Survival analysis of freezing stress in the North American native perennial flax, Linum lewisii.

Abstract. The expansive range of Lewis flax (Linum lewisii), an herbaceous perennial, exposes the species to a diversity of climatic conditions. As interest in the domestication and adoption of perennial crop alternatives grows and interest in this species for natural area restoration continues, the assurance of a commercial plant variety's ability to endure the full range of possible climatic extremes is paramount. This study examines the freezing tolerance of a geographically representative sampling of 44 Lewis flax accessions at winter temperature extremes experienced in the northern Great Plains of the USA. Survival analysis models were adapted to include temperature exposure, in replacement of ordinal time typically used in such models, to produce statistics evaluating reactions to extreme temperatures that Lewis flax would encounter in our field environments. Our results revealed Lewis flax is more freezing tolerant than previously reported, and revealed four accessions with significantly superior genetic freezing tolerance than the released 'Maple Grove' cultivar. Furthermore, regrowth analyses indicate variation among accessions not associated with survival, which could lead to improving regrowth rate and survival simultaneously. These findings and their methodology expand the understanding of Lewis flax adaptation for winter hardiness and offer an efficient, new model that can be used to evaluate freezing tolerance at ordinal temperatures without requiring extensive prior physiological knowledge for a species.

Heritable differences in abundance of bacterial rhizosphere taxa are correlated with fungal necrotrophic pathogen resistance.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

Assessment of biogeographic variation in traits of Lewis flax (Linum lewisii) for use in restoration and agriculture.

Lewis flax (Linum lewisii) is widely distributed across western North America and is currently used in native ecosystem restoration. There is also growing interest in de novo domestication of Lewis flax as a perennial oilseed crop. To better understand this species and facilitate both restoration and domestication, we used common gardens to assess biogeographical variation in a variety of seed and growth traits from 37 flax accessions, consisting of 35 wild populations from the Intermountain West region, the pre-variety germplasm Maple Grove (L. lewisii) and the cultivar 'Appar' (L. perenne) and related this variation to collection site geography and climate. Results from linear mixed models suggest there is extensive phenotypic variation among populations of Lewis flax within the Intermountain West. Using a multivariate approach, we identify a key suite of traits that are related to latitude and climate and may facilitate adaptation, including flowering indeterminacy, seed mass and stem number. These traits should be taken into account when considering the release of new germplasm for restoration efforts. We also find that Lewis flax seed contains desirably high amounts of alpha-linolenic acid and is otherwise mostly indistinguishable in fatty acid composition from oil-type varieties of domesticated flax (L. usitatissimum), making it a strong candidate for domestication. This study provides fundamental knowledge for future research into the ecology and evolution of Lewis flax, which will inform its use in both restoration and agriculture.

Genetic mapping of a pollinator preference trait: Nectar volume in sunflower (Helianthus annuus L.).

Although high pollinator visitation is crucial to ensure the yields of pollinator-dependent crops, the quantitative trait loci (QTL) controlling nectar volume in sunflower (Helianthus annuus L.), a pollinator preference trait, have yet to be identified. To address this, a recombinant inbred line mapping population, derived from lines with contrasting nectar volume, was used to identify loci responsible for the phenotype. As a result, linkage mapping and QTL analysis discovered major loci on chromosomes 2 and 16 that are associated with variation in nectar volume in sunflower. Increased nectar volume is also associated with increased sugars and total energy available per floret. The regions on chromosomes 2 and 16 associated with the nectar phenotype exhibit indications of chromosome structural variation, such that the phenotype is associated with rearrangements affecting regions containing hundreds of genes. Candidate genes underlying QTL on chromosomes 9 and 16 are homologous to genes with nectary function in Arabidopsis. These results have implications for sunflower breeding, to enhance pollination efficiency in sunflower, as well as current and future studies on sunflower evolution.

Aberrant RNA splicing due to genetic incompatibilities in sunflower hybrids.

