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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Sunflower and Plant Biology Research » Research » Research Project #424876

Research Project: Sunflower Genetic Improvement with Genes from Wild Crop Relatives and Domesticated Sunflower

Location: Sunflower and Plant Biology Research

2015 Annual Report

1a. Objectives (from AD-416):
Objective 1: Identify gaps and acquire new wild species to fill gaps or deficiencies in the sunflower germplasm collection. Objective 2: Identify insect pests and pathogens of sunflower, develop effective screening methods to optimize assessment of resistance to sunflower pathogens, determine mechanisms of plant resistance, phenotype germplasm for resistance to major insect pests and pathogens, and introgress insect and disease resistance genes from the wild species into diverse cultivated germplasm. Subobjective 2A: Identify and monitor pathogens. Subobjective 2B: Develop effective screening procedures for Phomopsis and insect damage. Subobjective 2C: Identify and assess mechanisms of insect resistance. Subobjective 2D: Transfer disease resistance, insect resistance, and other agronomic traits from wild Helianthus species into cultivated sunflower and improve methods for interspecific hybridization and release of pre-breeding germplasm. Subobjective 2E: Characterize and map resistance to pathogens and insect pests, and other agronomic traits. Objective 3: Develop sunflower germplasm with high yield, high oil content, and desirable fatty acid concentrations, as well as novel resistance genes for diseases and insects. Subobjective 3A: Develop new inbred lines with novel fatty acid compositions, such as low saturated fatty acids, and high oleic acid. Subobjective 3B: Pyramid disease and insect resistance genes with high yield and oil content in a common germplasm.

1b. Approach (from AD-416):
Currently, there are a number of factors that reduce sunflower yield including a host of insects and diseases that require careful and costly management practices, reducing profitability. Research is proposed to reduce the input costs by developing durable pest resistance, herbicide resistance, oil content and quality increasing the oil per acre yield of sunflower. Specifically, we will collect wild sunflower relatives to broaden the crop’s genetic base. This germplasm will be phenotyped for resistance to major insect pests and pathogens, cytoplasmic male sterility, and fertility restoration. Methods for improving interspecific hybridization will focus on techniques to detect introgressed alien chromosome segments in interspecific crosses using the genomic fluorescence in situ hybridization technique. Interspecific gene transfer will be evaluated using molecular markers for desirable agronomic traits such as resistance genes to rust and downy mildew. Interspecific germplasm with useful genes will be introgressed into cultivated sunflower and released as enhanced pre-breeding germplasm. Current diseases will be monitored for shifts in virulence and races, and for newly emerged diseases. A field test will be developed to reliably test for the newly emerged Phomopsis stem canker pathogen. An efficient non-destructive screening method will be developed for detecting damage of banded sunflower moth, sunflower moth, red sunflower seed weevil, and sunflower stem weevil. Insect resistance mechanisms will be identified and assessed for sunflower moth. Resistance genes for pathogens and insects, and other agronomic traits will be characterized and mapped. DNA markers for selected traits will be developed and used for marker-assisted breeding. Enhanced sunflower germplasm with high yield, high oil content, and desirable fatty acids concentration, as well as novel resistance genes for diseases and insects will be developed and released. Accomplishing these objectives will provide producers with improved sunflower that will provide a stable supply of high quality oil and confectionery sunflower, improving on-farm profitability and providing the consumer with a reliable domestic supply of a healthy oil, a staple in the American diet.

