Research Project: Genetic Enhancement of Sunflower Yield and Tolerance to Biotic Stress

Location: Sunflower and Plant Biology Research

2023 Annual Report

Objectives
OBJECTIVE 1: Develop and release sunflower germplasm and inbred lines with enhanced yield potential, desirable oil traits, or resistance to crop pests (insects and pathogens), along with effective molecular markers. Subobjective 1A: Develop genetic markers for Phomopsis, rust, and downy mildew resistance. Subobjective 1B: Characterize genetic and pathogenic variation in Phomopsis populations in North Central sunflower growing regions. Subobjective 1C: Evaluate diverse interspecific germplasm for resistance to Phomopsis, rust, and downy mildew. Subobjective 1D: Develop pre-breeding and advanced germplasm with novel traits or combinations of agronomically important traits. OBJECTIVE 2: Identify and characterize traits associated with resistance to insect pests and improved sunflower-pollinator interactions, and evaluate their effectiveness in insect management systems. Subobjective 2A: Evaluate susceptibility of sunflowers to insect pests and develop genetic markers for host plant resistance traits. Subobjective 2B: Assess variation and develop genetic markers for traits associated with pollinator visitation. OBJECTIVE 3: Design and deploy tools that allow for more efficient prediction of crop breeding traits and pest outbreaks, plus development of new crop varieties and cropping systems that allow farmers to address rapidly changing environmental conditions in ND (NP 301, C1, PS1A and 1B).

Approach
The economic impact of sunflower production in the United States is at least $1.5 billion per year. In the primary sunflower production areas, sunflower must compete with genetically-modified crops like corn and soybean that can be easier to produce or have more consistent yields. To maintain its position as a valuable rotational crop and ensure a consistent supply of heart-healthy oil, both maximum yield and consistency of yield must be improved. Losses from diseases and insect pests, along with related costs of management, are primary challenges for improving sunflower yields. Proposed research aims to improve resistance to diseases and insect pests and combine these traits with herbicide resistance, improved oil content and quality to create a more competitive crop. Specific objectives are to: (1) develop genetic markers for resistance to three major sunflower pathogens, (2) understand genetic and pathogenic variation for a disease that has recently increased in incidence and severity, (3) search for new sources of disease resistance from crop wild relatives of cultivated sunflower, (4) identify and characterize traits that will provide resistance to insect pests or improve sunflower-pollinator interactions (which positively contribute to yields), and (5) combine desired traits for pest resistance with other important agronomic traits to create superior germplasm. Success in these objectives will allow higher, more consistent yields and reduce costs of production, contributing to a stable supply of oil and non-oil sunflowers that supports profitable farming.

