Unveiling the genetic basis of Sclerotinia head rot resistance in sunflower.

BACKGROUND: Sclerotinia sclerotiorum is a necrotrophic fungus that causes Sclerotinia head rot (SHR) in sunflower, with epidemics leading to severe yield losses. In this work, we present an association mapping (AM) approach to investigate the genetic basis of natural resistance to SHR in cultivated sunflower, the fourth most widely grown oilseed crop in the world. RESULTS: Our association mapping population (AMP), which comprises 135 inbred breeding lines (ILs), was genotyped using 27 candidate genes, a panel of 9 Simple Sequence Repeat (SSR) markers previously associated with SHR resistance via bi-parental mapping, and a set of 384 SNPs located in genes with molecular functions related to stress responses. Moreover, given the complexity of the trait, we evaluated four disease descriptors (i.e, disease incidence, disease severity, area under the disease progress curve for disease incidence, and incubation period). As a result, this work constitutes the most exhaustive AM study of disease resistance in sunflower performed to date. Mixed linear models accounting for population structure and kinship relatedness were used for the statistical analysis of phenotype-genotype associations, allowing the identification of 13 markers associated with disease reduction. The number of favourable alleles was negatively correlated to disease incidence, disease severity and area under the disease progress curve for disease incidence, whereas it was positevily correlated to the incubation period. CONCLUSIONS: Four of the markers identified here as associated with SHR resistance (HA1848, HaCOI_1, G33 and G34) validate previous research, while other four novel markers (SNP117, SNP136, SNP44, SNP128) were consistently associated with SHR resistance, emerging as promising candidates for marker-assisted breeding. From the germplasm point of view, the five ILs carrying the largest combination of resistance alleles provide a valuable resource for sunflower breeding programs worldwide.

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.

Virulence Characterization and Identification of Maize Lines Resistant to Puccinia sorghi Schwein. Present in the Argentine Corn Belt Region.

Puccinia sorghi Schwein., the causal agent of maize common rust, is an endemic disease in the Argentine Corn Belt region. Virulence surveys of the pathogen population within the region have not been performed; thus, the understanding of the pathogen population is low and it is difficult to deploy resistance genes that could be effective at controlling the disease. In total, 58 single-uredinial isolates derived from infected maize leaves collected in different locations throughout the Argentine Corn Belt region during 2010 to 2012 were tested on a set of 25 maize lines carrying different Rp genes. Maize lines Rp3-A and PIO19802 showed the lowest virulence frequencies (3.4 and 1.7%, respectively) for all tested isolates. Moreover, the combination in a single genotype of the resistance genes carried by lines Rp3-A and PIO19802 or either of these lines combined with the resistance genes from PIO12345 would confer resistance to all isolates tested. Virulent isolates on maize lines Rp-G, Rp1-K, and Rp-GI were most frequent in 2012. Twenty-four virulence phenotypes were identified, with phenotypes TCCG (17.2%), TTBB (15.5%), and TCFG (10.3%) being the most common throughout the region. Adult plant resistance associated with hypersensitive response was identified at vegetative stage 6 in maize lines PIO68752, PIO28427, and PIO36420.

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.

Genetic diversity in maize landraces from indigenous settlements of Northeastern Argentina.

In South America, native maize germplasm has been extensively studied particularly for the Andean region. However, relatively few genetic diversity studies include materials from the eastern region of the continent. Herein we present a genetic diversity characterization of four Popcorn maize landraces, maintained in indigenous settlements, from Northeastern Argentina (NEA). In addition, one Popcorn landrace from Northwestern Argentina (NWA) was incorporated for comparison. We characterized these landraces using ten microsatellite markers. For the whole data set, a total of 65 alleles were found, with an average of 7.22 alleles per locus. The average gene diversity was 0.370. Global fit to Hardy-Weinberg proportions was observed in all landraces. Global estimates of F (ST) revealed a significant differentiation among the populations. Individual Neighbor-joining clustering and Bayesian analyses allowed the recognition of most populations studied. Two main groups were distinguished by the Neighbor-joining clustering of populations. This grouping pattern would be consistent with a hypothesis of successive introductions of Popcorn in South America. The results presented will be useful to design strategies that maximize the utility of maize genetic resources.

