Basal stalk rot (BSR) is an important disease of sunflower (Helianthus annuus L.) in the cool and humid growing environment causing serious economic damage to the crop worldwide (Gulya et al., 1997). The disease is caused by the necrotrophic fungal pathogen, Sclerotinia sclerotiorum (Lib) de Bary, which infects over 400 broadleaf hosts including many important food crops (Boland and Hall, 1994; Bolton et al., 2006). In the United States alone, hundreds of millions of dollars’ worth of economic losses occur every year due to diseases caused by the S. sclerotiorum (Bolton et al., 2006). Throughout the entire growing season, sunflower suffers from three major forms of the disease caused by the fungus, BSR, also called Sclerotinia wilt, mid stalk rot (MSR), and head rot (HR). Under favorable growth conditions, the sclerotia (the compact mass of hardened fungal mycelium) germinates beneath the soil and the growing mycelia invade the sunflower roots to incite the BSR disease (unique to sunflower), in contrast, both MSR and HR are caused by the germination of airborne ascospore released from the apothecia and carried by wind and rain splashing on the sunflower leaves and capitula (Gulya et al., 1997).

The Northern Great Plains states of North Dakota, South Dakota, and Minnesota are the major sunflower production states of the United States, which together produced over 87% of the total sunflower in 2021 (USDA, 2021). Sclerotinia BSR is one of the predominant diseases of sunflower in this region (Gulya et al., 1989; Seiler et al., 2017a). The climatic condition of this region is highly favorable for BSR disease development during the sunflower growing season and an outbreak of the disease can cause serious economic loss for the growers (Gulya et al., 1989). Chemical and biological control of the BSR disease is limited and often not feasible due to the soilborne nature of the disease. Use of host genetic resistance is the most economical and effective approach for BSR management and has been used effectively in many breeding programs. Sunflower genetic resources are routinely evaluated for novel sources of BSR resistance in the cultivated and wild gene pools (Block et al., 2009, 2010, 2012; Talukder et al., 2014b; Seiler et al., 2017a).

Sclerotinia BSR resistance in sunflower is a polygenic trait governed by multiple small effect genes (Talukder et al., 2014c, 2016, 2021). Quantitative genetic variations for BSR resistance have been mapped in different segregating populations developed from both cultivated and wild sunflower species (Davar et al., 2010; Amouzadeh et al., 2013; Talukder et al., 2014c, 2016, 2021; Paknia et al., 2020). In the growth chamber under a controlled environment, Davar et al. (2010) identified seven QTL associated with BSR resistance on linkage groups (LGs) 1, 2, 4, 6, 8, 14, and 17 in a recombinant inbred line (RIL) population developed from the cross of PAC1/RHA 266, while Amouzadeh et al. (2013) identified five QTL on LGs 1, 3, 8, 10, and 17 in the same population using a different fungal isolate. Later, the population was evaluated in the field identifying five genomic regions associated with BSR resistance, two each on LGs 11 and 15, and one on LG14 (Paknia et al., 2020). Proportions of the phenotypic variance explained by each of the QTL in the controlled growth chamber study were in the range of 0.5%–8%, while they were higher for some QTL in the field study, ranging from 1.9% to 37.3% of the total variation. Six BSR resistance QTL were identified on LGs 4, 9, 10, 11, 16, and 17 in a RIL population derived from the cross HA 441/RHA 439 evaluated in the field for multiple years and locations (Talukder et al., 2016). The QTL on LGs 10 and 17 were the most stable across years and locations and explained 31.6% and 20.2%, respectively of the total phenotypic variations. Using a worldwide collection of open pollinated sunflower lines, Talukder et al. (2014c) identified two genes, HaCOI1-1 and HaCOI1-2, on LG14 strongly associated with Sclerotinia BSR resistance and the two genes are orthologous and paralogous to the Arabidopsis thaliana COI1 gene, respectively.