Progress Report
Subobjective 1A: Develop genetic markers for Phomopsis, rust, and downy mildew resistance. A cross between Phomopsis-susceptible (HA 89) and Phomopsis resistant (HA 378) parents was made to develop a population of 200 F6-derived recombinant inbred lines (RILs) to permit mapping of Phomopsis resistance. Molecular mapping was accomplished for the new rust resistance genes (R17 and R18) and downy mildew genes (Pl35, Pl36, Pl37, and Pl38) and DNA markers linked to these genes were also developed. Fine mapping was accomplished for the downy mildew resistance genes (Pl17–Pl20, and Pl34) and rust resistance genes (R12, R13a, and R16) with closely linked single nucleotide polymorphism markers to allow marker-assisted selection in sunflower breeding programs. Rust resistance gene R11 was cloned using ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing. Quantitative trait locus mapping for Phomopsis resistance was accomplished using a RIL population derived from the cross of HA 89 and Phomopsis-resistant HA-R3. Subobjective 1B: Characterize genetic and pathogenic variation in Phomopsis populations in North Central sunflower growing regions. A survey of Phomopsis stem canker disease lesions was conducted over two years across western Minnesota, North Dakota, and South Dakota and pathogen isolation was successful for 374 total stem lesions. Molecular species determination indicated that Diaporthe helianthi predominated, with D. gulyae identified in fewer than 10% of lesions and two samples identified as D. caulivora. Population analyses revealed a single, panmictic D. helianthi population across the major sunflower producing states. Stem lesion virulence tests of 20 isolates on 15 sunflower lines previously determined to possess stem lesioning resistance indicated that resistance in most lines is isolate-specific and identified five sunflower lines exhibiting broad-spectrum resistance to all tested isolates. Additional disease evaluations identified sunflower germplasm resources with distinct physiological forms of resistance to Phomopsis stem canker including stem lesion resistance, leaf lesion resistance, and resistance to movement of the pathogen from the petiole into the stem. Subobjective 1C: Evaluate diverse interspecific germplasm for resistance to Phomopsis, rust, and downy mildew. Multiple years of greenhouse and field screening for resistance to Phomopsis, rust, and downy mildew were completed for ˜ 100 interspecific sunflower germplasms. Screening for new sources of rust resistance identified eight promising amphiploid lines derived from six different perennial species showing high levels of resistance in segregating progeny (85 to 100% resistance). For resistance to Phomopsis, four interspecific amphiploids based on perennial sunflower crop wild relatives showed 80 to 100% resistance, while two germplasms based on wild annual sunflower species had 100% resistance. Other lines were found to be segregating for downy mildew resistance (up to 40% resistant plants), allowing for further selection, gene identification, and development of mapping populations for marker-assisted breeding. Subobjective 1D: Develop pre-breeding and advanced germplasm with novel traits or combinations of agronomically important traits. Over 2500 nursery rows of high yield, high oil, disease-, insect- and herbicide-resistant sunflower experimental lines were grown each year of the project plan in nurseries in Fargo, North Dakota, and about 1200 each year in our winter nursery in Chile. Of particular importance are several rust and downy mildew resistant sunflower lines released. During the five-year project period, 19 oilseed and confection germplasms (HA-DM6 to HA-DM16, HA-R14 to HA-R21) were released with resistance to one or multiple key sunflower pathogens. The releases were accompanied by validated DNA markers that permit the efficient use of germplasms in breeding programs that combine disease resistance with other key traits. Releases of inbred lines with resistance to the red sunflower seed weevil (HA 488) and the banded sunflower moth (HA 489) were also made. About 20 additional inbred lines developed for a balance of crop quality, yield, and resistance to biotic and abiotic stresses are in the process of being released. These releases also include a new class of early maturity lines that are adapted to northern climates, prevented plant in the Dakotas, and double crop in the central and southern plains. These lines are developing great commercial interest. Subobjective 2A: Evaluate susceptibility of sunflowers to insect pests and develop genetic markers for host plant resistance traits. Four years of field tests have provided two key results related to host plant resistance against the red sunflower seed weevil (RSSW). First, while some inbred lines previously-released by USDA-ARS are more susceptible to RSSW than others, a new line, HA 488, receives about 70% less damage than other inbred lines while also providing advantages (like greater oil content and self-pollination) over other potential sources of resistance. Second, when HA 488 is used as the female parent to create a sunflower hybrid, resistance from HA 488 appears to reduce RSSW damage by 40–50%. Preliminary efforts to map the resistance in HA 488 were not successful, but a second attempt with improvements to testing methods should enable association of resistance with a genetic marker. Subobjective 2B: Assess variation and develop genetic markers for traits associated with pollinator visitation. Data collected from hundreds of sunflower inbred lines were used to develop genetic markers for two traits, floret length and nectar volume, that help explain observed differences in pollinator preference. In a biparental population where high-nectar lines produced twice as much nectar as low-nectar lines (under field conditions), four markers on chromosomes 2 and 16 explained 63% of the variation in nectar volume. From a more diverse panel of inbred lines, floret length was found to vary from 6–10 mm, with shorter florets allowing bees easier access to their nectar. Four markers for floret length were found on chromosomes 1, 10, and 17. Because seed size is an important trait for sunflowers, the same lines were used to locate genes controlling seed length, width, and area. Three of the four loci that govern floret length were not located near any loci that affect seed size, meaning selection for improved pollination can be made without negative effects on seed quality.

Accomplishments
1. Genetic mapping of a pollinator preference trait in sunflower. Nectar is a floral reward that encourages pollination of crop plants by bees and other insects. Previous research with sunflowers (and many other crop species) shows bees prefer varieties with more nectar. ARS scientists in Fargo, North Dakota, and colleagues at the University of Colorado and North Dakota State University used a population made from parents with high or low nectar volume to find the location of genes that determine nectar volume. Four key markers were located, suggesting that breeding sunflowers to have increased nectar volume is feasible. Genes near some of the markers were similar to genes known to influence nectar in other plant species. The identification of markers for nectar volume provides a way for sunflower breeders to increase nectar rewards in sunflower hybrids, grown worldwide, with benefits to farmers and pollinators.

2. Sunflower lines with distinct forms of resistance to Phomopsis stem canker. Phomopsis stem canker, caused by the fungal pathogens Diaporthe helianthi and D. gulyae, is an important disease of sunflowers worldwide and has become more common in the northern Great Plains of the U.S. Identification of sunflower lines with resistance to the disease is crucial for breeding efforts to improve resistance in commercial sunflowers. ARS scientists in Fargo, North Dakota, discovered sunflower lines with distinct types of resistance to Phomopsis stem canker, including stem lesion resistance, leaf lesion resistance, and resistance to pathogen movement from the leaf into the stem. Combining these different forms of resistance in future breeding efforts will help create sunflower lines with long-lasting resistance to Phomopsis stem canker disease.