3. Progress Report:
An exploration of 2,450 miles was conducted to collect populations of annual Helianthus anomalus, H. deserticola, and other annual sunflower crop wild relatives as encountered in Utah and Arizona. This resulted in the collection of 20 accessions represented by eight H. anomalus, five H. deserticola, five H. petiolaris, and two H. annuus accessions added to the USDA National Plant Germplasm System wild crop relatives sunflower collection helping to fill gaps in the collection, making them available for research for improving cultivated sunflower, and preserving them for the future. A second year of testing was completed for 30 public inbred lines evaluated as part of a larger field trial for banded sunflower moth resistance. Moth damage to HA lines was relatively consistent between the two years and the best cultivated inbred lines appeared similar in damage to a resistance source, PI 494859. Phenotypic data was collected on the abundance of capitate glandular trichomes in an F4 population (from HA 300 × RHA 464); because the number of glandular trichomes correlates with the abundance of defensive compounds in sunflower florets, the corresponding genetic data should provide a way to more easily breed for chemical resistance to floret-feeding insects. Preliminary work on an F4 mapping population for resistance to red sunflower seed weevil was positive. With a cool period following (artificial) infestation of weevils, most larvae did not chew exit holes in pericarps, making a normal visual examination of achenes inadequate for damage assessment. However, an assessment of weevil damage using X-rays proved to be effective and indicated clear separation of weevil-susceptible and-resistant lines in the population. New interspecific crosses involving three accessions of perennial H. tuberosus, two accessions of H. strumosus, and one accession each of H. hirsutus and H. simulans were backcrossed with cultivated line HA 410. Previous crosses involving H. hirsutus, H. salicifolius, H. occidentalis, and H. divaricatus were advanced a generation to identify chromosome addition lines, plants with reduced vigor, cytoplasmic male-sterility, and plants with the same chromosome number as cultivated sunflower. Six accessions of wild annual H. annuus species previously reported to be resistant to downy mildew were crossed with lines NMS HA 89 (oil sunflower) and CMS CONFSCLB1 (confection sunflower). Twelve F1 hybrids were obtained for transferring new downy mildew resistance genes from wild species into cultivated sunflower. Germplasm PH3 released in 2004 for rust resistance and is now known to be resistant to all rust races in the U.S. The rust resistance gene has been named R14, with linked molecular markers that will help accelerate marker assisted breeding for rust resistant sunflower. Germplasm TX16R was also released in 2004 with resistance to all known U.S. downy mildew races. Mapping of the downy mildew resistance gene in TX16R has been completed. This will provide molecular markers for sunflower breeders to assist in the selection and development of durable rust and downy mildew-resistant hybrids. Cytoplasms of perennial Helianthus species cause vigor reduction in the absence of nuclear vigor restoration genes. An F3 mapping population confirmed the nuclear vigor restoration gene, and mapping of a commonly existing vigor restoration gene for perennial Helianthus cytoplasms was completed. This will aid in sunflower line development while using perennial Helianthus species cytoplasms to increase genetic diversity in the sunflower genome. Additionally, an F3 generation progeny confirmation involving a vigor restoration gene derived from H. giganteus has been completed with the mapping of the gene in progress. Mapping of a fertility restoration (Rf) gene restoring male-sterile cytoplasm for CMS ANN3 has been completed, providing markers to help utilize this wild H. annuus cytoplasm to diversify the narrow genetic base of cultivated sunflower. A new male-sterile cytoplasm was identified in backcross progenies of wild perennial H. salicifolius with cultivated sunflower line HA 410. An F2 mapping population for the fertility restoration gene from H. salicifolius was established and F3 progeny confirmation is in progress. Sclerotinia head rot resistance was mapped using an association mapping approach on a 260 population lines with 52 significant loci found. The 52 loci tended to cluster and in most breeding populations would act as a haplotype (closely linked genes). Single nucleotide polymorphism (SNP) markers were used to genotype four F2 populations previously used for SSR mapping to identify SNP markers linked to four rust R genes, R4, R5, R13a, and R13b. Of the 67 linkage group (LG) 2 SNP markers screened, two SNPs flanked R5 at a genetic distance of 0.6 cM and 1.2 cM, respectively. A total of 69 LG13 SNP markers were analyzed in the R4, R13a, and R13b populations. In the R4 consensus map, the R4 gene was flanked by seven SNP loci; three co-segregating SNPs are on one side (0.7 cM proximal) and four on the other side (0.6 cM distal). Similarly, SNP markers that are tightly linked to both R13a and R13b were identified. R13a was flanked by SNP markers at genetic distances of 0.4 and 0.2 cM. The SNP SFW00757 co-segregated with R13b, and another three co-segregating SNPs were 2.4 cM proximal to R13b. A mapping population of 140 F2 individuals was created between rust susceptible parent HA 89 and rust resistant parent HA-R8. Phenotyping of the 140 F3 families indicated that rust resistance was controlled by a single dominant gene from the resistant line HA-R8. Genotyping of the F2 population was conducted using genotyping by sequencing (GBS). The new R gene in HA-R8 was mapped to the upper end of LG8. The closest SNP marker linked to the R gene was at genetic distance of 0.2 cM. The rust resistance R2 gene was relocated from linkage group (LG) 9 to LG14. Based on phenotypic assessments and simple sequence repeat (SSR) analyses of the 117 F2 individuals derived from a cross of HA 89 with MC29 (USDA), R2 was mapped to LG14 of the sunflower genome, and not to the previously reported location on LG9. The closest SSR marker HT567 was located at 4.3 cM distal to R2. Furthermore, 36 selected SNP markers from LG14 were used to saturate the R2 region. Of the three closely linked markers, SFW00211 amplified an allele specific for the presence of R2 in a marker validation set of 46 breeding lines, and SFW01272 was also shown to be diagnostic for R2. Two double rust-resistant confection germplasms, HA-R12 and HA-R13 were developed by the pedigree breeding method and DNA marker-assisted selection, each containing two different rust resistance genes. HA-R12 is homozygous for both the R2 and R13a genes derived from MC29 and HA-R6, and HA-R13 is homozygous for the both the R5 and R13a genes derived from HA-R2 and HA-R6. Both lines have high levels of resistance to the predominant and the most virulent rust race currently identified in the U. S. These germplasms will be a welcome addition to the confection sunflower breeder’s efforts to provide urgently needed rust resistance genes that can be incorporated into finished commercial confection hybrids. Downy mildew resistant germplasm HA-DM1 is a BC2F3-derived BC2F4 oilseed maintainer selection from the cross of HA 89*2/NMS HA 89/Helianthus argophyllus accession PI 494573 developed by the backcross breeding method and DNA marker-assisted selection for the downy mildew resistance gene Pl18 introgressed from wild H. argophyllus PI 494573. The cross between NMS HA 89 and PI 494573 was made in 2009 and the selected resistant F1 plants were backcrossed twice to HA 89. The BC2F3-derived HA-DM1 is homozygous for the Pl18 gene verified by DNA markers, and immune to all known races of downy mildew, providing breeders with an effective and unique source of resistance against downy mildew in sunflower. A half-diallel genetic population for low saturated fatty acids in a high oleic background was analyzed. F2 seed was tested for variation in the low saturated fat trait using a cut-seed assay and gas chromatography. The seeds were planted in the field and will be self-pollinated for additional study as progeny lines in the F3 generation. Additional crosses of F3 plants are planned to further reduce saturated fat content. Parental materials of the diallel cross were released this year as genetic stocks HOLS1, HOLS2, HOLS3, and HOLS4. All were derived from low saturated fat plant introductions and mutagenesis stocks backcrossed to inbred line HA 466, a high oleic, imidazolinone resistant, Sclerotinia and Phomopsis resistant inbred line. Nearly 2000 nursery rows of high yield, high oil, disease, insect, and herbicide resistant sunflower experimental lines were grown in nurseries in Fargo, Puerto Rico, and Chile. Of these, several are candidates for release, including an early maturing, high yielding restorer line with high oleic acid, and a sunflower line with a simply inherited, dominant resistance to red sunflower seed weevil. The red sunflower seed weevil resistance is being mapped this year to release both germplasm and markers to the sunflower community. These planned releases for late FY15 are in addition to those already released and described for the purpose of gene pyramiding. This year, we have also genotyped the majority of the breeding lines for which testcross yield and disease resistance data have been accumulated, using a genotyping by sequencing (GBS) approach supplemented with whole-genome data of the parental stocks. The genomic data and historic phenotypic data sets will be modeled to determine the feasibility of genomic selection methods in hybrid sunflower breeding, as well as to make selections for traits in which useful markers have already been found, such as downy mildew.

