A consensus map of barley was constructed based on three reference doubled haploid (DH) populations and three recombinant inbred line (RIL) populations. Several sets of microsatellites were used as bridge markers in the integration of those populations previously genotyped with RFLP or with AFLP markers. Another set of 61 genic microsatellites was mapped for the Wrst time using a newly developed Xuorescent labelling strategy, referred to as A/T labelling. The Wnal map contains 3,258 markers spanning 1,081 centiMorgans (cM) with an average distance between two adjacent loci of 0.33 cM. This is the highest density of markers reported for a barley genetic map to date. The consensus map was divided into 210 BINs of about 5 cM each in which were placed 19 quantitative trait loci (QTL) contributing to the partial resistance to barley leaf rust (Puccinia hordei Otth) in Wve of the integrated populations. Each parental barley combination segregated for diVerent sets of QTLs, with only few QTLs shared by any pair of cultivars. Defence gene homologues (DGH) were identiWed by tBlastx homology to known genes involved in the defence of plants against microbial pathogens. Sixty-three DGHs were located into the 210 BINs in order to identify candidate genes responsible for the QTL eVects. Eight BINs were cooccupied by a QTL and DGH(s). The positional candidates identiWed are receptor-like kinase, WIR1 homologues and several defence response genes like peroxidases, superoxide dismutase and thaumatin.

Inheritance studies on the nonhost resistance of plants would normally require interspecific crosses that suffer from sterility and abnormal segregation. Therefore, we developed the barley-Puccinia rust model system to study, using forward genetics, the specificity, number, and diversity of genes involved in nonhost resistance. We developed two mapping populations by crossing the line SusPtrit, with exceptional susceptibility to heterologous rust species, with the immune barley cultivars Vada and Cebada Capa. These two mapping populations along with the Oregon Wolfe Barley population, which showed unexpected segregation for resistance to heterologous rusts, were phenotyped with four heterologous rust fungal species. Positions of QTL conferring nonhost resistance in the three mapping populations were compared using an integrated consensus map. The results confirmed that nonhost resistance in barley to heterologous rust species is controlled by QTL with different and overlapping specificities and by an occasional contribution of an R-gene for hypersensitivity. In each population, different sets of loci were implicated in resistance. Few genes were common between the populations, suggesting a high diversity of genes conferring nonhost resistance to heterologous pathogens. These loci were significantly associated with QTL for partial resistance to the pathogen Puccinia hordei and with defense-related genes.

We developed an evolutionary relevant model system, barley-Puccinia [corrected] rust fungi, to study the inheritance and specificity of plant factors that determine to what extent innate nonhost immunity can be suppressed. A mapping population was developed from a cross between an experimental barley line (SusPtrit) [corrected] with exceptional susceptibility to several heterologous [corrected] (nonhost) rust fungi and regular, immune, cv. Vada [corrected] Seedlings were inoculated with five heterologous [corrected] and two homologous (host) species of rust fungi. Resistance segregated quantitatively for each of the rust fungi. In total, 18 chromosomal regions were implicated. For each rust species, a different set of genes was effective. Of the 18 chromosomal regions, 11 were significantly effective to only one rust species and 7 were effective to more than one rust species, implying genetic linkage or pleiotropy. One resistance (R) gene for hypersensitive resistance to Puccinia hordei-secalini was mapped, suggesting occasional contribution of R genes to nonhost resistance in barley. Quantitative trait loci (QTLs) with effects to multiple rust fungi did not tend to be particularly effective to rust species that were phylogenetically related, as determined from their internal transcribed spacer sequence. We suggest that the QTLs described here play a role as specific and quantitative recognition factors that are specifically negated by the rust to successfully suppress innate immunity.

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.

