Optical and physical mapping with local finishing enables megabase-scale resolution of agronomically important regions in the wheat genome.

BACKGROUND: Numerous scaffold-level sequences for wheat are now being released and, in this context, we report on a strategy for improving the overall assembly to a level comparable to that of the human genome. RESULTS: Using chromosome 7A of wheat as a model, sequence-finished megabase-scale sections of this chromosome were established by combining a new independent assembly using a bacterial artificial chromosome (BAC)-based physical map, BAC pool paired-end sequencing, chromosome-arm-specific mate-pair sequencing and Bionano optical mapping with the International Wheat Genome Sequencing Consortium RefSeq v1.0 sequence and its underlying raw data. The combined assembly results in 18 super-scaffolds across the chromosome. The value of finished genome regions is demonstrated for two approximately 2.5 Mb regions associated with yield and the grain quality phenotype of fructan carbohydrate grain levels. In addition, the 50 Mb centromere region analysis incorporates cytological data highlighting the importance of non-sequence data in the assembly of this complex genome region. CONCLUSIONS: Sufficient genome sequence information is shown to now be available for the wheat community to produce sequence-finished releases of each chromosome of the reference genome. The high-level completion identified that an array of seven fructosyl transferase genes underpins grain quality and that yield attributes are affected by five F-box-only-protein-ubiquitin ligase domain and four root-specific lipid transfer domain genes. The completed sequence also includes the centromere.

Diversity analysis of cotton (Gossypium hirsutum L.) germplasm using the CottonSNP63K Array.

BACKGROUND: Cotton germplasm resources contain beneficial alleles that can be exploited to develop germplasm adapted to emerging environmental and climate conditions. Accessions and lines have traditionally been characterized based on phenotypes, but phenotypic profiles are limited by the cost, time, and space required to make visual observations and measurements. With advances in molecular genetic methods, genotypic profiles are increasingly able to identify differences among accessions due to the larger number of genetic markers that can be measured. A combination of both methods would greatly enhance our ability to characterize germplasm resources. Recent efforts have culminated in the identification of sufficient SNP markers to establish high-throughput genotyping systems, such as the CottonSNP63K array, which enables a researcher to efficiently analyze large numbers of SNP markers and obtain highly repeatable results. In the current investigation, we have utilized the SNP array for analyzing genetic diversity primarily among cotton cultivars, making comparisons to SSR-based phylogenetic analyses, and identifying loci associated with seed nutritional traits. RESULTS: The SNP markers distinctly separated G. hirsutum from other Gossypium species and distinguished the wild from cultivated types of G. hirsutum. The markers also efficiently discerned differences among cultivars, which was the primary goal when designing the CottonSNP63K array. Population structure within the genus compared favorably with previous results obtained using SSR markers, and an association study identified loci linked to factors that affect cottonseed protein content. CONCLUSIONS: Our results provide a large genome-wide variation data set for primarily cultivated cotton. Thousands of SNPs in representative cotton genotypes provide an opportunity to finely discriminate among cultivated cotton from around the world. The SNPs will be relevant as dense markers of genome variation for association mapping approaches aimed at correlating molecular polymorphisms with variation in phenotypic traits, as well as for molecular breeding approaches in cotton.

Cellular and molecular characterization of a stem rust resistance locus on wheat chromosome 7AL.

BACKGROUND: Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a major wheat disease which is mainly controlled through the release of resistant cultivars containing one or several resistance genes. Considerable effort has been put into the discovery of new resistance genes, but knowledge of their mechanisms of action is often lacking. In this study, the mechanism of resistance conferred by a recently discovered stem rust resistance locus on wheat chromosome 7AL was investigated through microscopic observations and RNA-sequencing, using the susceptible line Columbus and the independent, backcrossed, resistant lines Columbus-NS765 and Columbus-NS766. RESULTS: Microscopic observations of infected leaves revealed that the resistance conferred by the 7AL resistance locus was initiated 2 days post-inoculation, upon the fungus entry into the plant through the stoma. Resistance was manifested by death of guard and epidermal cells adjacent to an infection site. Occasionally, similar observations were made in the susceptible line, suggesting that the resistance response was the same in all genotypes, but enhanced in the resistant lines. Transcriptomic analysis, combined with assignment of genes to wheat chromosomes, revealed a disproportionately high number of differentially expressed genes were located on chromosomes 7AL and 6A. A number of genes annotated as cysteine-rich receptor-like kinases were located on chromosome 7AL. Closer investigation indicated that the encoded proteins were in fact putative receptor-like cytoplasmic kinases. One of the putative RLCK genes contained a SNP marker previously shown to co-segregate with the 7AL resistance locus. The results also indicated the presence of a large introgression on chromosome 6A in both resistant lines, but whether it has any role in the resistance response is unclear. CONCLUSIONS: This study represents the first investigation on the resistance mechanism conferred by the wheat 7AL stem rust resistance locus. The resistance response was associated with pre-haustorial cell death, and the transcriptome analysis suggested putative receptor-like cytoplasmic kinases as candidate resistance genes for further investigation.