Sunflower is one of the few crop species that originated and was domesticated in eastern North America (Blackman et al., 2011). There are 53 species for the genus Helianthus known to date, including 14 annual and 39 perennial species (Seiler et al., 2017b). All annual Helianthus species are diploid (2n = 2x = 34) while the perennial species have varied ploidy levels ranging from diploid to hexaploid with a few mixaploid species (Kane et al., 2013; Seiler et al., 2017b). Wild Helianthus species serve as potential sources of novel genes for several desirable traits, such as resistance to biotic and abiotic stresses, cytoplasmic male sterility, fertility restorer genes and other agronomic traits. Several of the novel genes from wild relatives have been utilized in cultivated sunflower development (for review, see Seiler et al., 2017b). Despite reports of high levels of Sclerotinia resistance existing in the secondary gene-pool, comprehensive studies have not been pursued to characterize and exploit the resistance in practical sunflower breeding programs. Only recently, introgression and molecular mapping of Sclerotinia resistance from wild species have been reported and germplasms with introgressed resistance have been released (Qi et al., 2016, 2018; Talukder et al., 2019a,b, 2021). Talukder et al. (2021) mapped 21 BSR resistance QTL in an advanced backcross (AB-QTL) population developed from the cross of HA 89 and an accession of the wild annual species H. argophyllus Torr. & Gray (silverleaf sunflower) using a high-density single nucleotide polymorphism (SNP) linkage map. The detected QTL were distributed on 11 sunflower chromosomes (LGs 2, 5, 6, 7, 8, 9, 10, 11, 14, 16, and 17) and each QTL explaining between 4.5% and 22.6% of the phenotypic variance. Remarkably, 13 of the 21 QTL had BSR resistance alleles derived from the H. argophyllus parent (Talukder et al., 2021).

Helianthus praecox Engelm. & A. Gray is a wild annual sunflower species with three subspecies: ssp. praecox, ssp. runyonii, and ssp. hirtus widespread in the state of Texas (Schilling, 2006; Turner, 2014). Helianthus praecox has previously been shown to possess high levels of resistance against Sclerotinia diseases (Škorić, 1987; Seiler, 1994, 2010; Rönicke et al., 2004; Christov and Velasco, 2008; Block et al., 2009, 2010; Christov, 2013). An AB-QTL population (Tanksley and Nelson, 1996) was developed by crossing and backcrossing of a H. praecox ssp. runyonii accession with cultivated sunflower lines. The objective for this study was to evaluate the population in the field and greenhouse and perform an in-depth inheritance analysis of BSR resistance segregating in the population using a high-density genetic map.

Like most host species, genetic control of Sclerotinia BSR resistance in sunflower is quantitative and governed by additive gene effects. No complete resistance source is known so far in the cultivated or the crop wild relatives. Traditionally, most sunflower breeding programs evaluate germplasm lines under inoculated/naturally infected field conditions for BSR resistance sources from the primary gene-pool and select resistant lines in multi-parent crossing schemes for inbred line development. However, due to the narrow genetic base of the cultivated sunflower (Cheres and Knapp, 1998), recombination of BSR resistance alleles is limited, leaving the crop vulnerable to Sclerotinia infection. Despite several known sources of Sclerotinia resistance in the wild sunflower gene-pools (for review, see Seiler et al. (2017b)), detailed analysis of resistance at the genomic level has not been attempted until recently (Qi et al., 2016, 2018; Talukder et al., 2019a,b, 2021). Recent advances in sunflower genomic resources (Badouin et al., 2017)² coupled with decreasing costs of next-generation sequencing (NGS) technology has enabled the discovery and tagging of DNA markers linked to the quantitative genetic variations for Sclerotinia resistance in sunflower. Introgression of Sclerotinia BSR resistance from wild H. praecox species into a cultivated sunflower background has been accomplished (Talukder et al., 2019a) and sunflower germplasm lines possessing higher level of BSR resistance have been developed and publicly released for use in BSR resistance breeding programs (Talukder et al., 2019b). Our current project is a follow-up research effort to molecularly dissect the transferred BSR resistance with respect to distribution, magnitude, and nature of expression under different growing environments.

The phenotypic responses to the BSR resistance in the AB-QTL population showed continuous distribution in both field and greenhouse evaluations (Figure 1; Supplemental Figure S1), typical nature for a quantitative trait controlled by numerous genes (Young, 1996). Despite seasonal variation in host resistance response to BSR disease, Spearman’s rank correlation between DI scores of the two seasons was highly significant (Figure 1D), validating the repeatability of the field screening trials. Similar seasonal variations were also observed when BSR field screening trials were conducted at the Carrington field site for a different sunflower mapping population (Talukder et al., 2021). Analysis of variance revealed that both genotype and genotype × environment interactions are highly significant for BSR DI measured in the field trials (Table 1). However, the variance component for the genotypes was much higher than the variance component for the genotype × environment interactions, signifying the greater contribution of the genetic makeups of the AB-QTL progeny lines to BSR resistance. This assumption was further supported by the moderately high (0.66) broad-sense heritability (H²) that measures the contribution of genetic factors in the observed phenotype.

