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Summary• Here, cytokinin-induced nitric oxide (NO) biosynthesis and cytokinin responses were investigated in Arabidopsis thaliana wild type and mutants defective in NO biosynthesis or cytokinin signaling components.• NO release from seedlings was quantified by a fluorometric method and, by microscopy, observed NO biosynthesis as fluorescence increase of DAR-4M AM (diaminorhodamine 4M acetoxymethyl ester) in different tissues.• Atnoa1 seedlings were indistinguishable in NO tissue distribution pattern and morphological responses, induced by zeatin, from wild-type seedlings. Wild-type and nia1,2 seedlings, lacking nitrate reductase (NR), responded to zeatin with an increase within 3 min in NO biosynthesis so that NR does not seem relevant for rapid NO induction, which was mediated by an unknown 2-(2-aminoethyl)2-thiopseudourea (AET)-sensitive enzyme and was quenched by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-1-oxy-3-oxide (PTIO). Long-term morphological responses to zeatin were severely altered and NO biosynthesis was increased in nia1,2 seedlings. As cytokinin signaling mutants we used the single-receptor knockout cre1/ahk4, three double-receptor knockouts (ahk2,3, ahk2,4, ahk3,4) and triple-knockout ahp1,2,3 plants. All cytokinin-signaling mutants showed aberrant tissue patterns of NO accumulation in response to zeatin and altered morphological responses to zeatin.• Because aberrant NO biosynthesis correlated with aberrant morphological responses to zeatin the hypothesis was put forward that NO is an intermediate in cytokinin signaling.

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BackgroundSunflower belongs to the largest plant family on earth, the genomically poorly explored Compositae. Downy mildew Plasmopara halstedii (Farlow) Berlese & de Toni is one of the major diseases of cultivated sunflower (Helianthus annuus L.). In the search for new sources of downy mildew resistance, the locus PlARG on linkage group 1 (LG1) originating from H. argophyllus is promising since it confers resistance against all known races of the pathogen. However, the mapping resolution in the PlARG region is hampered by significantly suppressed recombination and by limited availability of polymorphic markers. Here we examined a strategy developed for the enrichment of molecular markers linked to this specific genomic region. We combined bulked segregant analysis (BSA) with next-generation sequencing (NGS) and de novo assembly of the sunflower transcriptome for single nucleotide polymorphism (SNP) discovery in a sequence resource combining reads originating from two sunflower species, H. annuus and H. argophyllus.ResultsA computational pipeline developed for SNP calling and pattern detection identified 219 candidate genes. For a proof of concept, 42 resistance gene-like sequences were subjected to experimental SNP validation. Using a high-resolution mapping population, 12 SNP markers were mapped to LG1. We successfully verified candidate sequences either co-segregating with or closely flanking PlARG.ConclusionsThis study is the first successful example to improve bulked segregant analysis with de novo transcriptome assembly using next generation sequencing. The BSTA pipeline we developed provides a useful guide for similar studies in other non-model organisms. Our results demonstrate this method is an efficient way to enrich molecular markers and to identify candidate genes in a specific mapping interval.

Genotyping with a large number of molecular markers is a prerequisite to conduct genome-based genetic analyses with high precision. Here, we report the design and performance of a 25 K SNP genotyping array for sunflower (Helianthus annuus L.). SNPs were discovered based on variant calling in de novo assembled, UniGene-based contigs of sunflower derived from whole genome sequencing and amplicon sequences originating from four and 48 inbred lines, respectively. After inclusion of publically available transcriptome-derived SNPs, in silico design of the Illumina(®) Infinium iSelect HD BeadChip yielded successful assays for 22,299 predominantly haplotype-specific SNPs. The array was validated in a sunflower diversity panel including inbred lines, open-pollinated varieties, introgression lines, landraces, recombinant inbred lines, and F2 populations. Validation provided 20,502 high-quality bi-allelic SNPs with stable cluster performance whereby each SNP marker represents a single locus mostly in or near transcribed regions of the sunflower genome. Analyses of population structure and quantitative resistance to Sclerotinia midstalk rot demonstrate that this array represents a significant improvement over currently available genomic tools for genetic diversity analyses, genome-wide marker-trait association studies, and genetic mapping in sunflower.