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, ß-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include ß-1,4-glucosidases, ß-1,4-glucanases, ß-1,4-galactanases, a ß-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade ß-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of ß-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

miRNA regulation in the early development of barley seed.

BACKGROUND: During the early stages of seed development many genes are under dynamic regulation to ensure the proper differentiation and establishment of the tissue that will constitute the mature grain. To investigate how miRNA regulation contributes to this process in barley, a combination of small RNA and mRNA degradome analyses were used to identify miRNAs and their targets. RESULTS: Our analysis identified 84 known miRNAs and 7 new miRNAs together with 96 putative miRNA target genes regulated through a slicing mechanism in grain tissues during the first 15 days post anthesis. We also identified many potential miRNAs including several belonging to known miRNA families. Our data gave us evidence for an increase in miRNA-mediated regulation during the transition between pre-storage and storage phases. Potential miRNA targets were found in various signalling pathways including components of four phytohormone pathways (ABA, GA, auxin, ethylene) and the defence response to powdery mildew infection. Among the putative miRNA targets we identified were two essential genes controlling the GA response, a GA3oxidase1 and a homolog of the receptor GID1, and a homolog of the ACC oxidase which catalyses the last step of ethylene biosynthesis. We found that two MLA genes are potentially miRNA regulated, establishing a direct link between miRNAs and the R gene response. CONCLUSION: Our dataset provides a useful source of information on miRNA regulation during the early development of cereal grains and our analysis suggests that miRNAs contribute to the control of development of the cereal grain, notably through the regulation of phytohormone response pathways.

Evolution of care pathway for laparoscopic radical prostatectomy.

PURPOSE: We report results of the introduction of a laparoscopic radical prostatectomy (LRP) care pathway. This included the introduction of a transversus abdominis plane (TAP) local anesthetic block and other measures to reduce the impact of factors known to delay postoperative recovery. Outcomes including pain, analgesic requirements, complications, and length of stay are reported. PATIENTS AND METHODS: Two hundred consecutive patients undergoing LRP from 2008 to 2010 were prospectively studied. A detailed perioperative care pathway was developed and implemented. The pathway was modified after a pain audit to include bilateral transversus abdominis plane regional anesthetic blockade. Same day discharge criteria were applied to suitable patients. Demographics and perioperative and follow-up data were prospectively collected and recorded on a database. RESULTS: Overall, 78% of cases were discharged after 1 night stay; 14 patients were managed as true day cases without overnight stay. Operative time (P<0.0001), intraoperative blood loss (P=0.018), %≤ 1 day stay (P=0.0091), transfusion, and conversion rate (nil in latter 100 cases) all improved significantly in the second 100 group of patients compared with the first 100 cases. The introduction of TAP blocks led to significant reductions of mean intraoperative and postoperative opiate use (17.3 mg to 1.3 mg and 1.9 mg to 0.2 mg morphine, respectively) without any significant effect on perceived pain. True day cases did not experience a significantly different rate of complications than the whole cohort. CONCLUSIONS: Through a structured care pathway incorporating the TAP block, 1 night stay laparoscopic prostatectomy can be safely delivered with reduced inpatient stay costs. In selected patients, day-case prostatectomy is feasible.

Serbia: a country and a Cardiology Society formed in 2006.

The Cardiology Society of Serbia is at the centre of the country's progress in cardiovascular disease.