Lili Qi

Introduction: Sclerotinia sclerotiorum is a serious pathogen causing severe basal stalk rot (BSR) disease on cultivated sunflower (Helianthus annuus L.) that leads to significant yield losses due to insufficient resistance. The wild annual sunflower species H. petiolaris, commonly known as prairie sunflower is known for its resistance against this pathogen. Sunflower resistance to BSR is quantitative and determined by many genes with small effects on the resistance phenotype. The objective of this study was to identify loci governing BSR resistance derived from H. petiolaris using a quantitative trait loci (QTL) mapping approach.Methods: BSR resistance among lines of an advanced backcross population (AB-QTL) with 174 lines developed from a cross of inbred line HA 89 with H. petiolaris PI 435843 was determined in the field during 2017-2019, and in the greenhouse in 2019. AB-QTL lines and the HA 89 parent were genotyped using genotyping-by-sequencing and a genetic linkage map was developed spanning 997.51 cM and using 1,150 SNP markers mapped on 17 sunflower chromosomes.Results and discussion: Highly significant differences (pH²) of 0.61 for the tested resistance parameters. A total of 14 QTL associated with BSR resistance were identified on nine chromosomes, each explaining a proportion of the phenotypic variation ranging from 3.5% to 28.1%. Of the 14 QTL, eight were detected for BSR resistance in the field and six were detected under greenhouse conditions. Alleles conferring increased BSR resistance were contributed by the H. petiolaris parent at 11 of the 14 QTL.

Sunflower (Helianthus L.) production ranked third in global oilseed production in 2021. Downy mildew (DM), caused by the oomycete pathogen Plasmopara halstedii (Farl.) Berl. et de Toni, is a major constraint for worldwide sunflower production, causing significant yield losses in susceptible hybrids. The search for new sources of resistance to DM is the primary objective of all sunflower breeding programs. Germplasms HA‐DM15 (Reg. no. GP‐385, PI 702359) and HA‐DM16 (Reg. no. GP‐386, PI 702360) were developed by introgressing two new DM resistance genes, Pl₃₇ and Pl₃₈, from the wild sunflower species H. annuus and H. praecox into oilseed sunflower, respectively. The BC₁F₃‐derived HA‐DM15 harboring the Pl₃₇ gene was selected from the cross of HA 89//NMS HA 89/wild H. annuus PI 435417. The germplasm line HA‐DM16 carrying the Pl₃₈ gene is an improved oilseed derivative of Rf PRA‐417 with DM resistance originally derived from the wild H. praecox PRA‐417 from the cross of cytoplasmic male sterile (CMS) HA89*2/wild H. praecox PRA‐417. HA‐DM15/Pl₃₇ and HA‐DM16/Pl₃₈ are immue to the most predominant and virulent DM races idenfied in North America and Europe. Pl₃₇ and Pl₃₈ have been genetically mapped to sunflower chromosomes 4 and 2, respectively, with single nucleotide polymorphism markers linked to the both genes facilitating their transfer to elite sunflower lines by marker‐assisted selection, providing new and diverse sources for sunflower DM‐resistance breeding.

Rust and downy mildew (DM) are detrimental diseases in global sunflower (Helianthus annuus L.) production. Disease often results in yield loss and reduced seed quality. Host resistance to DM and rust is mediated by single dominant genes in sunflower. To better utilize genetic resistance, gene pyramiding is a common practice in crop breeding to extend the durability and longevity of resistance. Two confection sunflower germplasm lines, HA‐R20 (Reg. no. GP‐387, PI 702361) and HA‐R21 (Reg. no. GP‐388, PI 702362), were developed using the pedigree breeding method and DNA marker‐assisted selection and released by the USDA‐ARS Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station in September 2022. HA‐R20 is an F₃‐derived F₄ confection restorer selection from the cross of HA‐DM2 and HA‐R8, carrying rust resistance genes R₁₂ and R₁₅ and DM resistance gene Pl_Arg. HA‐R21 is an F₃‐derived F₄ confection maintainer selection from the cross of HA‐DM3 and HA‐R8, carrying rust resistance genes R_13a and R₁₅ and DM resistance gene Pl₁₇. Disease reaction and marker testing for DM and rust confirmed that both germplasms harbor DM and rust R genes in the homozygous condition and are resistant to all DM and rust races identified in North America to date. These multiple disease‐resistant lines will provide the sunflower industry with durable disease‐resistant sources for the continued improvement of the crop.

