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.

De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach.

BACKGROUND: Application of Single Nucleotide Polymorphism (SNP) marker technology as a tool in sunflower breeding programs offers enormous potential to improve sunflower genetics, and facilitate faster release of sunflower hybrids to the market place. Through a National Sunflower Association (NSA) funded initiative, we report on the process of SNP discovery through reductive genome sequencing and local assembly of six diverse sunflower inbred lines that represent oil as well as confection types. RESULTS: A combination of Restriction site Associated DNA Sequencing (RAD-Seq) protocols and Illumina paired-end sequencing chemistry generated high quality 89.4 M paired end reads from the six lines which represent 5.3 GB of the sequencing data. Raw reads from the sunflower line, RHA 464 were assembled de novo to serve as a framework reference genome. About 15.2 Mb of sunflower genome distributed over 42,267 contigs were obtained upon assembly of RHA 464 sequencing data, the contig lengths ranged from 200 to 950 bp with an N50 length of 393 bp. SNP calling was performed by aligning sequencing data from the six sunflower lines to the assembled reference RHA 464. On average, 1 SNP was located every 143 bp of the sunflower genome sequence. Based on several filtering criteria, a final set of 16,467 putative sequence variants with characteristics favorable for Illumina Infinium Genotyping Technology (IGT) were mined from the sequence data generated across six diverse sunflower lines. CONCLUSION: Here we report the molecular and computational methodology involved in SNP development for a complex genome like sunflower lacking reference assembly, offering an attractive tool for molecular breeding purposes in sunflower.

TREHALOSE PHOSPHATE SYNTHASE11-dependent trehalose metabolism promotes Arabidopsis thaliana defense against the phloem-feeding insect Myzus persicae.

Agricultural productivity is limited by the removal of sap, alterations in source-sink patterns, and viral diseases vectored by aphids, which are phloem-feeding pests. Here we show that TREHALOSE PHOSPHATE SYNTHASE11 (TPS11) gene-dependent trehalose metabolism regulates Arabidopsis thaliana defense against Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA). GPA infestation of Arabidopsis resulted in a transient increase in trehalose and expression of the TPS11 gene, which encodes a trehalose-6-phosphate synthase/phosphatase. Knockout of TPS11 function abolished trehalose increases in GPA-infested leaves of the tps11 mutant plant and attenuated defense against GPA. Trehalose application restored resistance in the tps11 mutant, confirming that the lack of trehalose accumulation is associated with the inability of the tps11 mutant to control GPA infestation. Resistance against GPA was also higher in the trehalose hyper-accumulating tre1 mutant and in bacterial otsB gene-expressing plants, further supporting the conclusion that trehalose plays a role in Arabidopsis defense against GPA. Evidence presented here indicates that TPS11-dependent trehalose regulates expression of the PHYTOALEXIN DEFICIENT4 gene, which is a key modulator of defenses against GPA. TPS11 also promotes the re-allocation of carbon into starch at the expense of sucrose, the primary plant-derived carbon and energy source for the insect. Our results provide a framework for the signaling function of TPS11-dependent trehalose in plant stress responses, and also reveal an important contribution of starch in controlling the severity of aphid infestation.

PAD4-dependent antibiosis contributes to the ssi2-conferred hyper-resistance to the green peach aphid.

Myzus persicae, commonly known as green peach aphid (GPA), is a sap-sucking insect with a broad host range. Arabidopsis thaliana responds to GPA infestation with elevated expression of the PHYTOALEXIN DEFICIENT4 (PAD4) gene. Previously, we had demonstrated that the loss of PAD4 gene function compromises Arabidopsis resistance to GPA. In contrast, a mutation in the Arabidopsis SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY2 (SSI2) gene, which encodes a desaturase involved in lipid metabolism, resulted in hyper-resistance to GPA. We demonstrate here that PAD4 is required for the ssi2-dependent heightened resistance to GPA. Based on electrical monitoring of insect behavior and bioassays in which the insect was given a choice between the wild type and the ssi2 mutant, it is concluded that the ssi2-conferred resistance is not due to deterrence of insect settling or feeding from the phloem of the mutant. Instead, hyper-resistance in the ssi2 mutant results from heightened antibiosis that curtails insect reproduction. Petiole exudates collected from uninfested ssi2 plants contain elevated levels of an activity that interferes with aphid reproduction in synthetic diets. PAD4 was required for the accumulation of this antibiotic activity in petiole exudates, supporting the role of PAD4 in phloem-based resistance. Because PAD4 expression is not elevated in the ssi2 mutant, we suggest that basal PAD4 expression contributes to this antibiosis.

