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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

2016 Annual Report

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.

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.

Progress Report
Objective 1-Acquire new wild species to fill gaps or deficiencies in the sunflower germplasm collection. An exploration of 2,250 miles was conducted to collect populations of crop wild relatives of sunflower on the barrier islands off the coast of Florida, and coastal Florida and Alabama. This resulted in the collection of 29 accessions represented by one annual Helianthus debilis ssp. debilis, eight annual H. debilis ssp. vestitus, four annual H. debilis ssp. cucumerifolius, four annual H. debilis ssp. tardiflorus, four perennial H. angustifolius, one perennial H. floridanus, two perennial H. heterophyllus, four perennial H. radula, and one perennial H. simulans. These additions to the USDA National Plant Germplasm System crop wild relatives sunflower genebank collection help to fill gaps in the collection, making them available for research for increasing the genetic diversity and improvement of cultivated sunflower, and preserving them for future generations. Objective 2B-Develop effective screening procedures for Phomopsis. Field screening of a recombinant inbred line population for Phomopsis is underway at three locations during the 2016 growing season. Objective 2C-Identify and assess mechanisms of insect resistance. A third year of testing was completed for 15 female lines along with developing inbred lines and putative sources of banded sunflower moth resistance in Casselton, ND. Results indicate wide variation in susceptibility to banded sunflower moth in released USDA inbred lines, but also show the best USDA lines are better than some resistance sources originally intended for use in future breeding. A newer elite line, HA 466, received very little banded moth damage indicating its potential for hybrid breeding or additional research on resistance to this pest. Research on possible resistance to the sunflower stem weevil using X-ray imaging found that most (90%) of the variation in the number of larvae found in stems of different cultivated sunflowers can be explained by the diameter of the inbred or hybrid stems. The influence of stem diameter on stem weevil infestation was later confirmed by planting hybrids with different within-row spacing to artificially create different stem sizes. Results of these trials suggest that host plant resistance may be an inefficient method to manage this pest. Objective 2D-Transfer disease resistance and other agronomic traits from wild species and release pre-breeding germplasm. In order to further characterize novel quantitative trait loci (QTL) for Sclerotinia stalk rot resistance derived from H. argophyllus (PI 494573), an advanced backcross population with 140 BC2F6 recombinant inbred lines was developed by single-seed descent. Two families resistant to Sclerotinia head rot have been selected for germplasm release. These lines had an average disease rating of 0.7, well below the 2.3 of the recurrent parent, and the 2.2 of the two resistant checks. Six families resistant to Sclerotinia stalk rot have also been selected for germplasm release. These lines had an average disease incidence of 4.7%, well below the 23% of the recurrent parent and 13% for the two resistant checks. Confirmation of wild species chromosome or chromosome segments conferring resistance using GISH, and crosses for QTL mapping of the resistance are in progress. Progress has been made in characterizing a white cotyledon trait, in somatic embryogenesis from corolla tubes of interspecific amphiploids between cultivated sunflower and wild crop relatives, and in the triploid F1 production of crosses between a wild H. nuttallii accession and cultivated sunflower. 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 crossed and backcrossed with cultivated line HA 410. Monosomic alien addition lines, with one additional chromosome of various wild species in a cultivated background were selected for further characterization and evaluation for disease resistance. Seeds of six interspecific amphiploids have been increased and agronomic characteristics collected for germplasm release. These amphiploids include cultivated lines P21 or HA 89 crossed with perennial Helianthus hirsutus, H. divaricatus and H. grosseserratus, H. strumosus, H. maximiliani, and H. nuttallii. These interspecific amphiploids have a full set of chromosomes (2n=34) from both wild species and the cultivated lines and have restored fertility for further crossing with the cultivated lines utilizing conventional plant breeding. Objective 2E-Characterize and map disease resistance genes and other agronomic traits. Molecular mapping of two downy mildew resistance genes, Pl17 from HA 458, and Pl18 from a sunflower wild species H. argophyllus was completed. Downy mildew (DM) resistance in the USDA inbred line HA 458 has been shown to be effective against all DM virulent races currently identified in the U.S. Phenotypic evaluation of 186 F2:3 families derived from a cross of HA 458 with HA 234 and bulked segregant analysis using 849 single sequence repeat (SSR) markers located the Pl17 resistance gene in linkage group 4, making it the first downy mildew gene discovered in this linkage group. Both SSR and single nucleotide polymorphism (SNP) markers linked to Pl17 were developed. Two flanking markers, SNP SFW04052 and SSR ORS963, delineated Pl17 in an interval of 3.0 cM, facilitating marker-assisted selection in sunflower breeding programs. A new dominant DM resistance gene (Pl18) transferred from wild Helianthus argophyllus into cultivated sunflower was mapped to linkage group (LG) 2 of the sunflower genome using bulked segregant analysis with 869 SSR markers. Since no other Pl gene has been mapped to LG2, this gene was novel and was designated as Pl18. SSR markers CRT214 and ORS203 flanked Pl18 at a genetic distance of 1.1 and 0.4 cM, respectively. Six co-segregating SNP markers were 1.2 cM distal to Pl18, and another four co-segregating SNP markers were 0.9 cM proximal to Pl18. This new gene is highly resistant to all DM races identified in the U.S. providing breeders with an additional gene to stack with other newly identified downy mildew genes, providing a new more durable source of resistance against this devastating pathogen. TX16R germplasm is resistant to all known U.S. sunflower downy mildew and rust races. Mapping of the downy mildew resistance gene in TX16R was completed last year, and mapping of the rust resistance gene was completed this year. Molecular markers linked to these resistance genes will enhance the development of downy mildew and rust resistant lines for the sunflower industry. Cytoplasms of perennial Helianthus species cause vigor reduction in the absence of nuclear vigor restoration genes. Following the mapping of a vigor restoration gene commonly existing in cultivated lines, a second vigor restoration gene existing in perennial Helianthus giganteus has also been mapped. This will facilitate sunflower line development when using cytoplasms of wild perennial Helianthus species for crop improvement. Two fertility restoration genes for restoring male-sterile cytoplasm of CMSGIG2 were mapped. The SSR markers linked to these genes will enhance the utilization of this cytoplasm to help diversify the use of the currently used single cytoplasm in sunflower. An F2 mapping population of CMSSAL1/RHA 801, a new perennial H. salicifolius male-sterile cytoplasm has been developed with an F3 progeny confirmation in progress. Fertility restoration genes for this new cms source were found in many cultivated lines, with 18 of 20 cultivated lines tested, with only HA 410 and HA 89 not possessing fertility restoration genes for this new cms source. This new cytoplasm will provide additional genetic diversity to the already narrow genetic base of sunflower. Objective 3A-Develop new inbred lines with novel fatty acid composition. A half-diallel genetic population for low saturated fatty acids in a high oleic background was analyzed. F3 seed was tested for variation in the low saturated fat trait using a single plant bulk assay and gas chromatography. The seeds were planted in the field and will be self-pollinated for additional study as progeny lines in the F4 generation. Crosses with a superior F3 individual, with 3.7% saturated fats and 93.6% oleic acid, have been made into several genetic backgrounds, including lines CM 595 and RHA 476, which are the parents for a new hybrid ‘Honeycomb NS’. Additional crosses of F4 plants are planned in the coming year to reduce saturated fat in the breeding program. Releases of the parents of the diallel were made last year, a year ahead of our original plan, in the form of HOLS1, HOLS2, HOLS3, and HOLS4. Objective 3B-Pyramid disease and insect resistance with high yield and oil content. 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 several Sclerotinia and Phomopsis resistant sunflower lines of both heterotic groups. We recently completed thorough genotyping of our experimental inbred lines dating back to 2007 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 this summer and fall to determine the feasibility of genomic selection methods for quantitative traits in hybrid sunflower breeding, as well as to make selections for traits in which useful markers have already been found, such as downy mildew. New releases this year include RHA 477, which is a downy mildew, imidazolinone herbicide tolerant, early maturing inbred, and RHA 478, RHA 479, RHA 480, and HA 481, which are Sclerotinia and Phomopsis resistant inbred lines with high yield and oil content potential.