Downy mildew (DM) and rust are two major global sunflower ( Helianthin annuus L.) diseases causing significant yield losses and reducing seed quality. Host plant resistance mediated by dominant race‐specific genes has been extensively used in sunflower production to control these diseases. However, the considerable variability of the DM and rust pathogens caused by mutation or recombination has changed the dynamics of the diseases, significantly increasing their incidence in recent years. This necessitates the development and release of sunflower germplasm with enhanced levels of disease resistance. Germplasm lines HA‐DM12 (Reg. no. GP‐382, PI 700006), HA‐DM13 (Reg. no. GP‐383, PI 700007), and HA‐DM14 (Reg. no. GP‐384, PI 700008), all with multiple DM and rust resistance, were developed using phenotypic evaluation and marker‐assisted selection. The three lines share a common DM gene ( Pl _Arg ) and a rust gene ( R ₁₂ ) and a combination of different DM genes ( Pl ₈ , Pl ₁₇ , and Pl ₁₈ , respectively). The triple‐gene pyramids—HA‐DM12 ( Pl ₈ Pl ₈ / Pl _Arg Pl _Arg / R ₁₂ R ₁₂ ), HA‐DM13 ( Pl ₁₇ Pl ₁₇ / Pl _Arg Pl _Arg / R ₁₂ R ₁₂ ), and HA‐DM14 ( Pl ₁₈ Pl ₁₈ / Pl _Arg Pl _Arg / R ₁₂ R ₁₂ )—confer comprehensive resistance to all races of DM and rust identified so far and can be used in sunflower breeding programs to develop hybrids with durable resistance to DM and rust.

Key message We finely mapped the rust resistance gene R₁₂ to a 0.1248-cM region, identified a potential R₁₂ candidate gene in the XRQ reference genome, and developed three diagnostic SNP markers for R₁₂. Abstract Rust is a devastating disease in sunflower that is damaging to the sunflower production globally. Identification and utilization of host-plant resistance are proven to be preferable means for disease control. The rust resistance gene R₁₂ with broad-spectrum specificity to rust was previously localized to a 2.4 Mb region on sunflower chromosome 11. To understand the molecular mechanism of resistance, we conducted whole-genome sequencing of RHA 464 (R₁₂ donor line) and reference genome-based fine mapping of the gene R₁₂. Overall, the 213 markers including 186 SNPs and 27 SSRs' were identified from RHA 464 sequences and used to survey polymorphisms between the parents HA 89 and RHA 464. Saturation mapping identified 26 new markers positioned in the R₁₂ region, and fine mapping with a large population of 2004 individuals positioned R₁₂ at a genetic distance of 0.1248 cM flanked by SNP markers C11_150451336 and S11_189205190. One gene, HanXRQChr11g0348661, with a defense-related NB-ARC-LRR domain, was identified in the XRQr1.0 genome assembly in the R₁₂ region; it is predicted to be a potential R₁₂ candidate gene. Comparative analysis clearly distinguished R₁₂ from the rust R₁₄ gene located in the vicinity of the R₁₂ gene on chromosome 11. Three diagnostic SNP markers, C11_147181749, C11_147312085, and C11_149085167, specific for R₁₂ were developed in the current study, facilitating more accurate and efficient selection in sunflower rust resistance breeding. The current study provides a new genetic resource and starting point for cloning R₁₂ in the future.

SUMMARY: Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R₁₁ in sunflower line HA‐R9 shows broad‐spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long‐read sequencing to clone the R₁₁ gene. Sequencing of a 60‐kb region spanning the R₁₁ locus from the R₁₁‐HA‐R9 rust‐resistant line and three EMS‐induced susceptible mutants facilitated the identification of R₁₁ and definition of induced mutations. The R₁₁ gene is predicted to have a single 3996‐bp open reading frame and encodes a protein of 1331 amino acids with CC‐NBS‐LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R₁₁ rust‐susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R₁₁‐HA‐R9 and R₁₁‐susceptible mutants revealed substantial differences in gene expression patterns associated with R₁₁‐mediated resistance at 7 days post‐inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

