Cristina Silvar

Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.

A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Recent advances in next-generation sequencing techniques and the development of genomics resources for crop plants with large genomes allow the detection of a large number of single nucleotide polymorphisms (SNPs) and their use in a high-throughput manner. However, such large numbers of SNPs are on the one hand not needed in some plant breeding projects and on the other hand not affordable in some cases, raising the need for fast and low-cost innovative techniques for marker detection. In marker selection in plant breeding programs, cleaved amplified polymorphic sequence (CAPS) markers still play a significant role as a complement to other high-throughput methods for SNP genotyping. New methods focusing on the acceleration of CAPS-based genotyping are therefore highly desirable. The combination of the classical CAPS method and a M13-tailed primer multiplexing assay was used to develop an agarose-gel-free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence-labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected in a capillary electrophoresis system. This method allowed the cost-effective genotyping of several SNPs in barley in a multiplexed manner at an overall low cost in a short period of time. This new method was efficiently combined with the simultaneous detection of simple sequence repeats in the same electrophoresis run, resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.

New Insights into Capsicum spp Relatedness and the Diversification Process of Capsicum annuum in Spain

The successful exploitation of germplasm banks, harbouring plant genetic resources indispensable for plant breeding, will depend on our ability to characterize their genetic diversity. The Vegetable Germplasm Bank of Zaragoza (BGHZ) (Spain) holds an important Capsicum annuum collection, where most of the Spanish pepper variability is represented, as well as several accessions of other domesticated and non-domesticated Capsicum spp from all over the five continents. In the present work, a total of 51 C. annuum landraces (mainly from Spain) and 51 accessions from nine Capsicum species maintained at the BGHZ were evaluated using 39 microsatellite (SSR) markers spanning the whole genome. The 39 polymorphic markers allowed the detection of 381 alleles, with an average of 9.8 alleles per locus. A sizeable proportion of alleles (41.2%) were recorded as specific alleles and the majority of these were present at very low frequencies (rare alleles). Multivariate and model-based analyses partitioned the collection in seven clusters comprising the ten different Capsicum spp analysed: C. annuum, C. chinense, C. frutescens, C. pubescens, C. bacatum, C. chacoense and C. eximium. The data clearly showed the close relationships between C. chinense and C. frutescens. C. cardenasii and C. eximium were indistinguishable as a single, morphologically variable species. Moreover, C. chacoense was placed between C. baccatum and C. pubescens complexes. The C. annuum group was structured into three main clusters, mostly according to the pepper fruit shape, size and potential pungency. Results suggest that the diversification of C. annuum in Spain may occur from a rather limited gene pool, still represented by few landraces with ancestral traits. This ancient population would suffer from local selection at the distinct geographical regions of Spain, giving way to pungent and elongated fruited peppers in the South and Center, while sweet blocky and triangular types in Northern Spain.

Towards positional isolation of three quantitative trait loci conferring resistance to powdery mildew in two Spanish barley landraces.

Three quantitative trait loci (QTL) conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper) and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family.

Resistance to powdery mildew in one Spanish barley landrace hardly resembles other previously identified wild barley resistances

Two major quantitative trait loci (QTLs) associated with resistance to powdery mildew (Blumeria graminis f. sp. hordei) were previously identified on chromosome 7H of the Spanish barley line SBCC097. The two QTLs seemed to share the same chromosomal position as the major genes mlt and Mlf, which were formerly described in Hordeum vulgare ssp. spontaneum-derived lines. In the present work, different lines that carry mlt (RS42-6*O), Mlf (RS137-28*E), or a combination of both (SI-4 and SI-6) were compared with SBCC097 to evaluate their relatedness at the phenotypic, cellular, and genetic levels. The resistance of the lines was characterised by inoculating them with a set of 27 isolates of B. graminis, which displayed a wide range of virulence. It was revealed that SBCC097 possessed a distinctive resistance spectrum. Microscopic assessment of the cytological development of the resistance response showed that SBCC097 clearly formed fewer well-established colonies and secondary hyphae than the other lines. This was confirmed by the infection type recorded after visual inspection. Genetic analyses of all five lines, based on markers flanking the QTLs derived from SBCC097, supported the macroscopic and microscopic data and pointed to the presence of a combination of novel genes or alleles in SBCC097, which may be included in the category of “intermediate-acting” genes, governing resistance mainly at the post-penetration stage.

