Diversity analysis and genomic prediction of Sclerotinia resistance in sunflower using a new 25 K SNP genotyping array.

KEY MESSAGE: We have developed a SNP array for sunflower containing more than 25 K markers, representing single loci mostly in or near transcribed regions of the genome. The array was successfully applied to genotype a diversity panel of lines, hybrids, and mapping populations and represented well the genetic diversity of cultivated sunflower. Results of PCoA and population substructure analysis underlined the complexity of the genetic composition of current elite breeding material. The performance of this genotyping platform for genome-based prediction of phenotypes and detection of QTL with improved resolution could be demonstrated based on the re-evaluation of a population segregating for resistance to Sclerotinia midstalk rot. Given our results, the newly developed 25 K SNP array is expected to be of great utility for the most important applications in genome-based sunflower breeding and research. ABSTRACT: 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.

BSTA: a targeted approach combines bulked segregant analysis with next- generation sequencing and de novo transcriptome assembly for SNP discovery in sunflower.

BACKGROUND: Sunflower 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 Pl(ARG)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 Pl(ARG) 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. RESULTS: A 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 Pl(ARG). CONCLUSIONS: This 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.

The effect of the bacterial effector protein harpin on transcriptional profile and mitochondrial proteins of Arabidopsis thaliana.

Here we report on the effect of the bacterial elicitor harpin from Pseudomonas syringae on Arabidopsis thaliana with an emphasis on transcriptional profiling and on changes of the mitochondrial proteome. Interestingly, of the currently about 400 identified mitochondrial proteins, transcriptional profiling by genome-wide DNA-microarray analyses revealed a total of 192 genes that showed significant changes in transcript abundance in response to the bacterial elicitor. The most dramatic changes were observed for the mitochondrial protein import apparatus of which 70% of all genes were induced. Proteomic analyses by 2D-PAGE and MALDI-TOF Mass Spectrometry for peptide fingerprint analysis confirmed the corresponding changes within the mitochondrial proteome in 28 cases. Strikingly, we found an accumulation of virtually all members of the TCA cycle. In sum, our results point to an involvement of mitochondria in the response of plant cells to the harpin elicitor, which becomes apparent both at the transcriptomic as well as proteomic level.

Transcriptional responses of Arabidopsis thaliana to the bacteria-derived PAMPs harpin and lipopolysaccharide.

Many plant-pathogen interactions are controlled by specific interactions between pathogen avirulence (avr) gene loci and the corresponding plant resistance R locus (gene-for-gene-hypothesis). Very often, this type of interaction culminates in a hypersensitive reaction (HR). However, recently pathogen-associated molecular patterns (PAMPs) such as flagellin or lipopolysaccharides (LPS) that are common to all bacteria have been shown to act as general elicitors of basal or innate immune responses in several plant species. Here, we summarize the genetic programs in Arabidopsis thaliana behind the LPS-induced basal response and the HR induced by harpin, respectively. Using Agilent Arabidopsis cDNA microarrays consisting of approximately 15,000 oligomers, changes in transcript accumulation of treated cells were monitored over a period of 24h after elicitor treatment. Analysis of the array data revealed significant responses to LPS (309 genes), harpin (951 genes) or both (313 genes). Concentrating our analysis on the genes encoding transcription factors, defence genes, cell wall biogenesis-related genes and signal transduction components we monitored interesting parallels, but also remarkably different expression patterns. Harpin and LPS induced an overlapping set of genes involved in cell wall biogenesis, cellular communication and signalling. The pattern of induced genes associated with cell rescue and general stress responses such as small heat-shock proteins was highly similar. In contrast, there is a striking difference regarding some of the most prominent, central components of plant defence such as WRKY transcription factors and oxidative burst-associated genes like NADPH oxidases, whose expression became apparent only after treatment with harpin. While both harpin and LPS can stimulate plant immunity in Arabidopsis, the PAMP LPS induces much more subtle host reactions at the transcriptome scale. The defence machinery induced by harpin resembles the known HR-type host responses leading to cell death after treatment with this elicitor. LPS is a weak inducer of basal resistance and induces a different pattern of genes. Strikingly the biggest overlap (40) of responding genes was found between the early harpin response (30min) and the late LPS response (24h).

Zeatin-induced nitric oxide (NO) biosynthesis in Arabidopsis thaliana mutants of NO biosynthesis and of two-component signaling genes.

* 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.

Conserved requirement for a plant host cell protein in powdery mildew pathogenesis.

In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.