Aims: In this study, a loop-mediated isothermal amplification (LAMP) assay has been developed and evaluated for the rapid and sensitive detection of Verticillium dahliae Kleb., the causal agent of vascular wilts in many economically important crops. Methods and Results: LAMP primers were designed based on a previously described RAPD marker, and the LAMP assay was applied for direct detection of V. dahliae grown on medium and from soil samples without DNA purification steps (direct-LAMP). Thirty-two agricultural soil samples from various olive orchards were collected, and the presence of pathogen was detected by LAMP, direct-LAMP and nested-PCR methods. The LAMP methodology could successfully detect V. dahliae with high specificity, and cross-reaction was not observed with different pathogenic and nonpathogenic fungi and bacteria. The LAMP assay was capable of detecting a minimum of 500 and 50 fg of purified target DNA per reaction of V. dahliae ND and D pathotypes, respectively. In contrast, nested-PCR could only detect 5 pg reaction À1 for both pathotypes. In artificially infested soil samples, the LAMP method detected 5 microsclerotia per gram of soil. Conversely, nested-PCR assay detected 50 microsclerotia g À1 soil. The detection ratios of LAMP and direct-LAMP protocols were better (26 and 24 positive samples out of 32 agricultural soils analysed, respectively) than that obtained for nested-PCR method (22 positive results). Moreover, direct-LAMP yielded positive detection of V. dahliae in agricultural soil samples within 60-80 min. Conclusions:The newly developed LAMP method was proved to be an effective, simple and rapid method to detect V. dahliae without the need for either expensive equipment or DNA purification. Significance and Impact of Study: This technique can be considered as an excellent standard alternative to plating and nested-PCR assays for the early, sensitive and low-cost detection of V. dahliae as well as other soilborne pathogens in the field.

No abstract

Alternate bearing (AB) refers to the tendency of trees to have an irregular crop load from 1 year (ON) to the next year (OFF). Despite its economic importance, it is not fully understood how gene networks and their related metabolic pathways may influence the irregular bearing in olive trees. To unravel molecular mechanisms of this phenomenon in olive (cv. Conservalia), the whole transcriptome of leaves and buds from ON and OFF-trees was sequenced using Illumina next generation sequencing approach. The results indicated that expressed transcripts were involved in metabolism of carbohydrates, polyamins, phytohormones and polyphenol oxidase (POD) related to antioxidant system. Expression of POD was increased in leaf samples of ON- versus OFF-trees. The expression pattern of the greater number of genes was changed more in buds than in leaves. Up-regulation of gene homologues to the majority of enzymes that were involved in photorespiration metabolism pathway in buds of ON-trees was remarkable that may support the hypotheses of an increase in photorespiratory metabolism in these samples. The results indicated changes in expression pattern of homologous to those taking part of abscisic acid and cytokinin synthesis which are connected to photorespiration. Our data did not confirm expression of homologue (s) to those of chlorogenic acid metabolism, which has been addressed earlier that have a probable role in biennial bearing in olive. Current findings provide new candidate genes for further functional analysis, gene cloning and exploring of molecular basses of AB in olive.

The high toxicity of cadmium (Cd) and its ready uptake by plants has become a major agricultural problem. To investigate the genetic architecture and genetic regulation of Cd tolerance in barley, we conducted quantitative trait loci (QTL) analysis in the phenotypically polymorphic Oregon Wolfe Barley (OWB) mapping population, derived from a cross between Rec and Dom parental genotypes. Through evaluating the Cd tolerance of 87 available doubled haploid lines of the OWB mapping population at the seedling stage, one minor and one major QTL were detected on chromosomes 2H and 6H, respectively. For chlorosis and necrosis traits, the major QTL explained 47.24% and 38.59% of the phenotypic variance, respectively. RNA-Seq analysis of the parental seedlings under Cd treatment revealed 542 differentially expressed genes between Cd-tolerant Rec and Cd-susceptible Dom genotypes. By analyzing sequence variations in transcribed sequences of the parental genotypes, 155,654 SNPs and 1,525 InDels were identified between the two contrasting genotypes and may contribute to Cd tolerance. Finally, by integrating the data from the identified QTLs and RNA-Seq analysis, 16 Cd tolerance-related candidate genes were detected, nine of which were metal ion transporters. These results provide promising candidate genes for further gene cloning and improving Cd tolerance in barley.