A Cluster of Nucleotide-Binding Site–Leucine-Rich Repeat Genes Resides in a Barley Powdery Mildew Resistance Quantitative Trait Loci on 7HL

Powdery mildew causes severe yield losses in barley production worldwide. Although many resistance genes have been described, only a few have already been cloned. A strong QTL (quantitative trait locus) conferring resistance to a wide array of powdery mildew isolates was identified in a Spanish barley landrace on the long arm of chromosome 7H. Previous studies narrowed down the QTL position, but were unable to identify candidate genes or physically locate the resistance. In this study, the exome of three recombinant lines from a high‐resolution mapping population was sequenced and analyzed, narrowing the position of the resistance down to a single physical contig. Closer inspection of the region revealed a cluster of closely related NBS‐LRR (nucleotide‐binding site–leucine‐rich repeat containing protein) genes. Large differences were found between the resistant lines and the reference genome of cultivar Morex, in the form of PAV (presence‐absence variation) in the composition of the NBS‐LRR cluster. Finally, a template‐guided assembly was performed and subsequent expression analysis revealed that one of the new assembled candidate genes is transcribed. In summary, the results suggest that NBS‐LRR genes, absent from the reference and the susceptible genotypes, could be functional and responsible for the powdery mildew resistance. The procedure followed is an example of the use of NGS (next‐generation sequencing) tools to tackle the challenges of gene cloning when the target gene is absent from the reference genome.

Assessing the Barley Genome Zipper and Genomic Resources for Breeding Purposes

The aim of this study was to estimate the accuracy and convergence of newly developed barley (Hordeum vulgare L.) genomic resources, primarily genome zipper (GZ) and population sequencing (POPSEQ), at the genome‐wide level and to assess their usefulness in applied barley breeding by analyzing seven known loci. Comparison of barley GZ and POPSEQ maps to a newly developed consensus genetic map constructed with data from 13 individual linkage maps yielded an accuracy of 97.8% (GZ) and 99.3% (POPSEQ), respectively, regarding the chromosome assignment. The percentage of agreement in marker position indicates that on average only 3.7% GZ and 0.7% POPSEQ positions are not in accordance with their centimorgan coordinates in the consensus map. The fine‐scale comparison involved seven genetic regions on chromosomes 1H, 2H, 4H, 6H, and 7H, harboring major genes and quantitative trait loci (QTL) for disease resistance. In total, 179 GZ loci were analyzed and 64 polymorphic markers were developed. Entirely, 89.1% of these were allocated within the targeted intervals and 84.2% followed the predicted order. Forty‐four markers showed a match to a POPSEQ‐anchored contig, the percentage of collinearity being 93.2%, on average. Forty‐four markers allowed the identification of twenty‐five fingerprinted contigs (FPCs) and a more clear delimitation of the physical regions containing the traits of interest. Our results demonstrate that an increase in marker density of barley maps by using new genomic data significantly improves the accuracy of GZ. In addition, the combination of different barley genomic resources can be considered as a powerful tool to accelerate barley breeding.

Deciphering the role of the phenylpropanoid metabolism in the tolerance of Capsicum annuum L. to Verticillium dahliae Kleb.

Verticillium dahliae is an economically relevant soilborne pathogen that causes vascular wilt in several crops, including pepper (Capsicum annuum). Fungal infection is usually visualized as a vascular browning, likely due to the onset of phenylpropanoid metabolism, which also seems to play a crucial role in the tolerance of some pepper varieties. In the current work, the potential function of distinct phenylpropanoid derivatives (suberin, lignin and phenolic compounds) in the pepper tolerance response against V. dahliae, was investigated. Histochemical and biochemical analyses ruled out suberin as a key player in the pepper-fungus interaction. However, changes observed in lignin composition and higher deposition of bound phenolics in infected stems seemed to contribute to the reinforcement of cell walls and the impairment of V. dahliae colonization. Most importantly, this is the first time that the accumulation of the hydroxycinnamic acid amide N-feruloyltyramine was reported in pepper stems in response to a vascular fungus. Fungitoxic activity for that hydroxycinnamate-tyramine conjugate was demonstrated as well.

Assessing the genetic diversity in onion (Allium cepa L.) landraces from northwest Spain and comparison with the European variability

ABSTRACT Fifteen onion landraces from Galicia (northwest Spain) were characterised with a set of 25 microsatellite markers and the genetic variability harboured by this collection was compared with the surrounding diversity by comparison with a representative panel of European onion landraces. Twenty markers were polymorphic allowing the detection of 121 alleles. Ninety-one of these were identified in the Galician group (average of 4.6 distinct alleles per locus) and 9% of the total number of alleles were recorded as unique alleles specifically contributed by onion landraces from northwest Spain. High values of observed and expected heterozygosities were detected for the majority of loci. Wright’s fixation index confirmed an excess of heterozygotes and a low level of inbreeding, suggesting that high levels of heterogeneity have accumulated within landraces. Multivariate and STRUCTURE analyses revealed that Galician onions possessed a specific genomic composition different from that found in European landraces.