Mapping of seedling resistance in barley to Puccinia striiformis f. sp. pseudohordei.

The barley grass stripe rust (BGYR) pathogen Puccinia striiformis f. sp. pseudohordei was first detected in Australia in 1997. While studies have established that it is virulent on wild barley grass, and can infect several barley cultivars, the basis of genetic resistance to this pathogen in barley is largely unknown. Understanding the genetic basis of host resistance and ensuring the selection of germplasm with multiple resistance genes are important to mitigate the potential impact of BGYR in barley production. Genetic analysis of seedling resistance to BGYR in two barley doubled haploid populations, Amaji Nijo/WI2585 (AN/WI) and Galleon/Haruna Nijo (GL/HN), indicated that resistance is governed by several genes. Marker regression analysis of the seedling resistance data from the AN/WI population detected a major QTL, BGYR_WI1 (resistance contributed by WI2585 with the closest marker explaining 52 % of the total phenotypic effect) on chromosome 1HS, flanked by the loci Xabg59 and Xabc310b at map positions 0.0 and 6.9 cM, respectively. Similarly, a major QTL, BGYR_HN1, (resistance contributed by Haruna Nijo with the closest marker explaining 70 % of the total phenotypic effect) was detected in the GL/HN population and was mapped to 1HS, flanked by the loci Xbcd135 and XHOR1 at map positions 12.8 and 24.5 cM, respectively. In addition, several minor loci that provided resistance against BGYR were detected in both populations. While defined QTL intervals were large, the analysis nonetheless provides new information on sources of major QTL controlling resistance to BGYR.

Loss of serum response factor induces microRNA-mediated apoptosis in intestinal smooth muscle cells.

Serum response factor (SRF) is a transcription factor known to mediate phenotypic plasticity in smooth muscle cells (SMCs). Despite the critical role of this protein in mediating intestinal injury response, little is known about the mechanism through which SRF alters SMC behavior. Here, we provide compelling evidence for the involvement of SRF-dependent microRNAs (miRNAs) in the regulation of SMC apoptosis. We generated SMC-restricted Srf inducible knockout (KO) mice and observed both severe degeneration of SMCs and a significant decrease in the expression of apoptosis-associated miRNAs. The absence of these miRNAs was associated with overexpression of apoptotic proteins, and we observed a high level of SMC death and myopathy in the intestinal muscle layers. These data provide a compelling new model that implicates SMC degeneration via anti-apoptotic miRNA deficiency caused by lack of SRF in gastrointestinal motility disorders.

Nucleotide sequence diversity in Velvet tobacco mottle virus: a virus with a unique Australian pathosystem.

Velvet tobacco mottle virus (VTMoV) is a naturally occurring mirid-transmitted sobemovirus of native velvet tobacco (Nicotiana velutina) plants in the Australian arid zone. We have sequenced the coding region of a typical field isolate of VTMoV (isolate I-17-04, satellite-plus) and show that it differed by nine polymorphisms from the previously sequenced atypical 'satellite-minus' variant VTMoV-K1 (represented here as L-K1-04), while retaining the same genomic and amino acid sequence motifs. We also report that although L-K1-04 was confirmed to be free of detectable satellite RNA by gel electrophoretic assay, the satellite sequence was detected in it by RT-PCR assay. Nucleotide sequence variation among the RNA-dependent RNA polymerase open reading frames of 15 field and laboratory isolates identified four phylogenetic groups, but these did not show a pattern related to site or time of sampling. This result would be consistent with nucleotide sequence variants of VTMoV being dispersed widely by migrating adult mirid vectors.

Mutation rate in Velvet tobacco mottle virus varies between genomic region and virus variant but is not influenced by obligatory mirid transmission.

Genomic mutation in plant viruses of cultivated plants is known to be influenced by virus, host and vector, but the factors influencing mutation in viruses of native plants in natural ecosystems are rarely studied. We have tested the effect of mode of transmission on mutation in Velvet tobacco mottle virus (VTMoV), a mirid-vectored sobemovirus associated with Nicotiana velutina, an Australian native xerophyte growing in a region isolated from anthropogenic influences. Two variants of VTMoV (K1 and R17) were passaged monthly in the alternative experimental plant host, N. clevelandii, for 2 years, either by mechanical inoculation or by transmission with the mirid Cyrtopeltis nicotianae. Sequence variations were scored after 24 passages in regions of the genome containing the open reading frames (ORFs) for the P1 and coat protein (CP). The mean mutation rate was 6.83 × 10(-4) nt/site year, but a higher overall rate was observed for the K1 (satellite -) than the R17 (satellite +) variant. The P1 ORF showed a higher frequency of non-synonymous mutations than the CP. No clear association was found between either mutation site or mutation rate and the mode of transmission, indicating that obligatory mirid transmission had not exerted a specific bottle-neck effect on sequence variation during the experimental time frame. Failure to detect any sequence motifs linked to vector transmission suggests that a specific capsid-stylet interaction is not required for transmission by mirids.

