Analysis of powdery mildew resistance in the Spanish barley core collection

24 Pag., 4 Tabl., 2 Fig. The definitive version is available at: www3.interscience.wiley.comThe Spanish Barley Core Collection, consisting of one hundred and fifty-nine landrace-derived inbred lines and sixteen cultivars, was characterized for resistance to powdery mildew (Blumeria graminis f. sp. hordei) using a set of 27 isolates with a wide spectra of virulences/avirulences on most of the genes expected to occur in Europe. No landrace-derived line and no cultivar were resistant to all the isolates but at least three landraces showed infection types below 2 for 23 isolates. Twenty-two landraces and one cultivar showed resistance against half of the isolates used. Eleven isolates were sufficient to separate the majority of resistance profiles. In total, thirty-four resistance spectra were detected and fourteen resistance genes/alleles were postulated alone or in combination: MlLa, Mlh, Mlg, Mla22, Mla7(Mlu), Mla7(Mlk), Mlk, Mla12, Mla9, Mla3, Mla6(Mla14), Mlra and Mla1. The majority of resistance spectra are composed only by one line. Resistance in twenty-one landrace-derived lines and four cultivars was based on either unidentified genes or combinations of known and unknown genes/alleles. Therefore, the SBCC may be a source for broadening the genetic base of powdery mildew resistance.This research was funded by projects AGL2004-05311 and AGL2007-63625, granted by the Spanish Ministry of Science and Innovation. C.S. holds an I3P-Doc contract from CSIC. C.S. was supported by mobility fellowships from DFG, CSIC and Fundación Caja Inmaculada.Peer reviewe

Resistance in pepper plants induced by Fusarium oxysporum f. sp. lycopersici involves different defence-related genes

Inoculation with Fusarium oxysporum f. sp. lycopersici (FOL) protects pepper plants from subsequent infection with Phytophthora capsici. In the present paper, the level of local and systemic protection achieved by plants induced with FOL was evaluated by quantifying the pathogen biomass and using real-time PCR. Differences in the amount of pathogen were found in stems and roots between FOL-treated and untreated plants, while pathogen biomass could not be detected in leaves of induced plants. Five defence-related genes coding for a PR-1 protein, a β-1,3-glucanase, a chitinase, a peroxidase and a sesquiterpene cyclase were up-regulated 48 h after treatment in all the tissues studied, and maximal mRNAs levels were found in leaves.This work was supported by grants from MEC (project AGF99-0301) and XUGA (project PGDIT03RAG10301). C.S. held a fellowship from MEC.Peer Reviewe

Development of a cost-effective pyrosequencing approach for SNP genotyping in barley

12 Pag., 1 Tabl., 1 Fig. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1439-0523An improved, efficient, reliable and cost-effective pyrosequencing protocol for single nucleotide polymorphism (SNP) genotyping in plants is described. Labelling of the PCR products, required for the single-stranded pyrosequencing assay, is carried out in a one-step PCR with a universal biotinalyted M13 primer of 19-bp. A ratio of 1 : 10 of tailed primer to M13 primer in a 15-μl reaction volume turned out to be best suited for a successful identification of different alleles based on known SNPs. This technology allows cost-effective SNP genotyping in plant genomes for loci of interest at an overall low cost in a short period of time and is therefore best suited for marker-based selection procedures.This study was partially carried out within project AGL2007-63625, funded by the Spanish Ministry for Science and Innovation. C.S. holds an I3P contract from CSIC. C.S. was supported by mobility fellowships from the Deutsche Forschungsgemeinschaft (DFG), CSIC, Fundacion Caja Inmaculada and COST Action FA0604 (Tritigen).Peer reviewe

Fine mapping and comparative genomics integration of two quantitative trait loci controlling resistance to powdery mildew in a Spanish barley landrace

