Breeding for Resistance to Target Spot of Tomato (Corynespora cassiicola), a New Threat to the Tomato Industry
E. Sierra-Orozco1*, J. Smeda1, K.V. Xavier1, G.E. Vallad1, & S.F. Hutton1
1GCREC, University of Florida. 14625 Co Rd 672, Wimauma, FL 33598
Fresh market tomato is a high value commodity in the southeastern US. Target spot of tomato (TS) caused by Corynespora cassiicola (Cc) has been an important disease for several decades but has become increasingly problematic in recent years. TS infects both foliage and fruit and can cause severe defoliation and reduction in marketable yield. There are currently no resistant cultivars available, and management relies entirely on fungicide applications. Moreover, recent work has demonstrated the insensitivity of Cc isolates to several commonly used fungicides, emphasizing the need for host resistance. The goals of this research project include identification of TS resistance in tomato wild relatives, characterization and mapping of major resistance genes/QTLs, and advancement of resistance into cultivated tomato. Resistance was identified in wild tomato accessions from S. pimpinellifolium, S. cheesmaniae and S. galapagense. In S. pimpinellifolium, a recombinant inbred line (RIL) population derived from NC EBR1 x LA2093 along with genome-wide marker information, was used to detect a single locus on chromosome 12 which explained at least 30% of phenotypic variation in two separate experiments. Further mapping in backcross populations confirmed the effect of the resistance locus. A similar locus was also detected from each of two S. galapagense accessions. Further research is needed to determine whether these sources confer alleles of the same gene(s) or separate but linked loci. A field trial to validate QTLs from different sources confirmed that the chromosome 12 locus from each source is effective under field conditions.
Predictive ability of genomic selection at the family level in Alfalfa
Mario H. M. L. Andrade*, Janam P. Acharya*, Juliana Benevenuto†, Ivone de Bem Oliveira**, Yolanda Lopez*, Patricio Munoz†, Marcio F. R. Resende Jr.†, and Esteban F. Rios*
*Agronomy Department, University of Florida, Gainesville, FL, 32611
†Horticultural Sciences Department, University of Florida, Gainesville, FL 32611
**Hortifrut North America, Inc., Estero, FL, United States.
Alfalfa (Medicago sativa L.) is one of most cultivated perennial forage legumes in subtropical and temperate regions, being the third most valuable commodity in the country. Its breeding process is usually done using a recurrent phenotypic selection scheme. However, yield improvement has stalled for the past years - increment of only 0.50% per year. Genomic selection (GS) can achieve higher genetic gains by shortening the breeding cycle and being more cost-effective. Our goal was to assess the predictive ability (PA) of GS at the family level in alfalfa dry matter yield (DMY) and canopy height (CH). We used data from an breeding population of 177 families (142 full-sibs and 35 half-sibs) planted in Citra, FL, in 2017 and harvested 11 times from (2018-2019). We assessed the PA of the genomic prediction model GBLUP using 114,945 SNPs, by genotyping family bulks, in predicting DMY and CH within each harvest. Two cross-validation schemes were used: 10-fold (CV10) (90/10) and leave-one-out (LOO) (5 sub-population/1 sub-population). The PA calculated using Pearson correlation between predicted and adjusted phenotypes. Both traits had similar trends in PA. The average PA for CV10 was 0.34 (0.15 to 0.51) and 0.32 (0.15 to 0.51) for CH and DMY respectively, and 0.21 (0.08 to 0.46) and 0.21 (-0.03 to 0.34) for LOO. The PA varied among harvests for both CV and traits. This showed the presence of GxE that affects PA. Another cause of the variation in PA was heritability of each trait in each harvest, as harvests with high heritability resulted in higher PA. The PA of the GS model reached in this study has the potential to reach higher genetic gains compared to phenotypic selection. Application of GS in alfalfa breeding could be facilitated by genotyping and phenotyping families in bulks.
