five SSR markers were found that segregate with the Pc2 allele. These markers are being utilized for fine mapping of the Pc2 in larger associating mapping panels. The most informative markers have been implemented in 2017 marker-assisted seedling selection to increase genetic gains for PhCR resistance in the University of Florida breeding program. This year we will continue to use the high-throughput DNA test for PhCR resistance breeding. Colletotrichum crown rot resistance To continue validating the major QTL, Cg1, for CCR resistance, we are conducting whole-genome SNP genotyping and pedigree-based marker-trait association analysis (in FlexQTL™ software) with the QTL validation populations (developed at 2015-2016 season). The results from this study will provide the precise location of Cg1 and the SNP haplotypes specific to the CCR resistance. Haplotypes for the identified QTL for CCR resistance will be manually constructed based on marker segregation and parental diplotypes using a Microsoft Excel based haplotyping tool. The predominant SNP haplotypes will be compared with those from the 2013-14 study to confirm the effect of the haplotypes on level of resistance. Constructing SNP haplotypes for the parents of the populations and other breeding germplasm will then allow us to select parental combinations for future crosses that are most likely to yield resistant individuals. Sequences of SNP probes in the QTL associated with CCR resistance will be aligned with F. vesca sp. bracteata v2.0 reference to determine the physical location of the QTL. To develop high-throughput markers for seedling selection for CCR resistance, HRM markers have been developed from the sequence of SNP array probes near the Cg1 locus for resistance to CCR. All progeny of five families used for the QTL discovery and validation were analyzed by the HRM markers linked to the CCR resistance locus, Cg1. The results from this study will be published in 2018. Construct BAC libraries from UF advanced selections to facilitate gene cloning and marker development. We constructed three BAC libraries for main breeding parents and/or advance selections. To identify the BAC clones possessing target traits such as disease resistance, flavor and fruit quality, probes specific to the genomic region were designed and hybridized. The identified BAC clones (each insert about 100 kb) will be sequenced by Illuminar NGS. The sequences will be aligned with F. x ananassa reference genome. The annotated genes in the target genomic region will be analyzed for their function, and candidate genes will be cloned for developing functional markers. In addition, the newly identified genetic elements from BAC clones such as protein-coding genes, expressed sequences, transposable elements, and simple sequence repeats will be greatly valuable for strawberry breeding program. Sequence UF cultivars/advanced selections for octoploid genome assembly and subgenome specific linkage map construction. It is technically challenging to develop a high quality reference genome for the octoploid F. x ananassa strawberry genome and generate a high-density subgenome specific linkage map. We are collaborating this project with the research groups at East Malling Research (EMR), UK, University of California strawberry breeding program, and Kazusa DNA Institute, Japan. Total five accessions, FL127, 11.28-34, 12.115-10, 14.101-225 and 13.26-134 possessing major QTL for disease resistance and fruit quality characteristics were sequenced using long-read paired end (approximately 450bp) and PacBio (approximately 20kb). Currently reference guided assembly has been conducting for several important target regions. Also, the massively parallel BAC sequencing will be done to clone important disease resistance genes such as Pc2 and Xf1. This approach will allow the subgenome, heterozygosity and complex genome assembly problems in octoploid strawberry. After sequencing and assembly completed, we will identify subgenome-specific SNPs cooperating with previous high-density linkage map and SNP marker information (Affymetrix Axiom IStraw90k and 35k SNP chip datasets). The identified subgenome-specific SNPs will be integrated into the high quality SNP linkage map developed from UCD and UF collaboration, and the consensus octoploid subgenome linkage map will be developed for UF breeding program. Develop gene-editing technology in octoploid strawberry to enhance the UF strawberry cultivar development program We are developing protocols to build the gene editing platform into elite varieties and advanced selections. The most recent cultivars, FL127 'Sweet Sensation', Florida Beauty, and Florida Brilliance will be tested for tissue culture optimization. CRISPR gene editing work using LF9 will be done as a collaboration with Dr. Kevin Folta. Tissue culture conditions has been optimized and now testing for transformation efficiency. This year we will develop the DNA free gene editing protocol using the most recent cultivar, Florida Brilliance. By collaboration with Dr. Alfred Huo at Mid-REC, CRISPR gene editing constructs will be developed for testing several candidate gene for Xf1 and Pc2.
Impacts
What was accomplished under these goals?