A SOS3 homologue maps to HvNax4, a barley locus controlling an environmentally sensitive Na+ exclusion trait.

Genes that enable crops to limit Na(+) accumulation in shoot tissues represent potential sources of salinity tolerance for breeding. In barley, the HvNax4 locus lowered shoot Na(+) content by between 12% and 59% (g(-1) DW), or not at all, depending on the growth conditions in hydroponics and a range of soil types, indicating a strong influence of environment on expression. HvNax4 was fine-mapped on the long arm of barley chromosome 1H. Corresponding intervals of ∼200 kb, containing a total of 34 predicted genes, were defined in the sequenced rice and Brachypodium genomes. HvCBL4, a close barley homologue of the SOS3 salinity tolerance gene of Arabidopsis, co-segregated with HvNax4. No difference in HvCBL4 mRNA expression was detected between the mapping parents. However, genomic and cDNA sequences of the HvCBL4 alleles were obtained, revealing a single Ala111Thr amino acid substitution difference in the encoded proteins. The known crystal structure of SOS3 was used as a template to obtain molecular models of the barley proteins, resulting in structures very similar to that of SOS3. The position in SOS3 corresponding to the barley substitution does not participate directly in Ca(2+) binding, post-translational modifications or interaction with the SOS2 signalling partner. However, Thr111 but not Ala111 forms a predicted hydrogen bond with a neighbouring α-helix, which has potential implications for the overall structure and function of the barley protein. HvCBL4 therefore represents a candidate for HvNax4 that warrants further investigation.

Prey life-history and bioenergetic responses across a predation gradient.

To evaluate the importance of non-consumptive effects of predators on prey life histories under natural conditions, an index of predator abundance was developed for naturally occurring populations of a common prey fish, the yellow perch Perca flavescens, and compared to life-history variables and rates of prey energy acquisition and allocation as estimated from mass balance models. The predation index was positively related to maximum size and size at maturity in both male and female P. flavescens, but not with life span or reproductive investment. The predation index was positively related to size-adjusted specific growth rates and growth efficiencies but negatively related to model estimates of size-adjusted specific consumption and activity rates in both vulnerable (small) and invulnerable (large) size classes of P. flavescens. These observations suggest a trade-off between growth and activity rates, mediated by reduced activity in response to increasing predator densities. Lower growth rates and growth efficiencies in populations with fewer predators, despite increased consumption suggests either 1) a reduction in prey resources at lower predator densities or 2) an intrinsic cost of rapid prey growth that makes it unfavourable unless offset by a perceived threat of predation. This study provides evidence of trade-offs between growth and activity rates induced by predation risk in natural prey fish populations and illustrates how behavioural modification induced through predation can shape the life histories of prey fish species.

Physical analysis of the complex rye (Secale cereale L.) Alt4 aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco.

Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library's suitability for investigating Al tolerance genes. The library provides 6 x genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus.

The impact of a contaminated city water supply on an emergency department.

In March 2007 Galway City and County's water supply was officially contaminated by cryptosporidiosis. The medical and nursing staff at the city's only Emergency Department had noted a rise in an atypical form of gastroenteritis in the preceding months. A retrospective audit of 11,723 charts from January 1st 2007 to 22nd March (day after contamination was confirmed) was performed to identify these patients. The number of potential gastroenteritis cases was 185 (incidence 1.6%), with a peak five weeks before the outbreak was confirmed. Half the patients were aged between 20 and 34. Pain (80%), nausea or vomiting (74%) and diarrhoea (66.5%) were the most frequent symptoms. The mean duration of symptoms at presentation was 2 days. Stool samples were sent for nine patients and four of these were tested for Cryptosporidium. Over a quarter (28.6%) of patients were admitted and almost three-quarters (69.7% +/- 7%) had a residential address in the affected area. Difficulty exists in the early identification of new outbreaks and many of the affected patients are not detected using routine surveillance or current capture methods.

An ALMT1 gene cluster controlling aluminum tolerance at the Alt4 locus of rye (Secale cereale L).