Development of PCR based genetic markers from whole genome sequencing of Finger limes (Citrus australasica)
Jaideep Kaur Deal, Kyle Weber and Manjul Dutt
University of Florida
The Australian finger lime (Citrus australasica) is a close relative of the commercially cultivated Citrus varieties. The fruit has a distinct finger-like shape with juice vesicles that separate into individual “pearls” resembling caviar, giving the nickname citrus caviar. In recent years Finger limes have gained importance due to their tolerance to Citrus greening (Huanglongbing disease; HLB), a bacterial disease that is devastating the Florida citrus industry. To effectively utilize finger limes as a genetic resource for the citrus industry, the underlying tolerance mechanism needs to be well understood. The objective of this study was to develop unique markers specific to the finger lime and which could help distinguish between finger limes and other citrus. Whole genome sequencing data was utilized to identify genetic markers, and many were validated using conventional PCR techniques. Whole genome sequencing was carried out for C. australasica cultivar VI697 using the Illumina HiSeq platform (150 bp paired end reads) to obtain a 30X coverage. Two reference genomes: citron (C. medica) and Valencia (C. sinensis) were used to align with the finger lime sequence. We identified 114163 potential SSR markers using the citron and 17841 markers using the Valencia genome sequence. We tested a total of 111 SSRs on C. australasica, C. australis, C. clementina and the hybrids Citrus x virgata (Sydney hybrid; C. australis x C. australasica) and C. clementina x C. australis respectively to identify a large number of polymorphic SSR markers.
Inheritance of Root-knot nematode Resistance in a Specialty Pepper Cultivar
Dominick Padilla1*, Donald W. Dickson2, and Bala Rathinasabapathi1
*Presenter’s e-mail: firstname.lastname@example.org
1Horticultural Sciences Department, University of Florida, Gainesville, FL
2Entomology and Nematology Department, University of Florida, Gainesville, FL
Southern root-knot nematode (Meloidogyne incognita; RKN) negatively affects the productivity of peppers (Capsicum annuum). To address this problem, the use of resistant varieties is an environmentally sound method for integrating with current management strategies. In our program, a breeding population was developed to obtain four advanced inbred lines with value-added traits related to fruit quality and stem architecture. A RKN resistance screening revealed that two of the siblines, ‘Ruby’ and ‘Jade’, exhibited high levels of resistance to RKN (M. incognita race 3), but two others ‘Jasper’ and ‘Topaz’ were susceptible. A segregation analysis of resistance in ‘Ruby’ was conducted for F2 and BC1 populations to characterize inheritance of resistance in this cultivar. When resistance was scored based on egg masses per gram root and categorized as ‘highly resistant’, ‘moderately resistant’ ‘moderately susceptible’ and ‘highly susceptible’, the data fitted to a model for two dominant factors controlling RKN resistance. Current research is in progress to genotype a population of phenotypically-characterized F2 population for single nucleotide polymorphism markers. Such marker data will be used for linkage analysis and mapping of the loci involved in RKN resistance in ‘Ruby’.
Diploid Sorghum Informs Targeted Mutagenesis for Altering Leaf Inclination Angle in Highly-Polyploid Sugarcane
Eleanor Brant1,2, Ayman Eid1,2, Mehmet Cengiz Baloglu1, and Fredy Altpeter1,2
1Agronomy Department, University of Florida, Gainesville, FL
2DOE Center for Advanced Bioenergy and Bioproducts Innovation, Gainesville, FL
Sugarcane is a prime feedstock for commercial production of biofuel and table sugar. Its highly polyploid genome is the most complex of any domesticated agricultural species (2n = 100–120), which complicates crop improvement by both traditional breeding and genome editing. Establishing protocols to achieve robust, specific, and efficient multiallelic editing in sugarcane has therefore remained challenging despite the flexibility and efficiency of RNA guided nucleases like CRISPR/Cas9. However, sorghum has a diploid genome, and its exons show a high level of sequence conservation to those of sugarcane. Therefore, our objectives were to; (1) assess if targeted mutagenesis in sorghum can inform genomic targets for modification of a specific trait in sugarcane, (2) assess if highly conserved sgRNA target sequences for LG1 support Cas9-mediated mutagenesis in both sorghum (single LG1 copy) and sugarcane (31 LG1 copies), and (3) introduce CRISPR/Cas9-mediated mutations into LIGULELESS1 (LG1) to explore if this alters leaf inclination angle in sugarcane for improved canopy level photosynthesis in high density plantings. Genome editing reagents were delivered into sugarcane calli (var. CP88-1762) alongside the nptII selectable marker via biolistic gene transfer. Guide RNAs previously shown to confer an upright leaf phenotype in sorghum were used. Transgenic lines were regenerated following selection and edits were confirmed via capillary electrophoresis and NGS sequencing. Sixteen sugarcane lines exhibiting between 9-100% lg1 knockout were obtained and phenotyped in a randomized block greenhouse trial. This work demonstrates efficient multiallelic editing in sugarcane and confirms the potential of sorghum as a model species for sugarcane gene editing. To add to this, results suggest lg1 knockout is a suitable strategy for creating upright leaf phenotypes in sugarcane, with several mutant lines showing significantly reduced leaf inclination angle.