Genome-scale studies have revealed divergent mRNA splicing patterns between closely related species or populations. However, it is unclear whether splicing differentiation is a simple byproduct of population divergence, or whether it also acts as a mechanism for reproductive isolation. We examined mRNA splicing in wild × domesticated sunflower hybrids and observed 45 novel splice forms that were not found in the wild or domesticated parents, in addition to 16 high-expression parental splice forms that were absent in one or more hybrids. We identify loci associated with variation in the levels of these splice forms, finding that many aberrant transcripts were regulated by multiple alleles with nonadditive interactions. We identified particular spliceosome components that were associated with 21 aberrant isoforms, more than half of which were located in or near regulatory QTL. These incompatibilities often resulted in alteration in the protein-coding regions of the novel transcripts in the form of frameshifts and truncations. By associating the splice variation in these genes with size and growth rate measurements, we found that the cumulative expression of all aberrant transcripts was correlated with a significant reduction in growth rate. Our results lead us to propose a model where divergent splicing regulatory loci could act as incompatibility loci that contribute to the evolution of reproductive isolation.

Breeding for sustainable oilseed crop yield and quality in a changing climate.

As the effects of climate change continue to alter crop-growing conditions year-to-year on both prime and marginal agricultural landscapes, we must consider the effects not only on yield but also on quality. This is particularly true for oilseed crops. In this review, we explore the importance of oilseeds in general and the specific uses of major oilseed crops including soybean, sunflower, canola, peanut, and cottonseed. We review the physiology of seed oil production, from the perspective of the plant's adaptation to environmental changes. Of particular importance is the role of temperature and water availability on oil synthesis. We then discuss how this influences genetic variation, phenotype variability due to environment, and the interaction of genetics and environment to affect composition and yield of vegetable oils. The ability to predict these effects using genomics and bioinformatics is an important new frontier for breeders to maximize stability of a desired fatty acid composition for their crop over increasingly extreme agricultural environments.

Genetic loci underlying quantitative resistance to necrotrophic pathogens Sclerotinia and Diaporthe (Phomopsis), and correlated resistance to both pathogens.

KEY MESSAGE: We provide results rooted in quantitative genetics, which combined with knowledge of candidate gene function, helps us to better understand the resistance to two major necrotrophic pathogens of sunflower. Necrotrophic pathogens can avoid or even benefit from plant defenses used against biotrophic pathogens, and thus represent a distinct challenge to plant populations in natural and agricultural systems. Sclerotinia and Phomopsis/Diaporthe are detrimental pathogens for many dicotyledonous plants, including many economically important plants. With no well-established methods to prevent infection in susceptible plants, host-plant resistance is currently the most effective strategy. Despite knowledge of a moderate, positive correlation in resistance to the two diseases in sunflower, detailed analysis of the genetics, in the same populations, has not been conducted. We present results of genome-wide analysis of resistance to both pathogens in a diversity panel of 218 domesticated sunflower genotypes of worldwide origin. We identified 14 Sclerotinia head rot and 7 Phomopsis stem canker unique QTLs, plus 1 co-located QTL for both traits, and observed extensive patterns of linkage disequilibrium between sites for both traits. Most QTLs contained one credible candidate gene, and gene families were common for the two disease resistance traits. These results suggest there has been strong, simultaneous selection for resistance to these two diseases and that a generalized mechanism for defense against these necrotrophic pathogens exists.

A Greenhouse Method to Evaluate Sunflower Quantitative Resistance to Basal Stalk Rot Caused by Sclerotinia sclerotiorum.

Resistance of sunflower to basal stalk rot (BSR) caused by the fungus Sclerotinia sclerotiorum is quantitative, controlled by multiple genes contributing small effects. Consequently, artificial inoculation procedures allowing sufficient throughput and resolution of resistance are needed to identify highly resistant sunflower germplasm resources and to map loci contributing to resistance. The objective of this study was to develop a greenhouse-based method for evaluating sunflower quantitative resistance to BSR that would be simple, space- and time-efficient, high throughput, high resolution, and correlated with field observations. Experiments were conducted with 5-week-old sunflower plants and Sclerotinia-infested millet seed as inoculum to assess the impact of pot size and temperature and to determine the most favorable inoculum rate and placement. Subsequently, an additional experiment was performed to assess the correlation of the greenhouse inoculation procedure with field results by using a panel of 32 sunflower genotypes with known field response to BSR previously determined in multiyear, multilocation artificially inoculated trials. Experimental observations indicated that the newly developed greenhouse inoculation procedure provided improved resolution to identify highly resistant genotypes and was strongly correlated with field observations. This method will be useful for screening of sunflower experimental and breeding materials, disease phenotyping of genetic mapping populations, and evaluation of resistance to different pathogen isolates.

