Fine Mapping and Candidate Gene Analysis of Pm36, a Wild Emmer-Derived Powdery Mildew Resistance Locus in Durum Wheat.

Powdery mildew (PM) is an economically important foliar disease of cultivated cereals worldwide. The cultivation of disease-resistant varieties is considered the most efficient, sustainable and economical strategy for disease management. The objectives of the current study were to fine map the chromosomal region harboring the wild emmer PM resistance locus Pm36 and to identify candidate genes by exploiting the improved tetraploid wheat genomic resources. A set of backcross inbred lines (BILs) of durum wheat were genotyped with the SNP 25K chip array and comparison of the PM-resistant and susceptible lines defined a 1.5 cM region (physical interval of 1.08 Mb) harboring Pm36. The genetic map constructed with F2:3 progenies derived by crossing the PM resistant line 5BIL-42 and the durum parent Latino, restricted to 0.3 cM the genetic distance between Pm36 and the SNP marker IWB22904 (physical distance 0.515 Mb). The distribution of the marker interval including Pm36 in a tetraploid wheat collection indicated that the positive allele was largely present in the domesticated and wild emmer Triticum turgidum spp. dicoccum and ssp. dicoccoides. Ten high-confidence protein coding genes were identified in the Pm36 region of the emmer, durum and bread wheat reference genomes, while three added genes showed no homologous in the emmer genome. The tightly linked markers can be used for marker-assisted selection in wheat breeding programs, and as starting point for the Pm36 map-based cloning.

Mapping Powdery Mildew (Blumeria graminis f. sp. tritici) Resistance in Wild and Cultivated Tetraploid Wheats.

Wheat is the most widely grown crop and represents the staple food for one third of the world's population. Wheat is attacked by a large variety of pathogens and the use of resistant cultivars is an effective and environmentally safe strategy for controlling diseases and eliminating the use of fungicides. In this study, a collection of wild and cultivated tetraploid wheats (Triticum turgidum) were evaluated for seedling resistance (SR) and adult plant resistance (APR) to powdery mildew (Blumeria graminis) and genotyped with a 90K single nucleotide polymorphism (SNP) array to identify new sources of resistance genes. The genome-wide association mapping detected 18 quantitative trait loci (QTL) for APR and 8 QTL for SR, four of which were identical or at least closely linked to four QTL for APR. Thirteen candidate genes, containing nucleotide binding sites and leucine-rich repeats, were localized in the confidence intervals of the QTL-tagging SNPs. The marker IWB6155, associated to QPm.mgb-1AS, was located within the gene TRITD1Av1G004560 coding for a disease resistance protein. While most of the identified QTL were described previously, five QTL for APR (QPm.mgb-1AS, QPm.mgb-2BS, QPm.mgb-3BL.1, QPm.mgb-4BL, QPm.mgb-7BS.1) and three QTL for SR (QPm.mgb-3BL.3, QPm.mgb-5AL.2, QPm.mgb-7BS.2) were mapped on chromosome regions where no resistance gene was reported before. The novel QTL/genes can contribute to enriching the resistance sources available to breeders.

Evaluation of the use of sonication of retrieved implants for the diagnosis of prosthetic joint infection in a routine setting.

In order to evaluate the usefulness of sonication of retrieved implants for the diagnosis of prosthetic joint infection (PJI) in a large group of patients in a routine setting, we designed a 3-year retrospective study. Patients were classified into two groups: those meeting the clinical criteria of PJI and those that did not (control group). Two hundred patients and 276 samples were included. The types of infection were early (n = 44), delayed (n = 53), positive intraoperative cultures (n = 13) and late-acute (n = 8). The culture sensitivities of sonicate fluid, periprosthetic tissue, synovial fluid and combination of periprosthetic tissue and/or synovial fluid were 69.5, 52.8, 54.8 and 60.2%, respectively. The specificities were 97.6, 90.3, 93.0 and 89.9%, respectively. Sonicate fluid culture of implants was more sensitive than peri-implant tissue, synovial fluid and combination of periprosthetic tissue and/or synovial fluid for all infection types, though it was especially useful in delayed infection: 91.3% vs. 60.0% (p = 0.0015), 63.2% (p = 0.0005) and 66.7% (p = 0.0001), respectively. When sonicate fluid culture of implants was performed in addition to conventional cultures, the sensitivity increased significantly in total (from 60.2 to 77.1%) and delayed PJI (from 45.1 to 71.7%). On the other hand, for early PJI, sonicate fluid culture of prosthesis was not superior to conventional diagnostic methods.