Accelerating the Design- Build-Test-Learn (DBTL) Cycles in the Metabolic Engineering of Oilcane via Inducible Expression
Moni Qiande1, Dang Viet Cao1,2, Hui Liu2,3, John Shanklin2,3, and Fredy Altpeter1,2
1University of Florida - IFAS, Gainesville, FL; 2DOE Center for Advanced Bioenergy and Bioproducts Innovation; 3Brookhaven National Laboratory, Upton, NY
Sugarcane is an ideal target crop to fuel the emerging bioeconomy. It combines superior biomass production and photosynthetic efficiency with hyperaccumulation of sucrose in its stem, which offers great prospects for diversion to alternative products. We recently reported the generation of oilcane, a sugarcane which has been metabolically engineered for hyperaccumulation of triacylglycerol (TAG) in its vegetative biomass. Inducible promoters may allow lipid production at will, at a time when tissue culture or critical stages of plant development are already completed. Since sugarcane tolerates elevated temperatures between 40° and 45°C for an extended period of time, we explored different heat shock promoters (HSP) and the combination of heat inducible and constitutively expressed lipogenic factors to accelerate DTBL cycles. Data describing transgene expression and resulting TAG accumulation before and after activation of different lipogenic factors and their combinations will be reported.
Marker Assisted Backcrossing for Virus Resistance in Squash
Swati Shrestha, Yuqing Fu, Geoffrey Meru
University of Florida
Breeding disease resistant varieties is cornerstone for disease management. Aphid-transmitted viruses, Zucchini Yellow Mosaic Virus (ZYMV) and Papaya Ringspot Virus (PRSV-W) are major limitation to squash production world-wide. Natural source of resistance to ZYMV and PRSV-W has been reported in wild unadapted genotype from Nigeria called Nigerian Local. Resistance alleles from the genotype can be utilized for development of virus resistant squash varieties, however, lack of tightly linked markers to resistant sources restricts marker assisted selection for rapid generation of resistant lines. In the current study, whole genome re-sequencing based bulked segregant QTL analysis method named BSA QTL-seq was used to identify quantitative trait loci (QTL) and DNA markers associated with ZYMV and PRSV-W resistance in Nigerian Local. F2 mapping populations were developed by crossing Nigerian Local (resistant) X Butterbush (susceptible) and phenotyped for ZYMV and PRSV-W resistance by artificial viral inoculation in separate experiments. BSA QTL-seq method identified four QTL associated with ZYMV resistance on Chromosome 2 (QtlZYMV-C02), 4 (QtlZYMV-C04), 8 (QtlZYMV-C08), 20 (QtlZYMV-C20) and single QTL associated with PRSV-W resistance on Chromosome 9 (QtlPRSV-C09) with p value < 0.05. After QTL identification fourteen Kompetitive allele specific (KASP) SNP markers were developed for ZYMV resistance and thirteen KASP markers were developed for PRSV-W resistance from their respective QTL regions identified. The markers were tested for association with ZYMV and PRSV-W resistance in diverse genotypes. Three KASP markers significantly associated with ZYMV resistance and two KASP markers associated with PRSV-W resistance were identified (Kruskal-Wallis p value < 0.05). Findings of the study will facilitate marker assisted back-crossing for rapid introgression of virus resistance in elite squash lines and allow further understanding of genomics underlying ZYMV and PRSV-W resistance in squash.