33 Pags., 1 Tabl., 4 Figs. The definitive version, with Supplementals Materials, is available at: http://www.springerlink.com/content/0040-5752/The intervals containing two major quantitative trait loci (QTL) from a Spanish barley landrace conferring broad spectrum resistance to Blumeria graminis were subjected to marker saturation. First, all the available information on recently developed marker resources for barley was exploited. Then, a comparative genomic analysis of the QTL regions with other sequenced grass model species was performed. As a result of the first step, 32 new markers were added to the previous map and new flanking markers closer to both QTL were identified. Next, syntenic integration revealed that the barley target regions showed homology with regions on chromosome 6 of rice (Oryza sativa), chromosome 10 of Sorghum bicolor and chromosome 1 of Brachypodium distachyon. A nested insertion of ancestral syntenic blocks on Brachypodium chromosome 1 was confirmed. Based on sequence information of the most likely candidate orthologous genes, 23 new barley unigene-derived markers were developed and mapped within the barley target regions. The assessment of colinearity revealed an inversion on chromosome 7HL of barley compared to the other three grass species, and nearly perfect colinearity on chromosome 7HS. This two-step marker enrichment allowed for the refinement of the two QTL into much smaller intervals. Inspection of all predicted proteins for the barley unigenes identified within the QTL intervals did not reveal the presence of resistance gene candidates. This study demonstrates the usefulness of sequenced genomes for fine mapping and paves the way for the use of these two loci in barley breeding programs.This work was funded by the Spanish Ministry of Science and Innovation (projects GEN2006-28560-E, AGL2007-63625, and Plant-KBBE ExpResBar, EUI2009-04075), and co-funded by the European Regional Development Fund. CS held an I3P contract from CSIC. CS was supported by mobility fellowships from the Deutsche Forschungsgemeinschaft (DFG OR72/5-1), CSIC, Fundación Caja Inmaculada and COST Action FA0604 (Tritigen).Peer reviewe

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

23 Pags., 2 Tabls., 3 Figs. The definitive version is available at: http://link.springer.com/journal/10658wo 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.C.S. held a mobility fellowship from the University of Coruña. Part of the results were obtained within the project ExpResBar funded by the German Federal Ministry of Education and Research under grant number 0315702B within the KBBE-II call, and by the Spanish Ministry of Science and Innovation, grant number EUI2009-04075.Peer reviewe

Development of diagnostic markers and physical mapping for the Rrs1 resistance locus against scald

Rhynchosporium secalis, the causal agent of scald, is still one of the most important foliar diseases of barley. Its high genetic variability and recombination frequency enable it to rapidly overcome monogenic resistances. To date four major scald resistance genes have been identified in cultivated barley (Hordeum vulgare ssp. vulgare), and another four in wild barley (Hv. spontaneum or Hv. bulbosum). The most abundant and effective one is the Rrsl resistance locus, mapping near the centromeric region of chromosome 3H. Aim of the project is the fine mapping of the Rrsl locus and the development of diagnostic markers.This project was funded by the GABI-Plant-KBBE II Project, ExpResBar” under the grant 0315702C and the StMELF.Peer Reviewe

Identification of quantitative trait loci for resistance to powdery mildew in a Spanish barley landrace

30 Pag., 1 Tabl., 5 Fig. The definitive version is available at: http://www.springerlink.com/content/1380-3743/The Spanish landrace-derived inbred line SBCC97, together with other lines from the Spanish Barley Core Collection, displays high resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei. The objective of this study was to map quantitative trait loci (QTLs) for resistance to powdery mildew in a recombinant inbred line population derived from a cross between SBCC97 and the susceptible cultivar ‘Plaisant’. Phenotypic analysis was performed using four B. graminis isolates, and genetic maps were constructed with mainly simple sequence repeat (SSR) markers, following a sequential genotyping strategy. Two major QTLs with large effects were identified on chromosome 7H, and they accounted for up to 45% of the total phenotypic variance. The alleles for resistance at each QTL were contributed by the Spanish parent SBCC97. One locus was mapped to the short arm of chromosome 7HS, and was flanked by the resistance gene analogue (RGA) marker S9202 and the SSR GBM1060. This corresponded to the same chromosomal region in which a major race-specific resistance gene from Hordeum vulgare ssp. spontaneum, designated as mlt, had been identified previously. The second QTL was linked tightly to marker EBmac0755, and it shared its chromosomal location with the qualitative resistance gene Mlf, which has only been described previously in the wild ancestor H. spontaneum. This is the first report of these two QTLs occurring together in cultivated barley, and it paves the way for their use in barley breeding programs that are designed to transfer resistance alleles into elite cultivars.This work was supported by the Spanish Ministry of Education and Research (Project AGL2007-63625), and by the European Regional Development Fund. C.S. holds an I3P contract from CSIC. H.D. was supported by a Masters fellowship from IAMZ-CIHEAM.Peer reviewe