Genetic Mapping of Loci for Resistance to Stem Rust in a Tetraploid Wheat Collection.

Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a major biotic constraint to wheat production worldwide. Disease resistant cultivars are a sustainable means for the efficient control of this disease. To identify quantitative trait loci (QTLs) conferring resistance to stem rust at the seedling stage, an association mapping panel consisting of 230 tetraploid wheat accessions were evaluated for reaction to five Pgt races under greenhouse conditions. A high level of phenotypic variation was observed in the panel in response to all of the races, allowing for genome-wide association mapping of resistance QTLs in wild, landrace, and cultivated tetraploid wheats. Twenty-two resistance QTLs were identified, which were characterized by at least two marker-trait associations. Most of the identified resistance loci were coincident with previously identified rust resistance genes/QTLs; however, six regions detected on chromosomes 1B, 5A, 5B, 6B, and 7B may be novel. Availability of the reference genome sequence of wild emmer wheat accession Zavitan facilitated the search for candidate resistance genes in the regions where QTLs were identified, and many of them were annotated as NOD (nucleotide binding oligomerization domain)-like receptor (NLR) genes or genes related to broad spectrum resistance.

The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments.

BACKGROUND: In plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding. RESULTS: Twenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait. CONCLUSIONS: The availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

Evaluation of a commercial multiplex PCR (Unyvero i60®) designed for the diagnosis of bone and joint infections using prosthetic-joint sonication.

BACKGROUND: The development of sonication protocols over the last few years has improved the sensitivity of conventional cultures for the diagnosis of prosthetic-joint infection (PJI). However, the development of a new, specifically designed kit for the molecular diagnosis of PJI could provide a major improvement in this field. METHODS: Prostheses retrieved from patients who underwent implant removal from May 2014 to May 2015 were sent for culture, and processed according to a previously defined protocol that included sonication. Furthermore, 180 microlitres of sonication fluid were used to carry out the multiplex PCR test (Unyvero i60 system®). A comparison of the sensitivity, specificity, positive (PPV) and negative (NPV) predictive value, was performed. The study was approved by the Clinical Research Ethics Committee. RESULTS: The analysis included 88 prostheses from 68 patients (1.29 prostheses/patient). The type of prostheses studied were knee (n=55), total hip (n=26), partial hip (n=5), and shoulder (n=2). Twenty-nine patients were diagnosed with a PJI (15 delayed, 12 acute, and 2 haematogenous infections). In 24 cases, the result of the PCR was positive, all but 1 corresponding to patients with clinical criteria of PJI. Nine resistance mechanisms were detected from 5 samples. The Unyvero i60 system® showed slightly better results than traditional culture in terms of specificity and PPV. CONCLUSIONS: The Unyvero i60 system® may play a role in rapid diagnosis of PJI, due to its high specificity and PPV. However, despite these results, cultures have to be performed to detect organisms not detected by the system.

Development of a High-Density SNP-Based Linkage Map and Detection of QTL for ß-Glucans, Protein Content, Grain Yield per Spike and Heading Time in Durum Wheat.