1. Collection of sunflower crop wild relatives. Solving pest problems 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 and perennial sunflower crop wild relatives’ germplasm from Florida and Alabama. Populations of the beach, Florida, narrow-leaf, variable-leaf, ray-less, and muck sunflower were added to the USDA National Plant Germplasm System wild crop relatives sunflower genebank collection. The germplasm collected has the potential to develop disease resistant sunflower that can be grown on marginal agricultural areas and also to lessen the impact of changing environmental conditions. The beach sunflower has recently been identified as highly resistant to the most virulent race of broomrape, one of the leading threats to sustained global sunflower production. 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.

2. Durable sunflower rust resistance. In the U.S., oilseed sunflower is challenged by a serious foliar leaf rust disease that has been increasingly prevalent in much of the production area with the development of new virulent races. Few suitable inbred 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 developed and released a germplasm derived from the annual crop wild relative of sunflower that incorporates a single dominant gene with resistance to all known races of North American rust with related molecular marker for marker-assisted breeding. Interestingly, this germplasm also contains a single dominant gene with resistance to all known races of another devastating sunflower pathogen, downy mildew with related molecular markers. Stacking these genes provides the sunflower industry with an opportunity to develop durable resistant sunflower lines for these pathogens in an environmentally friendly manner, sustaining sunflower production in large portions of the U.S., improving net returns for sunflower growers, and providing food processors with an abundant source of healthy oil for the American consumers.

