Rust caused by the fungus Puccinia helianthi and downy mildew (DM) caused by the obligate pathogen Plasmopara halstedii are two of the most globally important sunflower diseases. Resistance to rust and DM is controlled by race-specific single dominant genes. The present study aimed at pyramiding rust resistance genes combined with a DM resistance gene, using molecular markers. Four rust resistant lines, HA-R3 (carrying the R₄ gene), HA-R2 (R₅), HA-R8 (R₁₅), and RHA 397 (R_13b), were each crossed with a common line, RHA 464, carrying a rust gene R₁₂ and a DM gene Pl_Arg. An additional cross was made between HA-R8 and RHA 397. Co-dominant simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers linked to the target genes were used to discriminate between homozygotes and heterozygotes in F₂ populations. Five pyramids with different combinations of rust resistance genes were selected in the homozygous condition through marker-assisted selection, and three of them were combined with a DM resistance gene Pl_Arg: R₄/R₁₂/Pl_Arg, R₅/R₁₂/Pl_Arg, R_13b/R₁₂/Pl_Arg, R₁₅/R₁₂, and R_13b/R₁₅. The pyramiding lines with the stacking of two rust and one DM genes were resistant to all known races of North American sunflower rust and all known races of the pathogen causing DM, potentially providing multiple and durable resistance to both rust and DM. A cluster of 12 SNP markers spanning a region of 34.5 Mb on chromosome 1, which co-segregate with Pl_Arg, were tested in four populations. Use of those markers, located in a recombination suppressed region in marker selection, is discussed.

Phomopsis stem canker (PSC) caused by Diaporthe helianthi is increasingly becoming a global threat for sunflower production. In this study, the genetic basis of PSC resistance was investigated in a recombinant inbred line (RIL) population developed from a cross between HA 89 (susceptible) and HA-R3 (resistant). The RIL population was evaluated for PSC disease incidence (DI) in seven screening trials at multiple locations during 2016–2018. The distribution of PSC DI in the RIL population was continuous, confirming a polygenic inheritance of the trait. A moderately high broad-sense heritability (H², 0.76) was estimated for the trait across environments. In the combined analysis, both the genotype and the genotype × environment interactions were highly significant. A linkage map spanning 1505.33 cM was constructed using genotyping-by-sequencing derived markers. Marker–trait association analysis identified a total of 15 quantitative trait loci (QTL) associated with PSC resistance on 11 sunflower chromosomes, each explaining between 5.24 and 17.39% of the phenotypic variation. PSC resistance QTL were detected in two genomic regions each on chromosomes 3, 5, 13, and 17, while one QTL each was detected in the remaining seven chromosomes. Tightly linked single nucleotide polymorphism (SNP) markers flanking the PSC resistance QTL will facilitate marker-assisted selection in PSC resistance sunflower breeding.

Downy mildew (DM) is one of the severe biotic threats to sunflower production worldwide. The inciting pathogen, Plasmopara halstedii, could overwinter in the field for years, creating a persistent threat to sunflower. The dominant genes Pl₁₈ and Pl₂₀ conferring resistance to known DM races have been previously mapped to 1.5 and 1.8 cM intervals on sunflower chromosomes 2 and 8, respectively. Utilizing a whole-genome resequencing strategy combined with reference sequence-based chromosome walking and high-density mapping in the present study, Pl₁₈ was placed in a 0.7 cM interval on chromosome 2. A candidate gene HanXRQChr02g0048181 for Pl₁₈ was identified from the XRQ reference genome and predicted to encode a protein with typical NLR domains for disease resistance. The Pl₂₀ gene was placed in a 0.2 cM interval on chromosome 8. The putative gene with the NLR domain for Pl₂₀, HanXRQChr08g0210051, was identified within the Pl₂₀ interval. SNP markers closely linked to Pl₁₈ and Pl₂₀ were evaluated with 96 diverse sunflower lines, and a total of 13 diagnostic markers for Pl₁₈ and four for Pl₂₀ were identified. These markers will facilitate to transfer these new genes to elite sunflower lines and to pyramid these genes with broad-spectrum DM resistance in sunflower breeding.

Meiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition. We cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses. The Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.

The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiosis. Here, we cloned two Rec8 -like homoeologous genes (homoeoalleles) from tetraploid wheat ( TtRec8-A1 and TtRec8-B1 ) and one from hexaploid wheat ( TaRec8-D1 ), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat ( TaRec8-A1 and TaRec8-B1 ) and the one in Aegilops tauschii ( AetRec8-D1 ) by comparative analysis. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B ( TtRec8-B1 and TaRec8-B1 ) and those from the subgenomes A and D ( TtRec8-A1, TaRec8-A1 , and TaRec8-D1 ). TtRec8-A1 expressed dominantly over TtRec8-B1 , but comparably to TaRec8-D1 . Therefore, the Rec8 homoeoalleles from the subgenomes A and D may be functionally more active than the one from the subgenome B in wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in the polyploid, and imply the distinction of the wheat subgenome B from other subgenomes in the origin and evolution. HIGHLIGHT: This work revealed the structural and expression patterns of the Rec8 -like homoeologous genes in polyploid wheat, implying a unique origin and evolutionary route of the wheat B subgenome.