1. Development of high throughput genotyping platform development in strawberry breeding. For effective marker-assisted selection to screening large seedling populations, the new genotyping method is required in our breeding program. We established the high resolution melting (HRM) method for genotyping UF breeding germplasm in 2015. HRM is the high-throughput method to detect genetic variations and mutations in PCR reactions without running agarose gels and sequencing. HRM technology allows the processing of high sample numbers more conveniently with lower cost than any traditional methods. Also, multiple alleles for target traits such as fruit quality or disease resistance can be detected by HRM. Using this HRM-based genotyping system, approximately 16,500 seedlings were screened for peach aroma (γ-decalactone) and disease resistance (Phythophthora crown rot, bacterial angular leaf spot, and anthracnose) in 2015, 30,000 in 2016, and 35,000 in 2017 breeding program. 2. Development of breeding populations and field evaluation for Phytophthora crown rot resistance. We identified QTL for PhCR resistance using recent advanced DNA knowledge from strawberry and developed the QTL conferring for PhCR resistance loated in the linkage group 7D, and developed molecular markers tightly linked to the resistance locus. These results were published to peer review journals. My student (Younghee Noh) has defined the major QTL region for PhCR resistance located on the distal end of LG 7D, Pc2. To better characterize the 1.5 Mb Pc2 region, Younghee Noh developed markers for fine mapping. Using the SNP whole genome genotyping and sequencing data, a total of 24 SNP-based high resolution melting (HRM) markers were developed and further 63 SSR markers were selected for the Pc2 region. All the markers were tested on a set of 24 strawberry varieties or breeding selections that inherited putative functional allele combinations of Pc2pc2 (heterozygous resistant) or pc2pc2 (susceptible) based on SNP haplotypes from the QTL analysis. The seven HRM and five SSR markers were found that segregate with the Pc2 allele. These markers are being utilized for fine mapping of the Pc2 in larger segregating F1 populations. The most informative markers has been developed and implemented in marker-assisted seedling selection to increase genetic gains for PhCR resistance in the University of Florida breeding program. 3. Research on Colletotrichum crown rot resistance in strawberry. Continual development of new strawberry varieties with disease resistance is critical for nursery and fruit growers. Colletotrichum crown rot (CCR) caused by Colletotrichum gloeosporioides is one of the most common and destructive diseases in strawberry growing regions of the southeastern United States and particularly in Florida. My MS student, Ashlee Anciro, with our breeding team's support planted a total of 248 lines from the five full-sib families at the experimental field in Gulf Coast Research and Education Center (GCREC) for the crown rot resistance study. In addition, my student collected DNA samples and sent for genome scanning using the Affymetrix Axiom IStraw90 SNP array. In 2017 study, we confirmed that a quantitative trait locus (QTL) on linkage group (LG) 6B, which we name FaRCg1, accounts for most of the genetic variation for resistance in these discovery populations. Additionally, significant marker effects in the QTL region were observed in two validation populations consisting of approximately 100 advanced selections and cultivars representative of the UF breeding germplasm. The discovery and validation of FaRCg1 have been determining for the genetic improvement of resistance to CCR. 4. Research on high-throughput marker development for strawberry angular leaf spot. Angular leaf spot (ALS) caused by Xanthomonas fragariae is the only major bacterial disease of cultivated strawberry (Fragaria × ananassa). Using a selective genotyping approach with the IStraw90 Axiom® SNP array and pedigree-based QTL detection software FlexQTL™, a single major-effect QTL was identified for angular leaf spot resistance. Based on SNP probe alignment to the wild diploid strawberry, F. vesca sp. bracteata, reference sequence, the resistance locus was localized at the 33.6 to 33.9 Mb region of LG6D. Recently, my student (Younghee Noh) successfully identified one high-throughput SNP-based high resolution melting (HRM) and two simple sequence repeat (SSR) markers with approximately 95% of selection efficiency and has used for marker-assisted selection in 2016 and 2017 in the UF strawberry breeding program. 