Targeted mutagenesis of apomictic tetraploid bahiagrass using CRISPR/Cas9
DAVID MAY1, Jennifer Gilby1, Sara Sanchez1, and Fredy Altpeter2. 1Agronomy Department, University of Florida - IFAS, Gainesville, FL; 2Agronomy Department, Plant Molecular and Cellular Biology Program, Genetics Institute, University of Florida - IFAS, Gainesville, FL.
New breeding technologies, including CRISPR/Cas, will enable trait introgression and metabolic engineering for improvement of tetraploid bahiagrass (Paspalum notatum Flüggé). An efficient protocol for targeted mutagenesis in bahiagrass was established by targeting magnesium-protoporphyrin IX chelatase (MgCh), which provided a rapid readout for the extent of multi-allelic editing. Guide RNAs (gRNAs), Cas9 and the NPTII selectable marker were delivered to ‘Argentine’ embryogenic callus via biolistics in two independent experiments. Visual identification followed by cleaved amplified polymorphic sequences (CAPS) assays and both Sanger and Illumina sequencing were used to identify a total of 9 edited lines, which were generated at efficiencies of 23 and 50% of the transgenic lines. Two of the edited lines displayed progression of mutagenesis over time which corresponded with a phenotypic progression that was visually apparent and is likely linked to somatic edits. Evaluation of alternative DNA repair mechanisms contributing to different editing outcomes will also be discussed. This gene editing protocol is currently being used for improvement of turf and forage quality and was also transferrable to diploid bahiagrass.
High-Throughput Phenotyping and Genomic Prediction Aid Yield Improvement in Alfalfa (Medicago sativa L.)
Anju Biswas*, Mario H. M. L. Andrade*, Claudio Fernandes Filho†, Aditya Singh‡, Diego Jarquin*, Patricio Munoz§, and Esteban F. Rios*
*Agronomy Department, University of Florida, Gainesville, FL, 32611
†Biology Department, Institute of Natural Sciences, Universidade Federal de Lavras, Lavras, MG, Brazil
‡Department of Agricultural and Biological Engineering, University of Florida, Gainesville, Florida, USA.
§Horticultural Sciences Department, University of Florida, Gainesville, Florida, USA
High-throughput phenotyping (HTP) and genomic prediction applied in plant breeding can lead to higher genetic gain for complex traits. Integrating phenomics and genomics in breeding pipelines is driven by developing non-destructive HTP platforms and advances in next-generation sequencing. Alfalfa (Medicago sativa L.) is a perennial forage legume grown on more than 30 million hectares worldwide and improving alfalfa dry matter yield (DMY) requires significant phenotyping efforts. We tested if aerial measurements of normalized difference vegetation index (NDVI) as secondary traits in genomic best linear unbiased prediction (GBLUP) models could increase the predictive ability for DMY using 145 full-sib and 34 half-sib alfalfa families. The experiment was established in November 2017, and DMY was harvested four times between August 2018 and January 2019. An unmanned aerial vehicle (UAV) with a multispectral camera was used for HTP before each harvest. NDVI was used as a response variable in a univariate model, as a secondary trait in a bivariate model, or as a covariate in univariate models. Predictive ability (PA) was estimated for models within and across harvests and for scenarios with various levels of DMY data (simulated in-silico by assigning 0% DMY for testing datasets and 0-100 % DMY from 50 % data in testing datasets)). Within harvests, NDVI increased predictive ability for DMY by 168% when used as a covariate, and 31% in bivariate models, on average. For across-harvests models, predictive ability increased when predicted one harvest from two or more harvests compared to predicted one harvest from one harvest. When training populations included multiple harvests, we did not see any significant difference for PA when we added more DMY (0-100%) data in the testing sets. Covariate models produced more precise predictive ability estimates than bivariate and univariate models. Thus, HTP can be used with genomic prediction models to improve genetic gain in alfalfa for DMY during early-generation breeding plots.
Optimization of marker density in genomic prediction in alfalfa (Medicago sativa L.)