Determination of Virulence Phenotypes of Plasmopara halstedii in the United States.

Downy mildew, caused by Plasmopara halstedii (Farl.) Berl. and de Toni, is an economically important disease in cultivated sunflowers, Helianthus annuus L. Resistance genes incorporated into commercial hybrids are used as an effective disease management tool, but the duration of effectiveness is limited as virulence evolves in the pathogen population. A comprehensive assessment of pathogen virulence was conducted in 2014 and 2015 in the U.S. Great Plains states of North Dakota and South Dakota, where approximately 75% of the U.S. sunflower is produced annually. The virulence phenotypes (and races) of 185 isolates were determined using the U.S. standard set of nine differentials. Additionally, the virulence phenotypes of 61 to 185 isolates were determined on 13 additional lines that have been used to evaluate pathogen virulence in North America and/or internationally. Although widespread virulence was identified on several genes, new virulence was identified on the Pl8 resistance gene, and no virulence was observed on the PlArg, Pl15, Pl17 and Pl18 genes. Results of this study suggest that three additional lines should be used as differentials and agree with previous studies that six lines proposed as differentials should be used in two internationally accepted differential sets. For effective disease management using genetic resistance, it is critical that virulence data be relevant and timely. This is best accomplished when pathogen virulence is determined frequently and by using genetic lines containing resistance genes actively incorporated into commercial cultivars.

Capitate Glandular Trichomes Fail to Provide Significant Resistance to Banded Sunflower Moth (Lepidoptera: Tortricidae).

Extracts from capitate glandular trichomes (CGT) of wild and cultivated sunflowers, Helianthus spp., have repellent or toxic effects on sunflower specialists and generalist herbivores less closely associated with sunflower. Though CGT have been primarily examined for their potential to provide partial resistance to the sunflower moth, Homoeosoma electellum Hulst (Lepidoptera: Pyralidae), a floret- and seed-feeding pest, the banded sunflower moth (Cochylis hospes Walsingham [Lepidoptera: Tortricidae]) is a similar species more common in the primary sunflower-producing states of North Dakota and South Dakota. Replicated field trials using partially inbred lines with low or high CGT densities were used to evaluate possible reductions to seed damage by C. hospes larvae in 2016-2017. Results failed to support the idea that CGT are a useful defense against larvae of C. hospes; the putative plant defense of high trichome density corresponded to slightly more, rather than less, insect damage. A test of a secondary explanation, that strength of sunflower hulls could help determine patterns of seed damage among tested lines, produced similarly negative results. Though timing of bloom differed between groups of most- and least-damaged lines, prior research and pheromone-trapping data suggest differences in plant maturity also cannot adequately explain the observed results. While the specific mechanisms remain unclear, significant differences in susceptibility to C. hospes exist for cultivated sunflower and limit losses from this primary insect pest.

Seed and floret size parameters of sunflower are determined by partially overlapping sets of quantitative trait loci with epistatic interactions.

KEY MESSAGE: Floret and seed traits are moderately correlated phenotypically in modern sunflower cultivars, but the underlying genetics are mostly independent. Seed traits in particular are governed in part by epistatic effects among quantitative trait loci. Seed size is an important quality component in marketing commercial sunflower (Helianthus annuus L.), particularly for the in-shell confectionery market, where long and broad seed types are preferred as a directly consumed snack food globally. Floret size is also important because corolla tube length was previously shown to be inversely correlated with pollinator visitation, impacting bee foraging potential and pollinator services to the plant. Commercial sunflower production benefits from pollinator visits, despite being self-compatible, and bees are required in hybrid seed production, where "female" and "male" inbred lines are crossed at field scale. Issues with pollination of long-seed confectionery sunflower suggest that there may be an unfavorable correlation between seed and floret traits; thus, our objective was to determine the strength of the correlation between seed and floret traits, and confirm any co-localization of seed and floret trait loci using genome-wide association analysis in the SAM diversity panel of sunflower. Our results indicate that phenotypic correlations between seed and floret traits are generally low to moderate, regardless of market class, a component of population substructure. Association mapping results mirror the correlations: while a few loci overlap, many loci for the two traits are not overlapping or even adjacent. The genetics of these traits, while modestly quantitative and influenced by epistatic effects, are not a barrier to simultaneous improvement of seed length and pollinator-friendly floret traits. We conclude that breeding for large seed size, which is required for the confectionery seed market, is possible without producing florets too long for efficient use by pollinators, which promotes bee foraging and associated pollination services.