5. Research on strawberry genome sequencing and assembly. Currently, our marker-assisted breeding is relying on the sequence information from F. vesca reference genome. Due the lack of an octoploid strawberry genome, the progress at identifying the causative genes and developing molecular markers underpinning important disease resistance, flavor and fruit quality are slow. Therefore, we can sequencing the whole genome of UF breeding accessions, which possess important breeding traits (ALS, PhCR, and CCR resistance; peach and grape aroma) for future cultivar development. The impact of this research is large for UF strawberry breeding program and further Florida strawberry industry. Total five accessions, FL127, 11.28-34, 12.115-10, 14.101-225 and 13.26-134 possessing major QTL for disease resistance and fruit quality characteristics need to be sequenced using long-read paired end (approximately 450bp) and PacBio (approximately 20kb), and an assembly using a novel haplotype selection procedure should be achieved. Also, the massively parallel BAC and fosmid clone sequencing can be done to allow the resolution of extremely long haplotypes. This approach allows the subgenome, heterozygosity and complex genome assembly problems. After sequencing and assembly completed, we can identify subgenome-specific SNPs cooperating with previous high-density linkage map and SNP marker information (Affymetrix Axiom IStraw90 SNP chip datasets). The identified subgenome-specific SNPs can be integrated into the high quality SNP linkage map developed by Kazusa group, and the consensus octoploid subgenome linkage map can be developed for UF breeding program. 6. Research on gene-editing system in strawberry. The genome-edited plants through Agrobacterium-mediated transformation contain foreign DNA sequences. It is not easy process to remove the A. tumefaciens-drived DNA sequences by breeding, especially species that reproduce or propagate asexually. Recently, the induction of targeted genome modification using protoplasts and regenerated from the protoplasts have been shown in Arabidopsis, lettuce, tobacco and rice. This method is known as RNA-guided endonuclease (RGEN), and is DNA-Free genome editing technique as there is no need for the help of any vector system. Plants developed from RGEN RNPs could be exempt from current GMO regulations. We are testing this protocol for UF breeding materials, one main cultivar FL127 'Sweet Sensation' and two advanced selections, FL12.121-5 and 12.55-220.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Whitaker, V. M., Lee, S., Osorio, S., Verma, J., Roach, J. A., Mangandi, J., Noh, Y.-H., Gezan, S. and Peres, N. 2016. Advances in Strawberry Breeding at the University of Florida. ISHS VIII International Strawberry Symposium, Quebec City, Canada. Aug 13-18, 2016.
Bassil, N., Amaya, I., Davis, T., Denoyes, B., Bernardo, R., Edger, P., Finn, C., Hardner, C., Hancock, J., Ivors, K., Knapp, S., Lee, S., Mangandi, J., Mahoney, L., Roach, J., Salinas, N., Verma, S., van de Weg, E., Whitaker, V., Peace, C. and Lezzoni, I. 2016. RosBREED: From genomics to DNA-informed breeding in strawberry. ISHS VIII International Strawberry Symposium, Quebec City, Canada. Aug 13-18.
Verma, S., Roach, J., Mangandi, J., Lee, S., Salinas, N., Bassil, N., Bink, M.C.A.M, van de Weg, E., Peace, C., Iezzoni, I. and Whitaker, V. 2016. DNA-informed strawberry breeding in RosBREED. Plant and Animal Genome Conference, (https://pag.confex.com/pag/xxiv/webprogram/Paper18784.html)
Frett, T., Clark, J., Sandefur, P., Jecman, A., Gasic, K., Peace, C. and Lee, S. 2016. Implementing marker-assisted selection (MAS) for bacterial spot [Xanthomonas Arboricola Pv. Pruni (Xap)] resistance in the University of Arkansas peach and nectarine breeding program. American Society for Horticultural Sciences Annual Conference, Atlanta, GA, August 8-11.
Roach, J., Verma, S., Anciro, A., Lee, S., Isobe, S., Jamieson, A., Bassil, N., Bink, M., van de Weg, E. and Whitaker, V. 2016. QTL discovery and marker development for strawberry angular leaf spot caused by Xanthomonas fragariae. PAG XXIV - Plant and Animal Genome Conference, (https://pag.confex.com/pag/xxiv/webprogram/Paper21811.html)
Noh, Y., Mangandi, J., Verma, S. and Whitaker, V., Cha, J. -S. and Lee, S. 2016. Development of high-throughput markers for Phytophthora crown rot resistance in strawberry. PAG XXIV - Plant and Animal Genome Conference, (https://pag.confex.com/pag/xxiv/webprogram/Paper19123.html)
Anciro, A. Mangandi, J. Verma, S. Whitaker, V. M. and Lee, S. 2016. Identification of quantitative trait loci and molecular markers for resistance to Colletotrichum crown rot in strawberry. Phytopathology. 106:6.