Aluminum toxicity is a major problem in agriculture worldwide. Among the cultivated Triticeae, rye (Secale cereale L.) is one of the most Al tolerant and represents an important potential source of Al tolerance for improvement of wheat. The Alt4 Al-tolerance locus of rye contains a cluster of genes homologous to the single-copy Al-activated malate transporter (TaALMT1) Al-tolerance gene of wheat. Tolerant (M39A-1-6) and intolerant (M77A-1) rye haplotypes contain five and two genes, respectively, of which two (ScALMT1-M39.1 and ScALMT1-M39.2) and one (ScALMT1-M77.1) are highly expressed in the root tip, typically the main site of plant Al tolerance/susceptibility. All three transcripts are upregulated by exposure to Al. High-resolution genetic mapping identified two resistant lines resulting from recombination within the gene cluster. These recombinants exclude all genes flanking the gene cluster as candidates for controlling Alt4 tolerance, including a homolog of the barley HvMATE Al-tolerance gene. In the recombinants, one hybrid gene containing a chimeric open reading frame and the ScALMT1-M39.1 gene each appeared to be sufficient to provide full tolerance. mRNA splice variation was observed for two of the rye ALMT1 genes and in one case, was correlated with a approximately 400-bp insertion in an intron.

Is ice right? Does cryotherapy improve outcome for acute soft tissue injury?

AIMS: The use of ice or cryotherapy in the management of acute soft tissue injuries is widely accepted and widely practised. This review was conducted to examine the medical literature to investigate if there is evidence to support an improvement in clinical outcome following the use of ice or cryotherapy. METHODS: A comprehensive literature search was performed and all human and animal trials or systematic reviews pertaining to soft tissue trauma, ice or cryotherapy were assessed. The clinically relevant outcome measures were (1) a reduction in pain; (2) a reduction in swelling or oedema; (3) improved function; or (4) return to participation in normal activity. RESULTS: Six relevant trials in humans were identified, four of which lacked randomisation and blinding. There were two well conducted randomised controlled trials, one showing supportive evidence for the use of a cooling gel and the other not reaching statistical significance. Four animal studies showed that modest cooling reduced oedema but excessive or prolonged cooling is damaging. There were two systematic reviews, one of which was inconclusive and the other suggested that ice may hasten return to participation. CONCLUSION: There is insufficient evidence to suggest that cryotherapy improves clinical outcome in the management of soft tissue injuries.

Haplotype characterization and markers at the barley Mlo powdery mildew resistance locus as tools for marker-assisted selection.

Recessive mlo alleles of the barley Mlo gene confer resistance to almost all known isolates of the powdery mildew fungal pathogen targeting barley (Hordeum vulgare). To characterize haplotypes present in the Mlo chromosomal region of cultivated Mlo and mlo barley genotypes, we conducted a polymorphism search in 3 predicted low-copy sequence regions adjacent to the Mlo gene by examining a sample of 4 Mlo and 3 mlo cultivars. Eight single-nucleotide polymorphisms (SNPs) and 1 insertion-deletion (indel) were detected, and easy to use PCR-based markers were developed for typing the SNPs. The PCR markers were used to characterize a collection of 46 Mlo and 25 mlo barley cultivars, identifying 3 distinct mlo-11 haplotypes, 1 mlo-9 haplotype, and 4 Mlo haplotypes. We summarized the haplotype and marker information obtained here and in a previous study to help breeders identify strategies for mlo marker-assisted selection. The ability of the markers to identify mlo-resistant genotypes in segregating populations was demonstrated using 2 resistance-characterized F2 populations derived by 3-way crosses.

High-resolution genetic mapping of the leaf stripe resistance gene Rdg2a in barley.

The dominant gene Rdg2a of barley conferring resistance to the hemi-biotrophic seed-borne pathogen Pyrenophora graminea is located in the distal region of chromosome arm 1 (7H)S. As the first step towards isolating the gene, a high-resolution genetic map of the region was constructed using an F(2) population of 1,400 plants (Thibaut Rdg2axMirco). The map included six classes of resistance gene analogues (RGAs) tightly associated with Rdg2a. Rdg2a was delimited to a genetic interval of 0.14 cM between the RGAs ssCH4 and MWG851. Additional markers were generated using the sequence from the corresponding region on rice chromosome 6, allowing delimitation of the Rdg2a syntenic interval in rice to a 115 kbp stretch of sequence. Analysis of the rice sequence failed to reveal any genes with similarity to characterized resistance genes. Therefore, either the rice-barley synteny is disrupted in this region, or Rdg2a encodes a novel type of resistance protein.

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping.

The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals.

Recombination between paralogues at the Rp1 rust resistance locus in maize.