3. Discovery of two sunflower downy mildew resistance genes. Downy mildew (DM) is one of the most serious widespread fungal diseases of sunflower that can cause significant yield losses of 50 to 80 percent in cool wet years. ARS researchers at Fargo, North Dakota discovered the two new DM resistance genes that are resistant to all virulent races currently identified in the U.S. Molecular mapping indicated these genes are different from all currently known DM resistance genes in sunflower. DNA markers linked to these genes called Pl17 and Pl18 were developed to facilitate marker-assisted selection in sunflower breeding programs. This offers the potential to control a major sunflower disease in an environmentally friendly manner, improving net returns for sunflower growers, and providing food processors with an abundant source of healthy oil for American consumers.

4. White mold head rot resistant sunflower. Sclerotinia white mold is the causal agent of a serious sunflower disease epidemic worldwide causing stalk, head, and mid-stem rot with the former two accounting for over 80% of the disease incidence. The genetics of resistance to head rot is quantitative, requiring many genes for control, which complicates the breeding effort. There is a lack of sunflower germplasm with high yield potential, good agronomic performance, and seed quality traits with Sclerotinia head rot resistance. ARS scientists at Fargo, North Dakota developed four fertility restoration germplasms with resistance to Sclerotinia head rot and good agronomic performance in an elite genetic background. These germplasms will provide sunflower breeders and producers with additional sources of genes to control a major disease limiting production and will help sustain sunflower production in an environmentally friendly manner.

5. White mold stalk rot resistant sunflower. Sclerotinia white mold is the causal agent of a serious sunflower disease epidemic worldwide causing stalk, head and, mid-stem rot with the former two accounting for over 80% of the disease incidence. The crop wild relatives are native to North America and are distributed over a large geographic area, which exposes them to a wide range of environmental conditions and disease organisms that coevolved with the crop, providing the opportunity to discover disease resistance genes in natural populations. The genetics of resistance to stalk rot is quantitative, requiring many genes for control, which complicates the breeding effort. ARS scientists at Fargo, North Dakota discovered high levels of resistance to stalk rot in several perennial crop wild relatives including Maximillian, Nuttall’s, California, woodland, and muck sunflowers. Six progeny families have been developed that have a disease incidence three times lower than the most resistant hybrids. These germplasms will provide sunflower breeders and producers with additional genes to diversify the genetic base of sunflower and will provide additional control for a major disease that limits production helping, to sustain sunflower production in an environmentally friendly manner.

6. 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 makes 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. ARS scientists at Fargo, North Dakota discovered six new female cytoplasms, derived from the wild perennial willow-leaf, Jerusalem artichoke, sawtooth, giant, and Maximillian sunflowers. Complementary male fertility restorer lines were also developed. The new cytoplasm lines and male fertility restoration lines can be used to diversify the currently used single cytoplasm as an alternative source for parental line development for hybrid sunflower, making it better able to withstand the ever-changing environment where it is grown.

7. Interspecific sunflower amphiploid genetic stocks. Interspecific amphiploids derived from crop wild relatives of sunflower help mine potential genes from a very large gene pool of 53 different species, especially the hard-to-cross perennial species. Amphiploids contain a full balanced set of chromosomes from both the crop wild relatives and the cultivated sunflower overcoming common fertility problems often encountered when making wide crosses. ARS scientists from Fargo, North Dakota, developed six genetic stocks derived from wild sunflower species. The value of these interspecific amphiploid genetic stocks is that they can act as a bridge in interspecific gene transfer, allowing for easier backcrossing with the cultivated sunflower to further broaden the genetic diversity of the sunflower crop, as well as to transfer specific target genes. The genetic stocks will also allow for the development of chromosome addition lines, with individual wild species chromosomes added to the background of cultivated sunflower lines, for genetic studies of wild species specific chromosomes. These amphiploids will give sunflower breeders increased access to more genetic diversity that has been previously extremely difficult to obtain using traditional breeding methodology.

8. Development of a sunflower line adapted to northern climates. Commercial hybrids for the northern growing zones in North America are few in number, with most of Canada limited to one suitable hybrid. In partnership with Agriculture and Agri-Food Canada, an ARS scientist at Fargo, North Dakota developed an inbred fertility restorer line, RHA 476, which is a high oleic line that produces very early maturing and higher yielding hybrids (when crossed to other early maturing, public lines) than the common commercial hybrids in the region. 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. Oils with high oleic acid 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.” Local seed growers have expressed interest in propagating seed of one testcross with especially high yield potential, CM 595/RHA 476, also known as ‘Honeycomb NS’. Joint development of hybrids using this line will expand production in more northern latitudes, and help sunflower adapt to the ever changing environmental conditions that it faces now and in the future.