Key message Two new downy mildew resistance genes, Pl₃₇ and Pl₃₈, were introgressed from wild sunflower species into cultivated sunflower and mapped to sunflower chromosomes 4 and 2, respectively Abstract Downy mildew (DM), caused by the oomycete pathogen Plasmopara halstedii (Farl.) Berl. & de Toni, is known as the most prevalent disease occurring in global sunflower production areas, especially in North America and Europe. In this study, we report the introgression and molecular mapping of two new DM resistance genes from wild sunflower species, Helianthus annuus and H. praecox, into cultivated sunflower. Two mapping populations were developed from the crosses of HA 89/H. annuus PI 435417 (Pop1) and CMS HA 89/H. praecox PRA-417 (Pop2). The phenotypic evaluation of DM resistance/susceptibility was conducted in the BC₁F₂-derived BC₁F₃ populations using P. halstedii race 734. The BC₁F₂ segregating Pop1 was genotyped using an Optimal GBS AgriSeq™ Panel consisting of 768 mapped SNP markers, while the BC₁F₂ segregating Pop2 was genotyped using a genotyping-by-sequencing approach. Linkage analysis and subsequent saturation mapping placed the DM resistance gene, designated Pl₃₇, derived from H. annuus PI 435417 in a 1.6 cM genetic interval on sunflower chromosome 4. Pl₃₇ co-segregated with SNP markers SPB0003 and C4_5738736. Similarly, linkage analysis and subsequent saturation mapping placed the DM resistance gene, designated Pl₃₈, derived from H. praecox PRA-417 in a 0.8 cM genetic interval on sunflower chromosome 2. Pl₃₈ co-segregated with seven SNP markers. Multi-pathotype tests revealed that lines with Pl₃₇ or Pl₃₈ are immune to the most prevalent and virulent P. halstedii races tested. Two germplasm lines, HA-DM15 with Pl₃₇ and HA-DM16 with Pl₃₈, were developed for use in sunflower DM-resistance breeding.

Downy mildew (DM), caused by the obligate pathogen Plasmopara halstedii (Farl.) Berl. & de Toni, is a worldwide yield‐limiting disease of sunflower (Helianthus annuus L.). To obtain durable DM resistance in sunflower hybrids, there is a need to stack new resistance genes into the best available germplasm. The DM resistance genes Pl₈, Pl₁₇, and Pl₁₈ were recently fine mapped to sunflower chromosomes 13, 4, and 2, respectively. The current study pyramided these genes into a single genotype by marker‐assisted selection. Three oilseed sunflower germplasms, HA‐DM9 (Reg. no. GP‐381, PI 700009), HA‐DM10 (Reg. no. GP‐379, PI 700004), and HA‐DM11 (Reg. no. GP‐380, PI 700005), are dual DM‐resistant germplasm lines developed and have been released by the USDA‐ARS, Sunflower and Plant Biology Research Unit in collaboration with the North Dakota Agricultural Experiment Station in 2021. The three lines, HA‐DM9 (Pl₈Pl₈/Pl₁₇Pl₁₇), HA‐DM10 (Pl₈Pl₈/Pl₁₈Pl₁₈), and HA‐DM11 (Pl₁₇Pl₁₇/Pl₁₈Pl₁₈), each stacked two different DM genes, providing broad resistance to all P. halstedii races identified in North America and Europe so far, and can be used in sunflower breeding programs to enhance DM resistance.

Rust and downy mildew (DM) are two important sunflower diseases that lead to significant yield losses globally. The use of resistant hybrids to control rust and DM in sunflower has a long history. The rust resistance genes, R_13a and R₁₆, were previously mapped to a 3.4 Mb region at the lower end of sunflower chromosome 13, while the DM resistance gene, Pl₃₃, was previously mapped to a 4.2 Mb region located at the upper end of chromosome 4. High-resolution fine mapping was conducted using whole genome sequencing of HA-R6 (R_13a) and TX16R (R₁₆ and Pl₃₃) and large segregated populations. R_13a and R₁₆ were fine mapped to a 0.48 cM region in chromosome 13 corresponding to a 790 kb physical interval on the XRQr1.0 genome assembly. Four disease defense-related genes with nucleotide-binding leucine-rich repeat (NLR) motifs were found in this region from XRQr1.0 gene annotation as candidate genes for R_13a and R₁₆. Pl₃₃ was fine mapped to a 0.04 cM region in chromosome 4 corresponding to a 63 kb physical interval. One NLR gene, HanXRQChr04g0095641, was predicted as the candidate gene for Pl₃₃. The diagnostic SNP markers developed for each gene in the current study will facilitate marker-assisted selections of resistance genes in sunflower breeding programs.