3. Cloning of a rust resistance gene R11. Rust is one of the most important diseases of sunflower worldwide. This disease is mainly managed through growing sunflowers with resistance genes (R genes). Understanding the location and function of R genes is important to successful breeding for resistance and management of rust. The rust R gene R11 in line HA-R9 shows resistance to most serious races of rust and was previously believed to be located on a small region in sunflower chromosome 13. ARS scientists in Fargo, North Dakota, identified the R11 gene as a single sequence of 3,996 base pairs and caused specific changes (mutations) in the gene which resulted in susceptibility to rust. Additional work suggests specific chemical pathways that may give resistance to rust and may allow improved use of this and other rust-resistance genes in sunflower breeding.

4. Stacking rust and downy mildew resistance genes from sunflower crop wild relatives. Rust and downy mildew (DM) are serious fungal diseases that lower crop yield and quality in confection sunflowers. The diseases are important worldwide, but have been more important in North America in recent years as the pathogens evolve to overcome resistance in the crop. Combining multiple genes (stacking) is a common practice in crop breeding make sure resistance lasts as long as possible. ARS scientists in Fargo, North Dakota, and colleagues at North Dakota State University developed two triple-stacked confection germplasms, HA-R20 and HA-R21, with two rust and one DM gene with resistance to the most common and serious races of rust and downy mildew. Genetic markers for the three genes were also provided to make breeding sunflowers that use these genes a more efficient process. These multiple disease-resistant lines will provide the sunflower industry with long-lasting sources of disease resistance for the continued improvement of the crop.

Review Publications
Prasifka, J.R., Yoshimura Ferguson, M.E., Fugate, K.K. 2023. Genotype and environment effects on sunflower nectar and their relationships to crop pollination. Journal of Pollination Ecology. 33(4):54-63. https://doi.org/10.26786/1920-7603(2023)719.
Liu, Z., Zhang, L., Seiler, G.J., Jan, C. 2023. Molecular mapping of the RF9 gene from RCMG 1 for CMS ANN3 derived from wild sunflower (Helianthus annuus L.). Molecular Breeding. 219.Article 46. https://doi.org/10.1007/s10681-023-03176-3.
Barstow, A., Prasifka, J.R., Attia, Z., Kane, N.C., Hulke, B.S. 2022. Genetic mapping of a pollinator preference trait: Nectar volume in sunflower (Helianthus annuus L.). Frontiers in Plant Science. 13(2022). https://doi.org/10.3389/fpls.2022.1056278.
Qi, L., Ma, G., Seiler, G.J. 2023. Registration of HA-DM9, HA-DM10, and HA-DM11 oilseed sunflower germplasms with dual resistance to sunflower downy mildew. Journal of Plant Registrations. 17(2):426-434. https://doi.org/10.1002/plr2.20283.
Qi, L., Talukder, M.I., Ma, G., Seiler, G. 2023. Introgression and targeting of the Pl37 and Pl38 genes for downy mildew resistance from wild Helianthus annuus and H. praecox into cultivated sunflower (Helianthus annuus L.). Theoretical and Applied Genetics. 136. Article 82. https://doi.org/10.1007/s00122-023-04316-y.
Shamimuzzaman, M., Ma, G., Underwood, W., Qi, L. 2023. Mutation and sequencing-based cloning and functional studies of the rust resistance gene R11 in sunflower (Helianthus annuus). Plant Journal. 115(2):480-493. https://doi.org/10.1111/tpj.16238.
Qi, L., Ma, G., Seiler, G.J. 2022. Registration of two oilseed sunflower germplasms, HA-R18 and HA-R19, resistant to sunflower rust. Journal of Plant Registrations. 16(3):649-655. https://doi.org/10.1002/plr2.20225.
Qi, L., Ma, G., Seiler, G.J. 2023. Registration of HA-DM12, HA-DM13 and HA-DM14 oilseed sunflower germplasms with resistance to sunflower downy mildew and rust. Journal of Plant Registrations. 1-13. https://doi.org/10.1002/plr2.20297.
Ma, G., Talukder, M.I., Song, Q., Li, X., Qi, L. 2023. Whole genome sequencing enables the molecular dissection and candidate gene identification of the rust resistance gene R12 in sunflower (Helianthus annuus L.). Journal of Theoretical and Applied Genetics. 136. Article 143. https://doi.org/10.1007/s00122-023-04389-9.
Rajamohan, A., Prasifka, J.R., Rinehart, J.P. 2022. Vitrification of lepidopteran embryos - a simple protocol to cryopreserve the embryos of the sunflower moth, Homoeosoma electellum. Insects. 13(10). Article 959. https://doi.org/10.3390/insects13100959.
Pantzke, S., Ferguson, B., Rajamohan, A., Rinehart, J.P., Prischmann-Voldseth, D., Prasifka, J.R. 2023. Thermal biology and overwintering behavior of the red sunflower seed weevil (Coleoptera: Curculionidae). Environmental Entomology. https://doi.org/10.1093/ee/nvad041.