4. Accomplishments
1. Stacking durable sunflower rust resistance genes. In confectionery sunflower grown as a seed crop in the U.S., leaf rust is a serious foliar disease that has been increasingly prevalent in much of the production area with the development of new virulent races. Few suitable inbred confection sunflower lines exist that have a high level of rust resistance, which poses risks of a potential disease epidemic from the use of a single resistance gene. ARS scientists at Fargo, North Dakota released two confectionery sunflower germplasms each incorporating two different single dominant rust resistance genes, R2 and R13a, and R5 and R13a, respectively. These genes will enhance durable rust resistance to this devastating disease in confectionery sunflower, sustaining sunflower production in large portions of the US, improving net returns for sunflower growers, and providing food processors with an abundant source of a healthy snack for the American consumers.

2. New high oleic acid reduced saturated fat sunflower lines. Oleic acid is a monounsaturated fat known to be beneficial in the human diet by increasing high-density lipoproteins cholesterol and reducing low density lipoproteins. Saturated fats are generally known to be neutral or negative to human health, depending on the length of the carbon chain. Oils with high oleic acid and low saturated fats will not require transesterification to increase oxidative stability, which is noteworthy since the FDA has now changed trans-fats to the status of “not generally recognized as safe.” ARS researchers at Fargo, North Dakota released four genetic stocks with very high levels of oleic acid and very low saturated fats in the seed oil. Use of these lines will provide the sunflower industry the opportunity to develop hybrids with higher quality oil allowing consumers a choice for selecting healthier oils in their diet.

3. Collection of sunflower crop wild relatives. Solving insect and disease pests and environmental stresses in the production of sunflower requires new sources of genetic diversity. ARS scientists at Fargo, North Dakota, and Ames, Iowa collected new annual sunflower crop wild relatives’ germplasm from Utah and Arizona. Populations of the desert, anomalus, and prairie sunflower were added to the USDA National Plant Germplasm System wild crop relatives sunflower collection. The germplasm collected has the potential to develop water stress tolerant sunflower that can be grown on marginal agricultural areas and also to lessen the impact of changing environmental conditions. The collection of the crop wild relatives not only makes them available for research related to the improvement of the sunflower crop, but also fills gaps in the collection and preserves them for future generations.

4. New female parent for hybrid sunflower production. Globally, sunflower is the fifth largest hybrid crop. It is currently based on a single female parent, CMS PET1, developed in 1969 derived from the wild prairie sunflower leaving sunflower with a very narrow genetic base. This potentially leaves sunflower very vulnerable to attack by pests similar to the disaster seen in corn with the outbreak of the southern corn leaf fungal blight in the 1970s. A newly identified female cytoplasm line, CMS SAL1, derived from the wild perennial willow-leaf sunflower and associated male restorer line is different from CMS PET1 and its associated male lines. The new CMS and male fertility restoration line can be used to diversify the currently used CMS PET1 cytoplasm as an alternative source for parental line development for hybrid sunflower.

5. Differences in defensive chemistry between wild and cultivated sunflowers. Sesquiterpene lactones (STL) are defensive compounds in sunflower and other related plants that provide natural defense from pests, including insects and pathogens. These compounds, contained in glandular hairs, are thought to be one reason that sunflower crop wild relatives are less damaged by floret-feeding insects such as larvae of the sunflower moth than the cultivated crop. An analysis of wild sunflowers, public inbred lines, and commercial hybrids showed that a group of closely related compounds were deficient in most cultivated germplasm, and that at least one of these compounds causes delayed development in sunflower moth larvae. An initial cross between the most STL-rich wild material, Plant Introduction, and a USDA inbred was made in an attempt to transfer the defensive chemistry of wild sunflowers into cultivated material. This offers the potential to control a major sunflower pest in an environmentally friendly manner.

Review Publications
Prasifka, J.R. 2015. Variation in the number of capitate glandular trichomes in wild and cultivated sunflower germplasm and potential for use in host plant resistance. Plant Genetic Resources. 13:68-74.

