Phenotyping Sunflower Genetic Resources for Sclerotinia Head Rot Response: Assessing Variability for Disease Resistance Breeding.

Sclerotinia head rot (SHR) is one of the most serious constraints to sunflower (Helianthus annuus L. var. macrocarpus) production worldwide. Here, we evaluated the response to SHR in a sunflower inbred panel from a large INTA germplasm collection, consisting of 137 inbred lines (ILs). Field trials were performed over five consecutive seasons using a twice-replicated randomized complete-block design. Disease incidence, disease severity, incubation period, and area under disease progress curve for disease incidence and severity were determined after controlled inoculation with the pathogen. Statistical analysis using mixed-effect models detected significant differences among ILs for all variables (P < 0.001). In addition, principal component analysis (PCA) and distance-based methods were used to classify the ILs according to their response to SHR, with ILs ALB2/5261 and 5383 emerging as the most resistant. Broad-sense heritability estimates ranged from 20.64% for disease severity to 10.58% for incubation period. The ample phenotypic variability of our collection, along with the moderate heritability estimates, highlight the importance of molecular breeding approaches to gain new insights into the genetic basis of sunflower resistance to SHR. The exhaustive phenotypic characterization presented here provides a reliable set of variables to comprehensively evaluate the disease and identifies two new sources of resistance to SHR.

Identification and characterization of contrasting sunflower genotypes to early leaf senescence process combining molecular and physiological studies (Helianthus annuus L.).

Leaf senescence is a complex mechanism ruled by multiple genetic and environmental variables that affect crop yields. It is the last stage in leaf development, is characterized by an active decline in photosynthetic rate, nutrients recycling and cell death. The aim of this work was to identify contrasting sunflower inbred lines differing in leaf senescence and to deepen the study of this process in sunflower. Ten sunflower genotypes, previously selected by physiological analysis from 150 inbred genotypes, were evaluated under field conditions through physiological, cytological and molecular analysis. The physiological measurement allowed the identification of two contrasting senescence inbred lines, R453 and B481-6, with an increase in yield in the senescence delayed genotype. These findings were confirmed by cytological and molecular analysis using TUNEL, genomic DNA gel electrophoresis, flow sorting and gene expression analysis by qPCR. These results allowed the selection of the two most promising contrasting genotypes, which enables future studies and the identification of new biomarkers associated to early senescence in sunflower. In addition, they allowed the tuning of cytological techniques for a non-model species and its integration with molecular variables.

Sunflower germin-like protein HaGLP1 promotes ROS accumulation and enhances protection against fungal pathogens in transgenic Arabidopsis thaliana.

KEY MESSAGE: The novel sunflower gene HaGLP1 is the first germin-like protein characterized from the family Asteraceae. It alters the host redox status and confers protection against Sclerotinia sclerotiorum and Rhizoctonia solani. Germin-like proteins (GLPs) are a large, diverse and ubiquitous family of plant glycoproteins belonging to the Cupin super family. These proteins have been widely studied because of their diverse roles in important plant processes, including defence. The novel sunflower gene HaGLP1 encodes the first germin-like protein characterized from the family Asteraceae. To analyse whether constitutive in vivo expression of the HaGLP1 gene may lead to disease tolerance, we developed transgenic Arabidopsis plants that were molecularly characterized and biologically assessed after inoculation with Sclerotinia sclerotiorum or Rhizoctonia solani. HaGLP1 expression in Arabidopsis plants conferred tolerance to S. sclerotiorum at the first stages of disease and interfered with R. solani infection, thus giving rise to significant protection against the latter. Furthermore, HaGLP1 expression in Arabidopsis plants elevated endogenous ROS levels. HaGLP1-induced tolerance does not appear to be related to a constitutive induction of the plant defence or the ROS-related genes examined here. In conclusion, our data suggest that HaGLP1 is an interesting candidate for the engineering of plants with increased fungal tolerance and that this gene could also be useful for the selection of naturally overexpressing sunflower genotypes for conventional breeding purposes.

Cytological characterization of sunflower by in situ hybridization using homologous rDNA sequences and a BAC clone containing highly represented repetitive retrotransposon-like sequences.

In the present work we report new tools for the characterization of the complete chromosome complement of sunflower (Helianthus annuus L.), using a bacterial artificial chromosome (BAC) clone containing repetitive sequences with similarity to retrotransposons and a homologous rDNA sequence isolated from the sunflower genome as probes for FISH. The rDNA signal was found in 3 pairs of chromosomes, coinciding with the location of satellites. The BAC clone containing highly represented retroelements hybridized with all the chromosome complement in FISH, and used together with the rDNA probe allowed the discrimination of all chromosome pairs of sunflower. Their distinctive distribution pattern suggests that these probes could be useful for karyotype characterization and for chromosome identification. The karyotype could be subdivided into 3 clear-cut groups of 12 metacentric pairs, 1 submetacentric pair, and 4 subtelocentric pairs, thus resolving previously described karyotype controversies. The use of BAC clones containing single sequences of specific markers and (or) genes associated with important agricultural traits represents an important tool for future locus-specific identification and physical mapping.

Molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of the cold-resistant Patagonian grass Bromus pictus associated with fructan accumulation under low temperatures.

Fructans are fructose polymers synthesized from sucrose in the plant vacuole. They represent short- and long-term carbohydrate reserves and have been associated with abiotic stress tolerance in graminean species. We report the isolation and characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) gene from a Patagonian grass species, Bromus pictus, tolerant to drought and cold temperatures. Structural and functional analyses of this gene were performed by Southern and Northern blot. Sugar content, quality and fructosyltransferase activity were studied using HPAEC-PAD (high-pH anion-exchange chromatography with pulsed amperometric detection), enzymatic and colorimetric assays. The putative 6-SFT gene had all the conserved motifs of fructosyl-transferase and showed 90% identity at the amino acid level with other 6-SFTs from winter cereals. Expression studies, and determination of sugar content and fructosyl-transferase activity were performed on five sections of the leaf. Bp6-SFT was expressed predominantly in leaf bases, where fructosyltransferase activity and fructan content are higher. Bp6-SFT expression and accumulation of fructans showed different patterns in the evaluated leaf sections during a 7 d time course experiment under chilling treatment. The transcriptional pattern suggests that the B. pictus 6-SFT gene is highly expressed in basal leaf sections even under control temperate conditions, in contrast to previous reports in other graminean species. Low temperatures caused an increase in Bp6-SFT expression and fructan accumulation in leaf bases. This is the first study of the isolation and molecular characterization of a fructosyltransferase in a native species from the Patagonian region. Expression in heterologous systems will confirm the functionality, allowing future developments in generation of functional markers for assisted breeding or biotechnological applications.