The evaluation of the AB-QTL population in the greenhouse is robust and uniform since sunflower plants are inoculated individually and a micro-climate conducive to the BSR disease development is maintained where disease avoidance is not possible (Underwood et al., 2021). Two traits, AUDPC and DR were measured in the greenhouse trials to assess BSR resistance in the AB-QTL progeny lines. As expected, the phenotypic distribution of both the traits were also continuous in the AB-QTL population (Figures 1E, F). However, unlike BSR DI measured in the field, the AUDPC and DR data were skewed toward higher AUDPC and lower DR values in the greenhouse. The apparent inconsistency might be due to the high disease pressure put on the young sunflower seedlings in the greenhouse trials. Interestingly, the Spearman’s rank correlation among the greenhouse measured traits and DI measured in the field were significant at varying levels (Table 2), suggesting a collinearity between the field and greenhouse evaluation of the HA 89/H. praecox AB-QTL population to some extent. Strong correlation between greenhouse and field evaluations have been previously observed when a panel of cultivated sunflower lines were evaluated for BSR disease (Underwood et al., 2021). Broad-sense heritability of the AUDPC (0.73) and DR (0.70) measured in the greenhouse test were also comparable to the heritability (H² = 0.66) of BSR DI measured in the field. It is also important to note that disease evaluations in this study were performed using a single isolate of S. sclerotiorum. Further research will be required to determine if genotype-isolate interactions are relevant to sunflower BSR resistance and to ensure that resistant genotypes and QTL identified in this study provide broad-spectrum resistance to a range of S. sclerotiorum isolates.

The linkage map developed for the HA 89/H. praecox AB-QTL population contained a total of 1,710 loci mapped on 17 sunflower chromosomes, which is not a very dense linkage map as one would expect from a biparental sunflower mapping population derived from two highly diverse parental lines (wild vs. cultivated). Segregation distortion is a common feature in many interspecific crosses (Zamir and Tadmor, 1986). In a backcross population involving wild species, most often the distorted ratios favor alleles from the cultivated recurrent parent (Rieseberg and Carney, 1998). Despite a high level of polymorphism between the parents at the DNA marker level, many of the markers were eventually removed from the linkage analysis due to severe segregation distortion. The total length of the current linkage map for the HA 89/H. praecox AB-QTL population was 1,802.95 cM, which is comparable to the SNP marker based high-density linkage maps reported in sunflower (Bowers et al., 2012; Talukder et al., 2014a, 2016, 2020, 2021; Gao et al., 2017; Zhou et al., 2018).

Due to the presence of significant genotype × environment interactions, QTL analyses of the Carrington field data were performed individually for each environment, as well as, in combination using phenotypic BLUPs (the best linear unbiased predictors) of two seasons DI data. The AUDPC and DR data collected in the greenhouse were also analyzed independently. A total of 19 QTL associated with BSR resistance on nine sunflower chromosomes were detected in the current study. Similarly, an earlier study also reported a large number of BSR resistance QTL identified in a population derived from a wild sunflower species (Talukder et al., 2021). The QTL alleles conferring BSR resistance in the AB-QTL population were mostly derived from the wild H. praecox parent (~84%), which is consistent with the previous observations where H. praecox derived introgression lines showed higher levels of resistance to Sclerotinia BSR disease than the cultivated sunflower lines (Talukder et al., 2019b). Among the 19 detected QTL, nine of them were detected in the field evaluation tests alone, including the QTL on LG 13, Qbsr-13.1, which explained the largest proportion of the phenotypic variance (16.99%) among all the QTL detected in this population. Similarly, seven QTL were detected only in the greenhouse test on LGs 1, 5, 9, 12, 13, and 14, of which the BSR resistance alleles for Qbsr-5.2 and Qbsr-12.2 were derived from the cultivated parent. Despite strong Spearman’s rank correlation between field and greenhouse traits, only three BSR resistance QTL were detected in both field and greenhouse environments on LGs 1 and 14 of the sunflower genome. Among these three ubiquitous QTL, Qbsr-1.1 and Qbsr-1.3 were detected on LG1 with BSR resistance contributing alleles derived from the wild H. praecox source. Both these QTL were mapped with high LOD values and explained moderate phenotypic effect for the BSR resistance in the AB-QTL population (Supplementary Table 3). Due to their stability across environments and novel resistance allele source, these two QTL would be the first choice to use in MAS breeding program designed for introgression of BSR-resistant into elite sunflower lines.