Noh, Y., Mangandi, J., Verma, S. and Whitaker, V., Isobe, S. and Lee, S. 2016. Application of high-throughput markers for Phytophthora crown rot resistance breeding in strawberry. Phytopathology. 106:12.
Noh, Y., Mangandi, J., Verma, S. and Whitaker, V., Isobe, S., Cha, J. -S. and Lee, S. 2016. Marker development and fine-mapping for the Phytophthora crown rot resistance locus, Pc1, in strawberry. Phytopathology. 106:85.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Free, H. C. A., Lee, S., Smith, B. G., Lu, F, Sedbrook, J. C., Sibout, R., Grabber, J. H., Runge, T. M., Mysore, K. S., Harris, P. J., Bartley, L. E. and Ralph, J. 2016. Monolignol-ferulate conjugates are naturally incorporated into plant lignins. Science Advances 2:e1600393
Lee, S., Whitaker, V. M. and Hutton, S. 2016. Mini Review: potential applications of nonhost resistance for crop improvement. Frontiers in Plant Science 7:997.
Jia, Y., Zhou, E., Lee, S. and Bianco, T. 2016. Coevolutionary dynamics of rice blast resistance gene Pi-ta and Magnaporthe oryzae avirulence gene AVR-Pita 1. Phytopathology 106(7): 676-683.
Roach, J. A., Verma, S., Peres, N. A., Jamieson, A. R., van de Weg, E. W., Bink, M. C.A.M., Bassil, N. V., Lee, S. and Whitaker, V. M. 2016. FaRXf1: a locus conferring resistance to angular leaf spot caused by Xanthomonas fragariae in octoploid strawberry. Theoretical and Applied Genetics 129(6):1191-1201.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Kaundal, A., Ramu, V., Oh, S., Lee, S., Pant, B., Lee, H-K., Rojas, C., Muthappa, S-K. and Mysore, K. S. 2017. General control non-repressible-4 (GCN4) destabilizes 14-3-3 and RIN4 complex to regulate stomatal aperture with implications on plant immunity. Plant Cell 29: 2233-2248.
Lee, S., Muthappa, S-K., Kang, M., Rojas, C., Tang, Y., Oh, S., Choudhury, S. R., Lee, H-K., Ishiga, Y., Allen, R. D., Pandey, S. and Mysore, K. S. 2017. The small GTPase, Nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity. Scientific Report 7: 9260.
Kang, M., Lee, S., Abdelmageed, H., Reichert, A., Lee, H-K., Fokar, M., Mysore, K. S. and Allen, R. D. 2017. Arabidopsis Stress Associated Protein 9 mediates biotic and abiotic stress responsive ABA signaling via the proteasome pathway. Plant, Cell and Environment 40(5): 702-716.
Noh, Y-H., Lee, S., Whitaker, V. M., Cearley, K. R. and Cha, J. 2017. A high-throughput marker-assisted selection system combining rapid DNA extraction and high-resolution melting analysis: strawberry as a model for fruit crops. Journal of Berry Research 7(1): 23-31.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Lee, S., Noh, Y., Mangandi, J., Verma, S. and Whitaker, V. M. 2017. Phytopathology 107:S5.1. https://doi.org/10.1094/PHYTO-107-12-S5.1
Deschamps, S., Folta, K. M., Lee, S., Whitaker, V. M. and Agehara, S. 2017. Optimization of Microenvironments by Multi-Colored Plastic Mulch Improves Earliness and Total Yield of Strawberry. FSHS 130th Annual Meeting in Tampa, Florida.
Lee, S., Noh, Y., Anciro, A., Verma, S., Yoo, C., Whitaker, V. M., Barbey, C. R. and Folta, K. M. 2017. Developing Superior Strawberry Cultivars with Advanced DNA Technologies. FSHS 130th Annual Meeting in Tampa, Florida.
Anciro, A., Mangandi, J., Verma, S., Osorio, L., Lee, S. and Whitaker, V. M. 2017. QTL discovery for resistance to Colletotrichum crown rot in strawberry (Fragaria � annanassa). PAG XXV - Plant and Animal Genome Conference.
Yoo, C., Whitaker, V. M. and Lee, S. 2017. Optimization of octoploid strawberry regeneration for the application of targeted genome editing. PAG XXV - Plant and Animal Genome Conference.
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