Genetic and phenotypic analyses indicate that resistance to flooding stress is uncoupled from performance in cultivated sunflower.

Given the rising risk of extreme weather caused by climate change, enhancement of abiotic stress resistance in crops is increasingly urgent. But will the development of stress-resistant cultivars come at the cost of yield under ideal conditions? We hypothesize that this need not be inevitable, because resistance alleles with minimal pleiotropic costs may evade artificial selection and be retained in crop germplasm. Genome-wide association (GWA) analyses for variation in plant performance and flooding response were conducted in cultivated sunflower, a globally important oilseed. We observed broad variation in flooding responses among genotypes. Flooding resistance was not strongly correlated with performance in control conditions, suggesting no inherent trade-offs. Consistent with this finding, we identified a subset of loci conferring flooding resistance, but lacking antagonistic effects on growth. Genetic diversity loss at candidate genes underlying these loci was significantly less than for other resistance genes during cultivated sunflower evolution. Despite bottlenecks associated with domestication and improvement, low-cost resistance alleles remain within the cultivated sunflower gene pool. Thus, development of cultivars that are both flooding-tolerant and highly productive should be straightforward. Results further indicate that estimates of pleiotropic costs from GWA analyses explain, in part, patterns of diversity loss in crop genomes.

Linkage Mapping and Genome-Wide Association Studies of the Rf Gene Cluster in Sunflower (Helianthus annuus L.) and Their Distribution in World Sunflower Collections.

Commercial hybrid seed production in sunflower currently relies on a single cytoplasmic male sterility (CMS) source, PET1 and the major fertility restoration gene, Rf1, leaving the crop highly vulnerable to issues with genetic bottlenecks. Therefore, having multiple CMS/Rf systems is important for sustainable sunflower production. Here, we report the identification of a new fertility restoration gene, Rf7, which is tightly linked to a new downy mildew (DM) resistance gene, Pl34 , in the USDA sunflower inbred line, RHA 428. The Rf7 gene was genetically mapped to an interval of 0.6 cM on the lower end of linkage group (LG) 13, while Pl34 was mapped 2.1 cM proximal to the Rf7. Both the genes are located in a cluster of Rf and Pl genes. To gain further insights into the distribution of Rf genes in the sunflower breeding lines, we used a genome-wide association study (GWAS) approach to identify markers associated with the fertility restoration trait in a panel of 333 sunflower lines genotyped with 8,723 single nucleotide polymorphism (SNP) markers. Twenty-four SNP markers on the lower end of LG13 spanning a genomic region of 2.47 cM were significantly associated with the trait. The significant markers were surveyed in a world collection panel of 548 sunflower lines and validated to be associated with the Rf1 gene. The SNP haplotypes for the Rf1 gene are different from Rf5 and the Rf7gene located in the Rf gene cluster on LG13. The SNP and SSR markers tightly flanking the Rf7 gene and the Pl34 gene would benefit the sunflower breeders in facilitating marker assisted selection (MAS) of Rf and Pl genes.

Using Nectar-Related Traits to Enhance Crop-Pollinator Interactions.