A review of published works revealed that Sclerotinia BSR resistance QTL were previously mapped on 15 of the 17 LGs in the sunflower genome except on LGs 12 and 13 (Davar et al., 2010; Talukder et al., 2016, 2021; Paknia et al., 2020). However, in our current study, BSR resistance QTL were mapped to two additional LGs 12 and 13 that were not previously reported in any published works (Supplementary Table 4). The QTL, Qbsr-12.1, Qbsr-12.3, Qbsr-13.1, Qbsr-13.2 and, Qbsr-13.3 mapped on these LGs had their BSR resistance alleles derived from the H. praecox and can be considered as a novel QTL source. Sequence comparison of the flanking markers in the sunflower genome revealed that Qbsr-4.2 was mapped in the same region, where the BSR resistance QTL Qbsr-4.1 was detected in the HA 441/RHA 439 population (Talukder et al., 2016). Similarly, the Qbsr-8.3 and Qbsr-14.3 QTL mapped in the current study on LGs 8 and 14, respectively, were located on the same genomic regions as Qbsr-8.2 and Qbsr-14.1 in the HA 89/H. argophyllus AB-QTL population (Talukder et al., 2021). The sunflower candidate gene, HaCOI1-2 paralogous to the A. thaliana COI1 gene (Talukder et al., 2014c) is present in the genomic region flanking by the significant SNP markers associated with BSR resistance QTL, Qbsr-14.3 on LG14. The other sunflower candidate gene, HaCOI1-1 significantly associated with Sclerotinia BSR (Talukder et al., 2014c) and HR (Filippi et al., 2020) resistance is located in the genomic region flanking by the SNP markers associated with the Qbsr-14.5 QTL mapped in the current study. Except for Qbsr-4.2, Qbsr-8.3, Qbsr-14.3, and Qbsr-14.5, all other QTL mapped in the current study are so far uniquely mapped to the HA 89/H. praecox AB-QTL population with BSR resistance alleles derived from the H. praecox parent and considered as a novel QTL source. A comprehensive comparison of BSR resistance QTL mapped in the RIL population derived from the cross PAC2/RHA266 (Davar et al., 2010; Amouzadeh et al., 2013; Paknia et al., 2020) with QTL detected in the current study could not be performed due to lack of sequence information of the flanking markers used in those studies.

Introgression of BSR resistance from wild species into cultivated sunflower background can certainly broaden the genetic diversity in the primary gene-pool of the crop (Qi et al., 2016, 2018; Talukder et al., 2019b,c). The use of the AB-QTL population approach has made a significant contribution in unraveling the valuable alleles from wild species sources (Wang and Chee, 2010; Talukder et al., 2021). It is clear that Sclerotinia BSR resistance in sunflower is a complex trait and controlled by many small-effect genes. QTL mapping provides a framework for marker-assisted selection of such a complex disease resistance trait. Molecular markers linked to the significant BSR resistance QTL identified in this study will provide a valuable resource in marker-based recurrent selection to develop BSR resistant sunflower (Johnson, 2001; Bernardo, 2004).

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: European Variation Archive (EVA) under the Project: PRJEB50764 and Analyses: ERZ5072953.

LQ and ZT conceived and designed the experiments. LQ developed the population and contributed to writing—review and editing. ZT, WU, CM, GS, LQ, XC, and XL performed the experiments. ZT, WU, and LQ analyzed the data. ZT prepared the original draft. All authors contributed to the article and approved the submitted version.

This research was supported by the USDA–ARS National Sclerotinia Initiative, grant number 3060-21220-031-00D and the USDA–ARS CRIS project no. 3060-2100-043-00D.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Mention of trade names or commercial products in this report is solely for providing specific information and does not imply recommendations or endorsement by the USDA. The USDA is an equal opportunity provider and employer.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.