A simple and sensitive silver-staining method for detecting AFLP markers in fungi

A simple and fast silver-staining method is described for detecting amplified fragment length polymorphism (AFLP) markers separated in denaturing polyacrylamide gels. The silver-staining method produced a good degree of resolution and sensitivity and could be widely applicable in AFLP analyses of fungi

Agronomic Characteristics, Malt Quality, and Disease Resistance of Barley Germplasm Lines with Partial Fusarium Head Blight Resistance

Fusarium head blight (FHB), incited by Fusarium graminearum Schwabe, has caused devastating losses in both yield and quality of barley (Hordeum vulgare L.) produced in the northern Great Plains from 1993 to 2003. Thirty-five barley germplasmlines with partial resistance to FHB have been identified in exotic and unadapted germplasm lines. Little is known about their agronomic characteristics, malt quality, and reaction to other diseases as compared to adapted cultivars. This information is needed so barley breeders can make informed decisions when planning crosses involving the resistant germplasm lines. The objective of this study was to compare the agronomic performance, malt quality, and disease reaction of barley germplasm lines with partial FHB resistance to cultivars grown in the northern Great Plains. Agronomic and malting data were collected on the 35 germplasm lines and five check cultivars grown in five environments in North Dakota from 1998 to 2000. Data for FHB severity and deoxynivalenol (DON, a mycotoxin produced by F. graminearum) accumulation were obtained for the same 40 entries grown in FHB-epidemic nurseries in North Dakota from 1997 to 1999. Seedling responses to foliar pathogens common in the northern Great Plains were determined in the greenhouse during fall 1997. None of the FHB-resistant barley germplasm lines had acceptable malt quality for all traits. Kernel plumpness, grain protein concentration, and malt extract were the traits impacted most severely. The FHB-resistant barley germplasm lines headed significantly later than the adapted barley cultivars. Most FHB-resistant germplasm lines were susceptible to the common foliar diseases of the northern Great Plains. At least four cycles of breeding will probably be necessary to develop FHB-resistant germplasm lines acceptable to producers and the malting and brewing industry

Heritability of Fusarium Head Blight Resistance and Deoxynivalenol Accumulation from Barley Accession CIho 4196

Fusarium head blight (FHB), incited by Fusarium graminearum Schwabe [telomorph Gibberella zea (Schwein)], has caused devastating losses to yield and quality of barley (Hordeum vulgare L.) produced in the upper U.S. Midwest from 1993 to 2000. Design of an efficient breeding strategy for developing FHB resistant cultivars is dependent on knowing (i) the heritability of FHB resistance and accumulation of deoxynivalenol (DON), a mycotoxin contaminant produced by F. graminearum and (ii) the correlated response of other traits during selection for reduced FHB. We conducted field studies in FHB disease nurseries using F4:5 and F4:6 families from the cross between the FHB susceptible six-rowed cultivar Foster and the resistant two-rowed accession CIho 4196 to gain knowledge in the areas listed above. Heritability of FHB severity and DON accumulation was 0.65 and 0.46, respectively. A moderately strong positive association between FHB severity and DON accumulation was observed (r = 0.62). FHB severity and DON accumulation were negatively associated with plant height, days to heading, spike angle, and spike density. The selection differentials calculated between the top F4:6 families selected for low FHB severity and the unselected F4:5 families were moderately high for FHB severity, DON accumulation, and days to heading. Less than 14% of the selected lines had six-rowed spikes. No difference in plant height was observed between the selected and unselected families. Thus, development of FHB resistant lines with acceptable DON accumulation and days to heading is obtainable. However, because no lines were as short as Foster, development of FHB resistant plants with acceptable plant height from a cross using CIho 4196 as a parent will be difficult

Association mapping of stem rust race TTKSK resistance in US barley breeding germplasm

KEY MESSAGE: Loci conferring resistance to the highly virulent African stem rust race TTKSK were identified in advanced barley breeding germplasm and positioned to chromosomes 5H and 7H using an association mapping approach. ABSTRACT: African races of the stem rust pathogen (Puccinia graminis f. sp. tritici) are a serious threat to barley production worldwide because of their wide virulence. To discover and characterize resistance to African stem rust race TTKSK in US barley breeding germplasm, over 3,000 lines/cultivars were assessed for resistance at the seedling stage in the greenhouse and also the adult plant stage in the field in Kenya. Only 12 (0.3 %) and 64 (2.1 %) lines exhibited a resistance level comparable to the resistant control at the seedling and adult plant stage, respectively. To map quantitative trait loci (QTL) for resistance to race TTKSK, an association mapping approach was conducted, utilizing 3,072 single nucleotide polymorphism (SNP) markers. At the seedling stage, two neighboring SNP markers (0.8 cM apart) on chromosome 7H (11_21491 and 12_30528) were found significantly associated with resistance. The most significant one found was 12_30528; thus, the resistance QTL was named Rpg-qtl-7H-12_30528. At the adult plant stage, two SNP markers on chromosome 5H (11_11355 and 12_31427) were found significantly associated with resistance. This resistance QTL was named Rpg-qtl-5H-11_11355 for the most significant marker identified. Adult plant resistance is of paramount importance for stem rust. The marker associated with Rpg-qtl-5H-11_11355 for adult plant resistance explained only a small portion of the phenotypic variation (0.02); however, this QTL reduced disease severity up to 55.0 % under low disease pressure and up to 21.1 % under heavy disease pressure. SNP marker 11_11355 will be valuable for marker-assisted selection of adult plant stem rust resistance in barley breeding. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00122-014-2297-8) contains supplementary material, which is available to authorized users