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.

Phloem-based resistance to green peach aphid is controlled by Arabidopsis PHYTOALEXIN DEFICIENT4 without its signaling partner ENHANCED DISEASE SUSCEPTIBILITY1.

Green peach aphid (GPA) Myzus persicae (Sülzer) is a phloem-feeding insect with an exceptionally wide host range. Previously, it has been shown that Arabidopsis thaliana PHYTOALEXIN DEFICIENT4 (PAD4), which is expressed at elevated levels in response to GPA infestation, is required for resistance to GPA in the Arabidopsis accession Columbia. We demonstrate here that the role of PAD4 in the response to GPA is conserved in Arabidopsis accessions Wassilewskija and Landsberg erecta. Electrical monitoring of aphid feeding behavior revealed that PAD4 modulates a phloem-based defense mechanism against GPA. GPA spends more time actively feeding from the sieve elements of pad4 mutants than from wild-type plants, and less time feeding on transgenic plants in which PAD4 is ectopically expressed. The activity of PAD4 in limiting phloem sap uptake serves as a deterrent in host-plant choice, and restricts aphid population size. In Arabidopsis defense against pathogens, all known PAD4 functions require its signaling and stabilizing partner EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1). Bioassays with eds1 mutants alone or in combination with pad4 and with plants conditionally expressing PAD4 under the control of a dexamethasone-inducible promoter reveal that PAD4-modulated defense against GPA does not involve EDS1. Thus, a PAD4 mode of action that is uncoupled from EDS1 determines the extent of aphid feeding in the phloem.

Premature leaf senescence modulated by the Arabidopsis PHYTOALEXIN DEFICIENT4 gene is associated with defense against the phloem-feeding green peach aphid.

Aphids, which are phloem-feeding insects, cause extensive loss of plant productivity and are vectors of plant viruses. Aphid feeding causes changes in resource allocation in the host, resulting in an increase in flow of nutrients to the insect-infested tissue. We hypothesized that leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf, could be utilized by plants to limit aphid growth. Using Arabidopsis (Arabidopsis thaliana) and green peach aphid (GPA; Myzus persicae Sulzer), we found that GPA feeding induced premature chlorosis and cell death, and increased the expression of SENESCENCE ASSOCIATED GENES (SAGs), all hallmarks of leaf senescence. Hypersenescence was accompanied by enhanced resistance against GPA in the Arabidopsis constitutive expresser of PR genes5 and suppressor of SA insensitivity2 mutant plants. In contrast, resistance against GPA was compromised in the phytoalexin deficient4 (pad4) mutant plant. The PAD4 gene, which is expressed at elevated level in response to GPA feeding, modulates the GPA feeding-induced leaf senescence. In comparison to the wild-type plant, GPA feeding-induced chlorophyll loss, cell death, and SAG expression were delayed in the pad4 mutant. Although PAD4 is associated with camalexin synthesis and salicylic acid (SA) signaling, camalexin and SA signaling are not important for restricting GPA growth; growth of GPA on the camalexin-biosynthesis mutant, pad3, and the SA deficient2 and NahG plants and the SA-signaling mutant, nonexpresser of PR genes1, were comparable to that on the wild-type plant. Our results suggest that PAD4 modulates the activation of senescence in the aphid-infested leaves, which contributes to basal resistance to GPA.