Rp1 is a complex rust resistance locus of maize. The HRp1-D haplotype is composed of Rp1-D and eight paralogues, seven of which also code for predicted nucleotide binding site-leucine rich repeat (NBS-LRR) proteins similar to the Rp1-D gene. The paralogues are polymorphic (DNA identities 91-97%), especially in the C-terminal LRR domain. The remaining family member encodes a truncated protein that has no LRR domain. Seven of the nine family members, including the truncated gene, are transcribed. Sequence comparisons between paralogues provide evidence for past recombination events between paralogues and diversifying selection, particularly in the C-terminal half of the LRR domain. Variants selected for complete or partial loss of Rp1-D resistance can be explained by unequal crossing over that occurred mostly within coding regions. The Rp1-D gene is altered or lost in all variants, the recombination breakpoints occur throughout the genes, and most recombinant events (9/14 examined) involved the same untranscribed paralogue with the Rp1-D gene. One recombinant with a complete LRR from Rp1-D, but the amino-terminal portion from another homologue, conferred the Rp1-D specificity but with a reduced level of resistance.

Sequence haplotypes revealed by sequence-tagged site fine mapping of the Ror1 gene in the centromeric region of barley chromosome 1H.

We describe the development of polymerase chain reaction-based, sequence-tagged site (STS) markers for fine mapping of the barley (Hordeum vulgare) Ror1 gene required for broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. hordei). After locating Ror1 to the centromeric region of barley chromosome 1H using a combined amplified fragment length polymorphism/restriction fragment-length polymorphism (RFLP) approach, sequences of RFLP probes from this chromosome region of barley and corresponding genome regions from the related grass species oat (Avena spp.), wheat, and Triticum monococcum were used to develop STS markers. Primers based on the RFLP probe sequences were used to polymerase chain reaction-amplify and directly sequence homologous DNA stretches from each of four parents that were used for mapping. Over 28,000 bp from 22 markers were compared. In addition to one insertion/deletion of at least 2.0 kb, 79 small unique sequence polymorphisms were observed, including 65 single nucleotide substitutions, two dinucleotide substitutions, 11 insertion/deletions, and one 5-bp/10-bp exchange. The frequency of polymorphism between any two barley lines ranged from 0.9 to 3.0 kb, and was greatest for comparisons involving an Ethiopian landrace. Haplotype structure was observed in the marker sequences over distances of several hundred basepairs. Polymorphisms in 16 STSs were used to generate genetic markers, scored by restriction enzyme digestion or by direct sequencing. Over 2,300 segregants from three populations were used in Ror1 linkage analysis, mapping Ror1 to a 0.2- to 0.5-cM marker interval. We discuss the implications of sequence haplotypes and STS markers for the generation of high-density maps in cereals.

The isolation and mapping of disease resistance gene analogs in maize.

Many of the plant disease resistance genes that have been isolated encode proteins with a putative nucleotide binding site and leucine-rich repeats (NBS-LRR resistance genes). Oligonucleotide primers based on conserved motifs in and around the NBS of known NBS-LRR resistance proteins were used to amplify sequences from maize genomic DNA by polymerase chain reaction (PCR). Eleven classes of non-cross-hybridizing sequences were obtained that had predicted products with high levels of amino acid identity to NBS-LRR resistance proteins. These maize resistance gene analogs (RGAs) and one RGA clone obtained previously from wheat were used as probes to map 20 restriction fragment length polymorphism (RFLP) loci in maize. Some RFLPs were shown to map to genomic regions containing virus and fungus resistance genes. Perfect cosegregation was observed between RGA loci and the rust resistance loci rp1 and rp3. The RGA probe associated with rp1 also detected deletion events in several rp1 mutants. These data strongly suggest that some of the RGA clones may hybridize to resistance genes.

The Yd2 gene for barley yellow dwarf virus resistance maps close to the centromere on the long arm of barley chromosome 3.

Barley yellow dwarf luteovirus (BYDV) causes serious yield losses in all cereals worldwide. The Yd2 gene from a number of Ethiopian barleys (Hordeum vulgare L.) has been the most effective means of providing resistance against BYDV in cultivated barley. Isolation of the Yd2 gene will enable characterisation of the molecular basis of the Yd2-BYDV interaction. This paper describes the first stage in a project to isolate the gene: the construction of a detailed linkage map of the Yd2 region. The map encompasses 27.6 centiMorgans (cM) of chromosome 3 and contains 19 RFLPs, 2 morphological marker loci, the centromere and Yd2. In the mapping population of 106 F2 individuals, Yd2 perfectly cosegregated with the RFLP loci Xwg889 and XYlp, which were located on the long arm, 0.5 cM from the centromere. The two morphological marker loci, uzu dwarfand white stripe j, both mapped distal to Yd2. The protein product of the gene at the XYlp locus will provide a convenient assay for the selection of Yd2 during the breeding of BYDV-resistant barley varieties.