Floral nectar and other reward facilitate crop pollination, and in so doing, increase the amount and breadth of food available for humans. Though abundance and diversity of pollinators (particularly bees) have declined over the past several decades, a concomitant increase in reliance on pollinators presents a challenge to food production. Development of crop varieties with specific nectar or nectar-related traits to attract and retain pollinating insects is an appealing strategy to help address needs of agriculture and pollinators for several reasons. First, many crops have specific traits which have been identified to enhance crop-pollinator interactions. Also, an improved understanding of mechanisms that govern nectar-related traits suggest simplified phenotyping and breeding are possible. Finally, the use of nectar-related traits to enhance crop pollination should complement other measures promoting pollinators and will not limit options for crop production or require any changes by growers (other than planting varieties that are more attractive or rewarding to pollinators). In this article, we review the rationale for improving crop-pollinator interactions, the effects of specific plant traits on pollinator species, and use cultivated sunflowers as a case study. Recent research in sunflower has (i) associated variation in bee visitation with specific floral traits, (ii) quantified benefits of pollinators to hybrid yields, and (iii) used genetic resources in sunflower and other plants to find markers associated with key floral traits. Forthcoming work to increase pollinator rewards should enable sunflower to act as a model for using nectar-related traits to enhance crop-pollinator interactions.

Genetic Architecture of Capitate Glandular Trichome Density in Florets of Domesticated Sunflower (Helianthus annuus L.).

Capitate glandular trichomes (CGT), one type of glandular trichomes, are most common in Asteraceae species. CGT can produce various secondary metabolites such as sesquiterpene lactones (STLs) and provide durable resistance to insect pests. In sunflower, CGT-based host resistance is effective to combat the specialist pest, sunflower moth. However, the genetic basis of CGT density is not well understood in sunflower. In this study, we identified two major QTL controlling CGT density in sunflower florets by using a F4 mapping population derived from the cross HA 300 × RHA 464 with a genetic linkage map constructed from genotyping-by-sequencing data and composed of 2121 SNP markers. One major QTL is located on chromosome 5, which explained 11.61% of the observed phenotypic variation, and the second QTL is located on chromosome 6, which explained 14.06% of the observed phenotypic variation. The QTL effects and the association between CGT density and QTL support interval were confirmed in a validation population which included 39 sunflower inbred lines with diverse genetic backgrounds. We also identified two strong candidate genes in the QTL support intervals, and the functions of their orthologs in other plant species suggested their potential roles in regulating capitate glandular trichome density in sunflower. Our results provide valuable information to sunflower breeding community for developing host resistance to sunflower insect pests.

A high-density SNP Map of sunflower derived from RAD-sequencing facilitating fine-mapping of the rust resistance gene R12.

A high-resolution genetic map of sunflower was constructed by integrating SNP data from three F2 mapping populations (HA 89/RHA 464, B-line/RHA 464, and CR 29/RHA 468). The consensus map spanned a total length of 1443.84 cM, and consisted of 5,019 SNP markers derived from RAD tag sequencing and 118 publicly available SSR markers distributed in 17 linkage groups, corresponding to the haploid chromosome number of sunflower. The maximum interval between markers in the consensus map is 12.37 cM and the average distance is 0.28 cM between adjacent markers. Despite a few short-distance inversions in marker order, the consensus map showed high levels of collinearity among individual maps with an average Spearman's rank correlation coefficient of 0.972 across the genome. The order of the SSR markers on the consensus map was also in agreement with the order of the individual map and with previously published sunflower maps. Three individual and one consensus maps revealed the uneven distribution of markers across the genome. Additionally, we performed fine mapping and marker validation of the rust resistance gene R12, providing closely linked SNP markers for marker-assisted selection of this gene in sunflower breeding programs. This high resolution consensus map will serve as a valuable tool to the sunflower community for studying marker-trait association of important agronomic traits, marker assisted breeding, map-based gene cloning, and comparative mapping.

Ovipositional preference and larval performance of the banded sunflower moth (Lepidoptera: Tortricidae) and its larval parasitoids on resistant and susceptible lines of sunflower (Asterales: Asteraceae).