High-density genetic linkage maps of crop species are particularly useful in detecting qualitative and quantitative trait loci for important agronomic traits and in improving the power of classical approaches to identify candidate genes. The aim of this study was to develop a high-density genetic linkage map in a durum wheat recombinant inbred lines population (RIL) derived from two elite wheat cultivars and to identify, characterize and correlate Quantitative Trait Loci (QTL) for ß-glucan, protein content, grain yield per spike and heading time. A dense map constructed by genotyping the RIL population with the wheat 90K iSelect array included 5444 single nucleotide polymorphism (SNP) markers distributed in 36 linkage groups. Data for ß-glucan and protein content, grain yield per spike and heading time were obtained from replicated trials conducted at two locations in southern Italy. A total of 19 QTL were detected in different chromosome regions. In particular, three QTL for ß-glucan content were detected on chromosomes 2A and 2B (two loci); eight QTL controlling grain protein content were detected on chromosomes 1B, 2B, 3B (two loci), 4A, 5A, 7A and 7B; seven QTL for grain yield per spike were identified on chromosomes 1A, 2B, 3A (two loci), 3B (two loci) and 6B; and one marker-trait association was detected on chromosome 2A for heading time. The last was co-located with a ß-glucan QTL, and the two QTL appeared to be negatively correlated. A genome scan for genomic regions controlling the traits and SNP annotated sequences identified five putative candidate genes involved in different biosynthesis pathways (ß-glucosidase, GLU1a; APETALA2, TaAP2; gigantea3, TaGI3; 14-3-3 protein, Ta14A; and photoperiod sensitivity, Ppd-A1). This study provides additional information on QTL for important agronomic traits that could be useful for marker-assisted breeding to obtain new genotypes with commercial and nutritional relevance.

DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.

BACKGROUND: Durum wheat (Triticum turgidum L.) is a cereal crop widely grown in the Mediterranean regions; the amber grain is mainly used for the production of pasta, couscous and typical breads. Single nucleotide polymorphism (SNP) detection technologies and high-throughput mutation induction represent a new challenge in wheat breeding to identify allelic variation in large populations. The TILLING strategy makes use of traditional chemical mutagenesis followed by screening for single base mismatches to identify novel mutant loci. Although TILLING has been combined to several sensitive pre-screening methods for SNP analysis, most rely on expensive equipment. Recently, a new low cost and time saving DHPLC protocol has been used in molecular human diagnostic to detect unknown mutations. RESULTS: In this work, we developed a new durum wheat TILLING population (cv. Marco Aurelio) using 0.70-0.85% ethyl methane sulfonate (EMS). To investigate the efficiency of the mutagenic treatments, a pilot screening was carried out on 1,140 mutant lines focusing on two target genes (Lycopene epsilon-cyclase, ε-LCY, and Lycopene beta-cyclase, ß-LCY) involved in carotenoid metabolism in wheat grains. We simplify the heteroduplex detection by two low cost methods: the enzymatic cleavage (CelI)/agarose gel technique and the denaturing high-performance liquid chromatography (DHPLC). The CelI/agarose gel approach allowed us to identify 31 mutations, whereas the DHPLC procedure detected a total of 46 mutations for both genes. All detected mutations were confirmed by direct sequencing. The estimated overall mutation frequency for the pilot assay by the DHPLC methodology resulted to be of 1/77 kb, representing a high probability to detect interesting mutations in the target genes. CONCLUSION: We demonstrated the applicability and efficiency of a new strategy for the detection of induced variability. We produced and characterized a new durum wheat TILLING population useful for a better understanding of key gene functions. The availability of this tool together with TILLING technique will expand the polymorphisms in candidate genes of agronomically important traits in wheat.

Cell wall traits as potential resources to improve resistance of durum wheat against Fusarium graminearum.