Fine-mapping of a powdery mildew QTL by exome sequencing

1 .pdf copy (A3) of the original poster presented by the Authors.Powdery mildew in barley is caused by the biotrophic fungus Blumeria graminis f. sp. hordei. Colonization of leaf surfaces results in severe yield losses in temperate latitudes worldwide. Up to date, most mildew resistance loci (Ml genes) have not been cloned, with the exception of mlo and Mla genes. Cloning efforts to identify them relied on cumbersome procedures, due to the lack of genomic resources and the large and highly repetitive nature of barley genome. In the last decade, new resources have been made available which accelerate barley research and breeding. In this work, we take advantage of those resources to fine map a powdery mildew resistance QTL on 7HL, through the development of a high-resolution mapping population followed by exome sequencing of recombinant lines with contrasting resistance phenotypes. Exome capture data allowed delimiting the physical position of the QTL to a single physical contig. The analysis of available genomic references led to the characterization of the gene composition in the region, revealing a cluster of NBS-LRRs taking up most of the QTL. We followed a non-standard pipeline to assemble reads from mixed mappings, causing heterozygous variants in NBS-LRR loci. Overall, 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 next generation sequencing tools to tackle the challenges of gene cloning when the template is absent from the reference.Peer reviewe

The Rrs1 locus and resistance against scald in barley.

Scald, caused by Rhynchosporium commune (formerly R. secalis ), is one of the most prevalent barley diseases worldwide. To date, four major scald resistance genes have been mapped in cultivated barley ( Hordeum vulgare ssp. vulgare ), and another four in wild barley ( Hv. spontaneum ) or Hv. bulbosum . The most abundant and effective one is the Rrs1 resistanc e locus, formerly known as Rh - Rh3Rh4 locus. It was mapped to the centromeric region of chromosome 3H. However, it is still not clear whether Rrs1 is a collection of several R - genes close to each other or several alleles of the same gene. A search for new r esistance sources revealed that Spanish landrace - derived lines SBCC145 and SBCC154 showed outstanding resistance to scald. To analyze the genetic basis in more detail two large DH mapping populations were developed crossing each donor line with cv. Beatrix . A large QTL in the centromeric region of chromosome 3H was found in both populations phenotyped for scald resistance in a well - established greenhouse test, therefore, confirmed this locus as the only resistance locus in both populations. To confirm and e nclose this locus, the “ Rrs1 region” has been saturated with all available SSR and SNP - markers and a consensus map was constructed. New markers for this region are developed based on the lllumina iSelect custom 9K barley chip, the barley genome zipper and a BSA analysis with AFLP. The genome zipper identified several candidate genes. Because of a low gene/cM density, an enrichment of candidate sequences in the region of Rrs1 is done by a BSTA (bulked segregant transcriptome analysis) with four normalized cD NA libraries and Illumina HiSeq in combination with a high resolution mapping program. For fine mapping and haplotyping of all the genes around the Rrs1 loci a mapping population comprising >10,000 F 2 from the cross SBCC145 x Beatrix has been constructed. F 2 screening of about 11,000 lines to select recombinant lines between two flanking markers has identified around 442 verified recombinant plants. The effective Rrs1 allele found and the closely linked markers developed are already useful tools for molecul ar breeding programs.Peer Reviewe

The Rrs1 resistance locus against scald in barley

Scald, caused by Rhynchosporium commune, is one of the most prevalent barley diseases worldwide. To date, four major scald resistance genes have been mapped in cultivated barley (Hordeum vulgare ssp. vulgare), and another four in wild barley (Hv. Spontaneum) or Hv. bulbosum. The most abundant and effective one is the Rrs1 resistance locus, which was mapped to the centromeric region of chromosome 3H. However, it is still not clear whether Rrs1 formerly known as Rh-Rh3Rh4 locus is a collection of several R-genes close to each other or several alleles of the same gene. A search for new resistance sources revealed that Spanish landrace-derived lines SBCC145 and SBCC154 showed outstanding resistance to scald. To analyze the genetic basis in more detail two large DH mapping populations were developed crossing each donor line with cv. Beatrix. A large QTL in the centromeric region of chromosome 3H was found in both populations, therefore, suggesting this locus as the only resistance locus in both populations. To confirm and enclose this locus, the “Rrs1 region” has been saturated with all available SSR and SNP-markers and a consensus map was constructed. New markers for this region are developed based on the lllumina iSelect custom 9K barley chip and candidate genes derived from the barley genome zipper. Because of a low gene/cM density, an enrichment of candidate sequences in the region of Rrs1 is done by a BSTA (bulked segregant transcriptome analysis) with four normalized cDNA libraries and Illumina HiSeq in combination with a high resolution mapping program. For fine mapping and haplotyping a mapping population comprising >10,000 F2 from the cross SBCC145 x Beatrix has been constructed. F2 screening of about 11,000 lines to select recombinant lines between two flanking markers has identified around 442 verified recombinant plants. The effective Rrs1 allele found and the closely linked markers developed are already useful tools for molecular breeding programs.Peer Reviewe