Review Publications
Mallinger, R.E., Werts, P., Gratton, C. 2015. Pesticide use within a pollinator-dependent crop has negative effects on the abundance and species richness of sweat bees, Lasioglossum spp., and on bumble bee colony growth. Journal of Insect Conservation. 19:999-1010. doi:10.1007/s10841-015-9816-z.
Ayaz, F.A., Colak, N., Topuz, M., Tarkowski, P., Jaworek, P., Seiler, G., Inceer, H. 2015. Comparison of nutrient content in fruit of commercial cultivars of eggplant (Solanum melongena L.). Polish Journal of Food and Nutrition Sciences. 65(4):251-259. doi:10.1515/pjfns-2015-0035.
Zhang, M., Liu, Z., Jan, C. 2016. Molecular mapping of a rust resistance gene R14 in cultivated sunflower line PH 3. Molecular Breeding. 36:32. doi:10.1007/s11032-016-0456-0.
Kantar, M.B., Sosa, C.C., Khoury, C.K., Castaneda-Alvarez, N.P., Achicanoy, H.A., Bernau, V., Kane, N.C., Marek, L., Seiler, G., Rieseberg, L.H. 2015. Ecogeography and utility to plant breeding of the crop wild relatives of sunflower (Helianthus annuus L.). Frontiers in Plant Science. doi: 10.3389/pls.2015.00841.
Qi, L.L., Foley, M.E., Cai, X.W., Gulya, T.J. 2016. Genetics and mapping of a novel downy mildew resistance gene, Pl18, introgressed from wild Helianthus argophyllus into cultivated sunflower (Helianthus annuus L.). Theoretical and Applied Genetics. 129:741-752. doi:10.1007/s00122-015-2662-2.
Harveson, R.M, Nelson, A., Mathew, F.M., Seiler, G.J. 2015. First report of Orobanche ludoviciana parasitizing sunflowers. Plant Health Progress. 16(4):216-217. doi:10.1094/PHP-BR-15-0043.
Prasifka, J.R., Spring, O., Conrad, J., Cook, L.W., Palmquist, D.E., Foley, M.E. 2015. Sesquiterpene lactone composition of wild and cultivated sunflowers and biological activity against an insect pest. Journal of Agricultural and Food Chemistry. 63(16):4042-4049. doi:10.1021/acx.jafc.5b00362.
Hulke, B.S., Grassa, C.J., Bowers, J.E., Burke, J.M., Qi, L., Talukder, Z.I., Rieseberg, L.H. 2015. A unified SNP map of sunflower (Helianthus annuus L.) derived from current genomic resources. Crop Science. 55:1696-1702. doi:10.2135/cropsci2014.11.0752.
Hulke, B.S., Gulya, T.J. 2015. Registration of the oilseed restorer sunflower germplasms RHA 472, RHA 473, RHA 474, and RHA 475, possessing resistance to Sclerotinia head rot. Journal of Plant Registrations. 2:232-238. doi:10.3198/jpr2014.12.0084crg.
Prasifka, J.R., Rinehart, J.P., Yocum, G.D. 2015. Nonconstant thermal regimes enhance overwintering success and accelerate diapause development for Smicronyx fulvus (Coleoptera: Curculionidae). Journal of Economic Entomology. 108(4):1804-1809. doi:10.1093/jee/tov173.
Prasifka, J.R., Bazzalo, M.E. 2016. Susceptibility of sunflower inbreds to Melanagromyza minimoides in Argentina and potential association with plant resistance traits. International Journal of Pest Management. 62(2):105-110.
Dehaan, L.R., Van Tassel, D.L., Anderson, J.A., Asselin, S.R., Barnes, R., Baute, G.J., Cattani, D.J., Culman, S.W., Dorn, K.M., Hulke, B.S., Kantar, M., Larson, S., Marks, M.D., Miller, A.J., Poland, J., Ravetta, D.A., Rude, E., Ryan, M.R., Wyse, D., Zhang, X. 2016. A pipeline strategy for grain crop domestication. Crop Science. 56:917-930.
Ma, G.J., Seiler, G.J., Markell, S.G., Gulya, T.J., Qi, L.L. 2016. Registration of two double rust resistant germplasms, HA-R12 and HA-R13 for confection sunflower. Journal of Plant Registrations. 10:69-74.
Qi, L.L., Seiler, G.J. 2016. Registration of an oilseed sunflower germplasm HA-DM1 resistant to sunflower downy mildew. Journal of Plant Registrations. 10:195-199.
Qi, L.L., Long, Y.M., Ma, G.J., Markell, S.G. 2015. Map saturation and SNP marker development for the rust resistance genes (R4, R5, R13a, and R13b) in sunflower (Helianthus annuus L.). Molecular Breeding. 35:196.