Seiler, G.J. 2015. Comparison of fatty acid composition of oil from original and regenerated populations of wild Helianthus species. Plant Genetic Resources: Characterization and Utilization. 13(1):83-89. DOI:10.1017/S1479262114000677.

Tinsley, N.A., Spencer, J.L., Estes, R.E., Prasifka, J.R., Schrader, P.M., French, B.W., Gray, M.E. 2015. Larval mortality and development for rotation-resistant and rotation-susceptible populations of the western corn rootworm on Bt corn. Journal of Applied Entomology. 139:46-54.

Qi, L.L., Ma, G.J., Long, Y.M., Hulke, B.S., Gong, L., Markell, S.G. 2015. Relocation of a rust resistance gene R2 and its marker-assisted gene pyramiding in confection sunflower (Helianthus annuus L.). Theoretical and Applied Genetics. 128(3):477-488. DOI:10.1007/s00122-014-2446-0.

Qi, L.L., Long, Y.M., Jan, C.C., Ma, G., Gulya, T.J. 2015. Pl17 is a novel gene independent of known downy mildew resistance genes in the cultivated sunflower (Helianthus annuus L.). Journal of Theoretical and Applied Genetics. 128(4):757-767. DOI:10.1007/s00122-015-2470-8.

Mathew, F.M., Prasifka, J.R., Gaimari, S.D., Shi, L., Markel, S.G., Gulya, T.J. 2015. Rhizopus oryzae associated with Melanagromyza splendida and stem disease of sunflowers (Helianthus annuus) in California. Plant Health Progress. 16(1):39-42. DOI:10.1094/PHP-RS-14-0042.

Seiler, G.J., Jan, C.C. 2014. Wild sunflower species as a genetic resource for resistance to sunflower broomrape (Orobanche cumana Wallr.). Helia. 37(61):129-139. DOI:10.1515/HELIA-2014-0013.

Jan, C.C., Liu, Z., Seiler, G.J., Velasco, L., Perez-Vich, B., Fernandez-Martinez, J. 2014. Broomrape (Orobanche cumana Wallr.) resistance breeding utilizing wild Helianthus species. Helia. 37(61):141-150.

Cainong, J.C., Bockus, W.W., Feng, Y., Chen, P., Qi, L., Sehgal, S.K., Danilova, T.V., Koo, D., Friebe, B., Gill, B.S. 2015. Chromosome engineering, mapping, and transferring of resistance to Fusarium head blight disease from Elymus tsukushiensis into wheat. Theoretical and Applied Genetics. 128(6):1019-1027. DOI:10.10.1007/S00122-015-2485-1.

Liu, Z., Cai, X., Seiler, G.J., Jan, C.C. 2014. Interspecific amphiploid-derived alloplasmic male sterility with defective anthers, narrow disk florets, and small ray flowers in sunflower. Plant Breeding. 133(6):742-747. DOI:10.1111/PBR.12216.

Qi, L., Gong, L., Markell, S.G., Seiler, G.J., Gulya Jr, T.J., Hulke, B.S. 2014. Registration of two confection sunflower germplasm Lines, HA-R10 and HA-R11, Resistant to sunflower rust. Journal of Plant Registrations. 8:329-333. DOI: 10.3198/jpr2014.02.0010crg.

Feng, J., Liu, Z., Seiler, G.J., Jan, C.C. 2015. Registration of cytoplasmic male-sterile oilseed sunflower genetic stocks CMS GIG2 and CMS GIG2-RV, and fertility restoration lines RF GIG2-MAX 1631 and RF GIG2-MAX 1631-RV. Journal of Plant Registrations. 9:125-127. DOI:10.3198/jpr2014.050029crgs.

Qi, L., Seiler, G.J. 2013. Registration of a male fertility restorer oilseed sunflower germplasm, HA-R9, resistant to sunflower rust. Journal of Plant Registrations. 7(3):353-357.

Pearson, T.C., Prasifka, J.R., Brabec, D.L., Haff, R.P., Hulke, B.S. 2014. Automated detection of insect-damaged sunflower seeds by X-ray imaging. Applied Engineering in Agriculture. 30(1):125-131.