Sipowicz P.1, Andrade M.H.M.L1, Fernandes Filho C.C 1, Resende M.F.R 2, Ferrao, L.F.V 2 and Rios E.F. 1
1Agronomy Dpt., University of Florida
2Horticultural Sciences Dpt.,University of Florida
Alfalfa (Medicago sativa L., 2n = 4x) given its forage nutritive value, ability to fix atmospheric nitrogen and high biomass yield is the most important perennial forage legume in the world. Historically, breeding efforts for yield in alfalfa have resulted in slower gains compared to other crops. Genomic selection has great potential to increase genetic gain for complex traits such as yield. The ability to predict the performance of genotypes for a given trait with genotypic information is key to the process of Genomic Selection. Optimization of marker data lowers resource allocation in the genomic prediction pipeline. The objective of this study was to optimize marker density for genomic prediction and compare the predictive ability (PA) among genotyping platforms. PA was measured for Sequence capture (SC) and a fixed array developed by Breeders Insight (BI) through 11 harvests in a population of 160 families. The model used for genomic prediction was GBLUP including the dominance effect (GDBLUP-D). PA ranged from 0.07 to 0.35 for SC and from 0.13 to 0.37 for BI. PA on average decreased when using less than 5000 and 1000 markers from SC and BI respectively. Subsets of markers outperformed the full set of markers in each harvest for both technologies. The optimal number of markers for genomic prediction was 1000 for BI and 5000 for SC. When using the full set of markers from each genotyping platform BI yielded higher PA for all the harvests compared to SC. Certain subsets outperformed the full set of markers in all harvests showing potential for the development of a pipeline to select a lower number of more informative markers to improve genomic prediction.
Identification of Transgressive Segregants for Resistance Against Bacterial Leaf Spot in Lettuce (Lactuca sativa)
Manzanero, Byron; Murray, Jesse; Peng, Hui; Sandoya, Germán
1Horticultural Sciences Department. Everglades Research and Education Centre, University of Florida IFAS, Belle Glade, FL
Corresponding author: Germán Sandoya email@example.com
Bacteria leaf spot (BLS) of lettuce (Lactuca sativa) caused by Xanthomonas hortorum pv vitians (Xhv) is a foliar disease capable of generating substantial yield losses. Lettuce production in Florida is concentrated in the Everglades Agricultural Area (EAA), encompassing approximately 10,000 acres and Xhv thrives in the warm, humid environment causing sporadic outbreaks. Management practices for BLS are limited as prophylactic measures with chemicals are not economical feasible due to the sporadic nature of the disease. Durable BLS resistant cultivars may be enhanced by pyramiding complementary resistance loci. BLS Resistant plant introductions (PI) 358001-1 and PI667690 are both resistant to strains of Xhv found in Florida. PI358001-1 possess a dominant single locus denominated Xcvr (Xhantomonas campestris vitians resistance), however, it is unknown if the Xcvr locus is found in PI667690. To identify if distinct resistance loci different than Xcvr, segregation analysis was conducted in F2 individuals & F3 families in a population obtained from a cross between PI358001-1 (R) x PI667690 (R), which were hypothesized to possess different single dominant resistant alleles. F2 individuals were infiltrated in a single trial with virulent strain L7 to determine the hypersensitive response (HR) and the F3 families were spray inoculated with strain L7; an additional spray inoculation experiment was conducted with strain Sc8B in two replications each. Segregation ratio of the F2 population was 316 individuals with compatible HR (resistant): 22 with incompatible HR (susceptible) reaction which was not significantly different (P >0.05) than the expected 15:1 ratio for the two independent single dominant locus model. DS ratings reported 183 resistant: 3 susceptible F3families. The segregation ratio once again was not significantly different than the expected 15:1 ratio for the F3 families (P >0.05) when spray inoculated with isolate L7, resulting in 183R: 1S F3-families. Results indicated all families inoculated with L7 were as resistant (P <0.05) as both parents, linear contrast and their RMEs values indicated that 21 families were significantly (P>0.05) more resistant than both parents against L7 strain. Similar results were obtained with strain SC8B, DS data indicated 183 resistant: 1 susceptible F3 families when inoculated with the strain Sc8B. Segregation ratio was not significantly different than the expected ratio 15 R: 1S. Linear contrast and their RMEs values indicated that 35 families were significantly (P>0.05) more resistant than both parents against SC8B strain. These results suggest the Xcvr gene possessed by PI358001-1 might be allelic to that of PI667690; additionally, Strain Sc8B appears to be more virulent than L7.