Banded sunflower moth, Cochylis hospes Walsingham, is one of the most destructive seed-feeding insect pests of sunflowers, causing significant economic yield losses in the northern Great Plains. In an attempt to understand host-plant resistance mechanisms for this pest, we field-tested, over several years, the effects of seven sunflower accessions, rated as resistant to C. hospes in previous screening trials, and a susceptible control (Par 1673-2), on the ovipositional preference and larval performance of C. hospes and its larval parasitoids. Of the resistant accessions, PI 494859 was the most preferred for oviposition, receiving a significantly greater number of eggs per head than did the susceptible Par 1673-2 in 2 of 3 yr. However, the numbers of larvae, and consequently the rate of seed infestation, found in PI 494859 heads were significantly lower than those in Par 1673-2 heads over all 3 yr. Female moths laid relatively few eggs on accessions PI 170385, 291403, and 251902, compared with on Par 1673-2, resulting in lower numbers of larvae per head and lower percentages of seed damaged. No association was observed between the concentrations of two diterpenoid alcohols or two diterpenoid acids in sunflower bracts and the numbers of eggs laid on the heads of the accessions. The number of banded sunflower moth larvae and the proportion of seeds damaged were positively correlated with kaurenoic acid concentrations and negatively correlated with kauranol concentrations. A positive association between resistance to larval feeding and parasitism was found in years 2006 and 2008, with resistant accessions having significantly greater proportions of parasitized larvae than did the susceptible Par 1673-2.

Candidate gene association mapping of Sclerotinia stalk rot resistance in sunflower (Helianthus annuus L.) uncovers the importance of COI1 homologs.

KEY MESSAGE: Functional markers for Sclerotinia basal stalk rot resistance in sunflower were obtained using gene-level information from the model species Arabidopsis thaliana. Sclerotinia stalk rot, caused by Sclerotinia sclerotiorum, is one of the most destructive diseases of sunflower (Helianthus annuus L.) worldwide. Markers for genes controlling resistance to S. sclerotiorum will enable efficient marker-assisted selection (MAS). We sequenced eight candidate genes homologous to Arabidopsis thaliana defense genes known to be associated with Sclerotinia disease resistance in a sunflower association mapping population evaluated for Sclerotinia stalk rot resistance. The total candidate gene sequence regions covered a concatenated length of 3,791 bp per individual. A total of 187 polymorphic sites were detected for all candidate gene sequences, 149 of which were single nucleotide polymorphisms (SNPs) and 38 were insertions/deletions. Eight SNPs in the coding regions led to changes in amino acid codons. Linkage disequilibrium decay throughout the candidate gene regions declined on average to an r (2) = 0.2 for genetic intervals of 120 bp, but extended up to 350 bp with r (2) = 0.1. A general linear model with modification to account for population structure was found the best fitting model for this population and was used for association mapping. Both HaCOI1-1 and HaCOI1-2 were found to be strongly associated with Sclerotinia stalk rot resistance and explained 7.4 % of phenotypic variation in this population. These SNP markers associated with Sclerotinia stalk rot resistance can potentially be applied to the selection of favorable genotypes, which will significantly improve the efficiency of MAS during the development of stalk rot resistant cultivars.

Identification of resistance to new virulent races of rust in sunflowers and validation of DNA markers in the gene pool.

Sunflower rust, caused by Puccinia helianthi, is a prevalent disease in many countries throughout the world. The U.S. Department of Agriculture (USDA)-Agricultural Research Service, Sunflower Research Unit has released rust resistant breeding materials for several decades. However, constantly coevolving rust populations have formed new virulent races to which current hybrids have little resistance. The objectives of this study were to identify resistance to race 336, the predominant race in North America, and to race 777, the most virulent race currently known, and to validate molecular markers known to be linked to rust resistance genes in the sunflower gene pool. A total of 104 entries, including 66 released USDA inbred lines, 14 USDA interspecific germplasm lines, and 24 foreign germplasms, all developed specifically for rust resistance, were tested for their reaction to races 336 and 777. Only 13 of the 104 entries tested were resistant to both races, whereas another six were resistant only to race 336. The interspecific germplasm line, Rf ANN-1742, was resistant to both races and was identified as a new rust resistance source. A selection of 24 lines including 19 lines resistant to races 777 and/or 336 was screened with DNA markers linked to rust resistance genes R(1), R(2), R(4u), and R(5). The results indicated that the existing resistant lines are diverse in rust resistance genes. Durable genetic resistance through gene pyramiding will be effective for the control of rust.