Identification of QTLs Associated with Fusarium Head Blight Resistance in Barley Accession CIho 4196

Fusarium head blight (FHB), incited by Fusarium graminearum Schwabe [teleomorph Gibberella zea (Schwein)], reduces quality of harvested barley (Hordeum vulgare L.) because of blighted kernels and the presence of deoxynivalenol (DON), a mycotoxin produced by the pathogen. CIho 4196, a two-rowed type, is one of the most resistant accessions known in barley; however, it possesses many undesirable agronomic traits. To better understand the genetics of reduced FHB severity and DON accumulation conferred by CIho 4196, a genetic map was generated using a population of recombinant inbred lines derived from a cross between Foster (a six-rowed malting cultivar) and CIho 4196. Quantitative trait loci (QTL) analyses were performed using data obtained from 10 field environments. The possible associations of resistance QTLs and various agronomic and morphological traits in barley also were investigated. The centromeric region of chromosome 2H flanked by the markers ABG461C and MWG882A (bins 6–10) likely (P\u3c0.001) contains two QTLs contributing to lower FHB severity and plant height, and one QTL each for DON accumulation, days to heading, and rachis node number. The QTL for low FHB severity in the bin 8 region explained from 3 to 9% of the variation, while the QTL in the bin 10 region explained from 17 to 60% of the variation. A QTL for DON accumulation that explained 9 to 14% of the variation was found in the bin 2 region of chromosome 4H. This may represent a new QTL not present in other FHB resistant sources. Resistance QTLs in the bin 8 region and bin 10 region of chromosome 2HL were provisionally designated Qrgz-2H-8 and Qrgz-2H-10, respectively. The QTL for DON accumulation in chromosome 4H was provisionally named QDON-4H-2

Exercise Blocks Ethanol-Induced Kappa Opioid Receptor Sensitization in Nucleus Accumbens and Ventral Tegmental Area

Exercise has been increasingly used as an adjunctive therapy in the treatment of alcohol use disorder (AUD). Despite this, the mechanism by which it influences the mesolimbic circuitry changes underlying alcohol addiction is not well understood. Previous studies have shown alcohol dependence to lead to upregulation of the Dynorphin-Kappa Opioid Receptor (KOR) system, making it a potential target for therapeutics. Thus, gaining a better understanding of these pathways will help develop evidence-based guidelines for integrating exercise into therapies for the treatment of AUD

Potential for re-emergence of wheat stem rust in the United Kingdom

This is the final version. Available on open access from Springer Nature via the DOI in this recordWheat stem rust, a devastating disease of wheat and barley caused by the fungal pathogen Puccinia graminis f. sp. tritici, was largely eradicated in Western Europe during the mid-to-late twentieth century. However, isolated outbreaks have occurred in recent years. Here we investigate whether a lack of resistance in modern European varieties, increased presence of its alternate host barberry and changes in climatic conditions could be facilitating its resurgence. We report the first wheat stem rust occurrence in the United Kingdom in nearly 60 years, with only 20% of UK wheat varieties resistant to this strain. Climate changes over the past 25 years also suggest increasingly conducive conditions for infection. Furthermore, we document the first occurrence in decades of P. graminis on barberry in the UK . Our data illustrate that wheat stem rust does occur in the UK and, when climatic conditions are conducive, could severely harm wheat and barley production.This project was funded by an institute development grant from the EI (Norwich, UK), an Industrial Partnership Award (BB/M025519/1) from the BBSRC, a European Research Council Starting Grant awarded to D.G.O.S. (number 715638), H2020 project EMPHASIS (number 634179), by the BBSRC Institute Strategic Programmes BB/J004553/1 and BB/P012574/1, the John Innes Foundation, and an African Women in Agricultural Research and Development (AWARD) fellowship to R.N.K

Mutagenesis of Puccinia graminis f. sp. tritici and selection of gain-of-virulence mutants

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance (Sr) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr-gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs. To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43, Sr44, or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43, Sr44, or Sr45, respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43. These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance (R) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis

Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons

Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance