Cytological and molecular characterization of wheat lines carrying leaf rust and stem rust resistance genes Lr24 and Sr24.

Previous studies showed that Australian wheat cultivars Janz and Sunco carry leaf rust and stem rust resistance genes Lr24 and Sr24 derived from Thinopyrum ponticum chromosome arm 3AgL. However, the size of the alien segments carrying Lr24 and Sr24 in the lines were not determined. In this study, we used non-denaturing fluorescence in situ hybridization (ND-FISH), genomic in situ hybridization (GISH), and PCR-based landmark unique gene (PLUG) markers to visualize the alien segments in Janz and Sunco, and further compared them with the segments in US cultivars Agent and Amigo. The fraction length (FL) of the alien translocation in Agent was 0.70-1.00, whereas those in Janz, Sunco, and Amigo were smaller, at FL 0.85-1.00. It was deduced that the alien gene RAg encoding for red grain color and rust resistance genes Lr24 and Sr24 on chromosome arm 3AgL were in bins of FL 0.70-0.85 and 0.85-1.00, respectively. We retrieved and extracted nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor genes corresponding to the region of Lr24 and Sr24 on chromosomes 3E, and 3J, 3Js and 3St from the reference genome sequences of Th. elongatum and Th. intermedium, respectively. A set of molecular markers developed for Lr24 and Sr24 from those extracted NBS-LRR genes will provide valuable information for fine mapping and cloning of these genes.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

Stem rust resistance in wheat is suppressed by a subunit of the mediator complex.

Stem rust is an important disease of wheat that can be controlled using resistance genes. The gene SuSr-D1 identified in cultivar 'Canthatch' suppresses stem rust resistance. SuSr-D1 mutants are resistant to several races of stem rust that are virulent on wild-type plants. Here we identify SuSr-D1 by sequencing flow-sorted chromosomes, mutagenesis, and map-based cloning. The gene encodes Med15, a subunit of the Mediator Complex, a conserved protein complex in eukaryotes that regulates expression of protein-coding genes. Nonsense mutations in Med15b.D result in expression of stem rust resistance. Time-course RNAseq analysis show a significant reduction or complete loss of differential gene expression at 24 h post inoculation in med15b.D mutants, suggesting that transcriptional reprogramming at this time point is not required for immunity to stem rust. Suppression is a common phenomenon and this study provides novel insight into suppression of rust resistance in wheat.

Delivery of cardiac resynchronization therapy via the left inferior phrenic vein: a case report.

BACKGROUND: The successful implantation of cardiac resynchronization therapy (CRT) may be prevented by anatomical variations that preclude the delivery of clinically effective left ventricular (LV) pacing from within the coronary sinus (CS) or its tributaries. Failure of lead delivery, suboptimal LV capture thresholds, or intractable phrenic nerve capture with accompanying diaphragmatic twitch is often encountered. Commonly employed alternative approaches to LV lead delivery, including epicardial, trans-septal, or transapical pacing are associated with significant morbidity. CASE SUMMARY: A 74-year-old man with ischaemic heart disease, prior mitral valve repair, long-standing atrial fibrillation, and severe symptomatic LV systolic dysfunction, underwent single chamber pacemaker upgrade to a CRT defibrillator. It was found not to be possible to place a CS lead during the procedure. Biventricular pacing was accomplished by the delivery of a pacing lead through the left inferior phrenic vein (LIPV). Satisfactory LV capture thresholds were obtained with the avoidance of clinically significant diaphragmatic stimulation. Following implantation, a marked clinical response to treatment was observed with improvement in both heart failure symptoms and LV ejection fraction. DISCUSSION: The LIPV is known to drain into the inferior vena cava in around one-third of examined subjects. In these individuals, LV lead delivery through the LIPV may provide an alternate route for the delivery of resynchronization therapy. This approach to the implantation of CRT may be considered when pacing via the CS or its branches are not achievable.

Chromosome Engineering Techniques for Targeted Introgression of Rust Resistance from Wild Wheat Relatives.

Hexaploid wheat has relatively narrow genetic diversity due to its evolution and domestication history compared to its wild relatives that often carry agronomically important traits including resistance to biotic and abiotic stresses. Many genes have been introgressed into wheat from wild relatives using various strategies and protocols. One of the important issues with these introgressions is linkage drag, i.e., in addition to beneficial genes, undesirable or deleterious genes that negatively influence end-use quality and grain yield are also introgressed. Linkage drag is responsible for limiting the use of alien genes in breeding programs. Therefore, a lot of effort has been devoted to reduce linkage drag. If a gene of interest is in the primary gene pool or on a homologous chromosome from species in the secondary gene pool, it can be introgressed into common wheat by direct crosses and homologous recombination. However, if a gene of interest is on a homoeologous chromosome of a species belonging to the secondary or tertiary gene pools, chromosome engineering is required to make the transfer and to break any linkage drag. Four general approaches are used to transfer genes from homoeologous chromosomes of wild relatives to wheat chromosomes, namely, spontaneous translocations, radiation, tissue culture, and induced homoeologous recombination. The last is the method of choice provided the target gene(s) is not located near the centromere where recombination is lacking or is suppressed, and synteny between the alien chromosome carrying the gene and the recipient wheat chromosome is conserved. In this chapter, we focus on the homoeologous recombination-based chromosome engineering approach and use rust resistance genes in wild relatives of wheat as examples. The methodology will be applicable to other alien genes and other crops.

Complementary resistance genes in wheat selection 'Avocet R' confer resistance to stripe rust.

KEY MESSAGE: Complementary genes for resistance to wheat stripe rust in an Avocet selection mapped to chromosome arms 3DL and 5BL. Susceptible Avocet selections lacked the 5BL gene due to a chromosomal deletion. This study reports the inheritance and genetic mapping of the YrA (temporary name of convenience to describe the specificity) seedling resistance to wheat stripe rust (caused by Puccinia striiformis f. sp. tritici; Pst) in a resistant selection of the Australian cv. Avocet [Avocet R (AvR)-AUS 90660]. Genetic analysis was performed on F2 populations and F3 generation families from crosses between wheats that carried and lacked the YrA resistance. Greenhouse seedling tests with two avirulent Pst pathotypes (104 E137 A- and 108 E141 A-) confirmed that the YrA resistance was inherited as two complementary dominant genes. Ninety-two doubled haploid (DH) lines from a cross between the Australian cv. Teal (Pst susceptible) and AvR were used for DArT-Seq genotypic analysis to map the seedling resistance. Marker-trait association analysis using 9035 DArT-Seq loci mapped the genes to the long arms of chromosomes 3D (3DL) and 5B (5BL), respectively. F2 populations from crosses between susceptible DH lines that carried either the 3DL or 5BL marker genotypes confirmed the complementary gene model. Fluorescence in situ hybridization (FISH) analysis determined that Teal carries a reciprocal T5B-7B translocation. FISH analysis also identified a 5BL chromosomal deletion in Avocet S relative to AvR that further validated the complementary gene model and possibly explained the heterogeneity of closely related wheats carrying the YrA resistance. The individual loci of the complementary YrA resistance were designated Yr73 (3DL) and Yr74 (5BL). Candidate single gene reference stocks will be permanently accessioned following cytological analysis to avoid the T5B-7B translocation.

The relationship of leaf rust resistance gene Lr13 and hybrid necrosis gene Ne2m on wheat chromosome 2BS.

KEY MESSAGE: Genetic and mutational analyses of wheat leaf rust resistance gene Lr13 and hybrid necrosis gene Ne2 m indicated that they are the same gene. Hybrid necrosis in wheat characterized by chlorosis and eventual necrosis of plant tissues in certain wheat hybrids is controlled by the interaction of complementary dominant genes Ne1 and Ne2 located on chromosome arms 5BL and 2BS, respectively. Multiple alleles at each locus can be identified by differences in necrotic phenotypes when varieties are crossed with a fixed accession of the other genotype. Some of at least five Ne2 alleles were described as s (strong), m (medium) and w (weak); alleles of Ne1 were similarly described. Ne2m causes moderate necrosis in hybrids with genotypes having Ne1s. Ne2 is located on chromosome arm 2BS in close proximity to Lr13. Most wheat lines with Ne2m carry Lr13, and all wheat lines with Lr13 appear to carry Ne2m. To further dissect the relationship between Lr13 and Ne2m, more than 350 crosses were made between cv. Spica (Triticum aestivum) or Kubanka (T. durum) carrying Ne1s and recombinant inbred lines or doubled haploid lines from three crosses segregating for Lr13. F1 plants from lines carrying Lr13 crossed with Spica (Ne1s) always showed progressive necrosis; those lacking Lr13 did not. Four wheat cultivars/lines carrying Lr13 were treated with the mutagen EMS. Thirty-five susceptible mutants were identified; eight were distinctly less glaucous and late maturing indicative of chromosome 2B or sub-chromosome loss. Hybrids of phenotypically normal Lr13 mutant plants crossed with Spica did not produce symptoms of hybrid necrosis. Thus, Lr13 and one particular Ne2m allele may be the same gene.

The powdery mildew resistance gene Pm8 derived from rye is suppressed by its wheat ortholog Pm3.

The powdery mildew resistance gene Pm8 derived from rye is located on a 1BL.1RS chromosome translocation in wheat. However, some wheat lines with this translocation do not show resistance to isolates of the wheat powdery mildew pathogen avirulent to Pm8 due to an unknown genetically dominant suppression mechanism. Here we show that lines with suppressed Pm8 activity contain an intact and expressed Pm8 gene. Therefore, the absence of Pm8 function in certain 1BL.1RS-containing wheat lines is not the result of gene loss or mutation but is based on suppression. The wheat gene Pm3, an ortholog of rye Pm8, suppressed Pm8-mediated powdery mildew resistance in lines containing Pm8 in a transient single-cell expression assay. This result was further confirmed in transgenic lines with combined Pm8 and Pm3 transgenes. Expression analysis revealed that suppression is not the result of gene silencing, either in wheat 1BL.1RS translocation lines carrying Pm8 or in transgenic genotypes with both Pm8 and Pm3 alleles. In addition, a similar abundance of the PM8 and PM3 proteins in single or double homozygous transgenic lines suggested that a post-translational mechanism is involved in suppression of Pm8. Co-expression of Pm8 and Pm3 genes in Nicotiana benthamiana leaves followed by co-immunoprecipitation analysis showed that the two proteins interact. Therefore, the formation of a heteromeric protein complex might result in inefficient or absent signal transmission for the defense reaction. These data provide a molecular explanation for the suppression of resistance genes in certain genetic backgrounds and suggest ways to circumvent it in future plant breeding.

Tissue doppler-derived contractile reserve is a simple and strong predictor of cardiopulmonary exercise performance across a range of cardiac diseases.

BACKGROUND: Resting echocardiographic measures of cardiac function such as left ventricular ejection fraction correlate poorly with exercise capacity. Assessment during exercise using measures less dependent on hemodynamic loading conditions, such as tissue Doppler imaging (TDI), may more accurately characterize the relationship between cardiac function and exercise capacity. METHODS AND RESULTS: One hundred one subjects with various cardiac diagnoses underwent exercise stress echocardiography with simultaneous cardiopulmonary gas exchange analysis. Standard two-dimensional, Doppler and spectral TDI parameters were assessed at both rest and peak exercise. Across all subjects the strongest relationship with peak oxygen uptake (pVO2 ) was with peak left ventricular systolic tissue velocity (S') during exercise (r = 0.84, P < 0.001). The strength of the relationship was greater than that observed with any other common echocardiographic measure of systolic or diastolic cardiac function. CONCLUSION: There is a very strong relationship between measurements of S' during exercise and exercise capacity. The previously observed poor correlation with standard measures of systolic and diastolic cardiac function may be explained both by the load dependence of parameters such as ejection fraction and by reliance on resting as opposed to exercise assessment.

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Genetic association of crown rust resistance gene Pc68, storage protein loci, and resistance gene analogues in oats.

Segregating F(3) families, derived from a cross between oat cultivar Swan and the putative single gene line PC68, were used to determine the association of seed storage protein loci and resistance gene analogues (RGAs) with the crown rust resistance gene Pc68. SDS-PAGE analysis detected three avenin loci, AveX, AveY, and AveZ, closely linked to Pc68. Their diagnostic alleles are linked in coupling to Pc68 and were also detected in three additional lines carrying Pc68. Another protein locus was linked in repulsion to Pc68. In complementary studies, three wheat RGA clones (W2, W4, and W10) detected restriction fragment length polymorphisms (RFLPs) between homozygous resistant and homozygous susceptible F(3) DNA bulks. Four oat homologues of W2 were cloned and sequenced. RFLPs detected with two of them were mapped using F(3) and F(4) populations. Clone 18 detected a locus, Orga2, linked in repulsion to Pc68. Clone 22 detected several RFLPs including Orga1 (the closest locus to Pc68) and three RGA loci (Orga22-2, Orga22-3, and Orga22-4) loosely linked to Pc68. The diagnostic RFLPs linked in coupling to Pc68 were detected by clone 22 in three additional oat lines carrying Pc68 and have potential utility in investigating and improving crown rust resistance of oat.

Rye-derived powdery mildew resistance gene Pm8 in wheat is suppressed by the Pm3 locus.

Genetic suppression of disease resistance is occasionally observed in hexaploid wheat or in its interspecific crosses. The phenotypic effects of genes moved to wheat from relatives with lower ploidy are often smaller than in the original sources, suggesting the presence of modifiers or partial inhibitors in wheat, especially dilution effects caused by possible variation at orthologous loci. However, there is little current understanding of the underlying genetics of suppression. The discovery of suppression in some wheat genotypes of the cereal rye chromosome 1RS-derived gene Pm8 for powdery mildew resistance offered an opportunity for analysis. A single gene for suppression was identified at or near the closely linked storage protein genes Gli-A1 and Glu-A3, which are also closely associated with the Pm3 locus on chromosome 1AS. The Pm3 locus is a complex of expressed alleles and pseudogenes embedded among Glu-A3 repeats. In the current report, we explain why earlier work indicated that the mildew suppressor was closely associated with specific Gli-A1 and Glu-A3 alleles, and predict that suppression of Pm8 involves translated gene products from the Pm3 locus.

Wheat stripe rust resistance genes Yr5 and Yr7 are allelic.

Stripe rust is one of the most destructive diseases of wheat. Breeding for resistance is the most economical and environmentally acceptable means to control stripe rust. Genetic studies on resistance sources are very important. Previous inheritance studies on Triticum aestivum subsp. spelta cv. album and wheat cultivar Lee showed that each possessed a single dominant gene for stripe rust resistance, i.e., Yr5 and Yr7, respectively. Both were located on the long arm of chromosome 2B, but due to the complexities caused by genetic background effects there was no clear evidence on the allelism or linkage status of these genes. Our study, involving an intercross of Avocet S near-isogenic lines possessing the genes, provided clear evidence for allelism or extremely close linkage of Yr5 and Yr7 based on phenotypic and molecular studies.