Complex mutational patterns and size homoplasy at maize microsatellite loci.

Microsatellite markers have become one of the most popular tools for germplasm characterization, population genetics and evolutionary studies. To investigate the mutational mechanisms of maize microsatellites, nucleotide sequence information was obtained for ten loci. In addition, Single-Strand Conformation Polymorphism (SSCP) analysis was conducted to assess the occurrence of size homoplasy. Sequence analysis of 54 alleles revealed a complex pattern of mutation at 8/10 loci, with only 2 loci showing allele variation strictly consistent with stepwise mutations. The overall allelic diversity resulted from changes in the number of repeat units, base substitutions, and indels within repetitive and non-repetitive segments. Thirty-one electromorphs sampled from six maize landraces were considered for SSCP analysis. The number of conformers per electromorph ranged from 1 to 7, with 74.2% of the electromorphs showing more than one conformer. Size homoplasy was apparent within landraces and populations. Variation in the amount of size homoplasy was observed within and between loci, although no differences were detected among populations. The results of the present study provide useful information on the interpretation of genetic data derived from microsatellite markers. Further efforts are still needed to determine the impact of these findings on the estimation of population parameters and on the inference of phylogenetic relationships in maize investigations.

Population structure of the boll weevil in cotton fields and subtropical forests of South America: a bayesian approach.

The main goal of this contribution is to investigate the genetic structure of boll weevil populations from South America (Argentina and Brazil) and to make further comparisons with a putative source population from USA. Samples were collected in a Paranaense forest under reserve protection, cotton fields and non-cultivated areas. Data from anonymous molecular markers were analysed using both traditional methods of population genetics and Bayesian approaches. Results help to support a previous hypothesis on the presence of two lineages of boll weevil populations in South America: one with characteristics of recent invaders and the other with characteristics of ancient populations. The sample from Urugua-í Provincial Park (Misiones, Argentina) shows the highest percentage of polymorphic loci, the highest values of mean heterozigosity, and the largest number of population-specific alleles, all being typical features of ancient populations. Furthermore, the Urugua-í sample shows two gene pools occurring in sympatry, probably as a consequence of a secondary contact. The remaining samples reveal not only lower percentages of polymorphic loci and heterozygosity values, but also an almost negligible presence of specific alleles. Bayesian methods also suggest the occasional migration of some individuals of ancient lineages from their natural habitats in fragments of the Paranaense forest into cotton fields, and vice versa.

Molecular phylogeny of Larrea and its allies (Zygophyllaceae): reticulate evolution and the probable time of creosote bush arrival to North America.

Nucleotide sequences of Rubisco Large Subunit (rbcL) and the internal transcribed spacers (ITS) of nrDNA were obtained for the five species of Larrea and one species each of Bulnesia (ITS only) and Plectrocarpa (rbcL only). Parsimony analyses were conducted, including sequences from seven genera of Zygophyllaceae reported by other authors-Kallstroemia, Zygophyllum, Augea, Fagonia, Pintoa, Guaiacum, and Porlieria. The main conclusions of the present study are (1) the Argentine endemic Plectrocarpa tetracantha belongs to the subfamily Larreoideae (New World Clade); (2) all three phylogenies obtained from rbcL, ITS, and combined data sets show a close relationship between the tetraploid L. cuneifolia (sect. Bifolium) and the diploid multifoliolate pair L. nitida-L. ameghinoi (sect. Larrea), which could result from a possible intersectional hybrid origin of the tetraploid; (3) L. divaricata (sect. Bifolium) and L. tridentata (sect. Bifolium) form a highly supported monophyletic group, which agrees with previous cytogenetic and molecular evidence; and (4) the rate of nucleotide substitution of rbcL was estimated based on geological and fossil records. Under the molecular clock hypothesis, nucleotide sequence divergence between L. divaricata and L. tridentata suggests a Late Neogene (8.4 to 4.2 mybp) time of arrival of the diploid ancestors of L. tridentata to North American deserts.