BACKGROUND: Fusarium graminearum, one of the causal agents of Fusarium Head Blight (FHB, scab), leads to severe losses in grain yield and quality due to the production of mycotoxins which are harmful to human and livestock. Different traits for FHB resistance in wheat were identified for common wheat (Triticum aestivum L.) while the sources of FHB resistance in durum wheat (Triticum turgidum ssp. Durum), one of the cereals most susceptible to F. graminearum infection, have not been found. New lines of evidence indicate that content and composition of cell wall polymers affect the susceptibility of the wall to degrading enzymes produced by pathogens during infection and can play a role in the outcome of host-pathogen interactions. The objective of our research is to identify potential cell wall biochemical traits linked to Fusariosis resistance to be transferred from a resistant common wheat to a susceptible durum wheat line. RESULTS: A detailed analysis of cell wall composition in spikes isolated from a highly resistant common wheat accession "02-5B-318", a breeding line derived from the FHB-resistant Chinese cv. Sumai-3 and a high susceptible durum wheat cv. Saragolla was performed. Significant differences in lignin monolignols composition, arabinoxylan (AX) substitutions and pectin methylesterification were found between resistant and susceptible plants. We isolated and characterized a pectin methylesterase gene WheatPME1, which we found being down regulated in the FHB-resistant line and induced by fungal infection in the susceptible wheat. CONCLUSIONS: Our results indicate cell wall traits differing between the FHB sensitive and resistant wheat genotypes, possibly related to FHB-resistance, and identify the line 02-5B-318R as a potential resource of such traits. Evidence suggests that WheatPME1 is involved in wheat response to F. graminearum.

A high-density, SNP-based consensus map of tetraploid wheat as a bridge to integrate durum and bread wheat genomics and breeding.

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor (wild emmer, T. turgidum ssp. dicoccoides). The consensus map harboured 30 144 markers (including 26 626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631 cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes.

Phytochemical composition and anti-inflammatory activity of extracts from the whole-meal flour of Italian durum wheat cultivars.

In this study, the quali-quantitative composition of hydrophilic (phenolic acids) and lipophilic (isoprenoids) extracts from whole-meal flour of five elite Italian durum wheat cultivars was determined. Significant differences in the content of bioactive compounds were observed among the wheat extracts, in particular concerning the content of bound phenolic acids, lutein and ß-tocotrienols. The cultivars Duilio and Svevo showed the highest amount of phenolic acids and isoprenoids, respectively. Extracts were evaluated for their anti-inflammatory activity on HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor ß1 (TGF-ß1). Durum wheat extracts significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids and at 0.2 µg/mL of isoprenoids. Conversely, the secretion of the anti-inflammatory mediator TGF-ß1 was not modified by neither hydrophilic nor lipophilic extracts. These results provide further insight into the potential of durum wheat on human health suggesting the significance of varieties with elevated contents of bioactive components.

[The method of selection of the residents in Spain. Analysis of the examination MIR and offer of a new methodology]. / El método de selección de los residentes en España. Análisis del examen MIR y propuesta de una nueva metodología.

INTRODUCTION: The method for selecting medical graduates for residency positions has a strong influence on teaching and learning strategies in medical schools. The methodology currently used in Spain does not seem appropriate for ranking the candidates or improving curriculum development. Thus, and taking into account the most consistent methodologies used in the United Kingdom and USA, we have designed a new method to be used in our country. OBJECTIVES: To analyze the limitations of the methodology used in Spain, and propose a new one aimed to improve the accuracy of selection itself and avoiding the negative influence of the current method on curricular development. In addition, we emphasize the necessity of improving teaching and learning in the clinical context to assure that graduating students reach an adequate level of clinical competence. CONCLUSIONS: The method for selecting candidates to residency post currently used in Spain, which relies mainly on testing theoretical knowledge, should be changed for an alternative methodology taking into account student,s performance through the course and assessing his/her ability for clínical contextualization of knowledge and level of clinical competence.

Genetic basis of qualitative and quantitative resistance to powdery mildew in wheat: from consensus regions to candidate genes.

BACKGROUND: Powdery mildew (Blumeria graminis f. sp. tritici) is one of the most damaging diseases of wheat. The objective of this study was to identify the wheat genomic regions that are involved in the control of powdery mildew resistance through a quantitative trait loci (QTL) meta-analysis approach. This meta-analysis allows the use of collected QTL data from different published studies to obtain consensus QTL across different genetic backgrounds, thus providing a better definition of the regions responsible for the trait, and the possibility to obtain molecular markers that will be suitable for marker-assisted selection. RESULTS: Five QTL for resistance to powdery mildew were identified under field conditions in the durum-wheat segregating population Creso × Pedroso. An integrated map was developed for the projection of resistance genes/ alleles and the QTL from the present study and the literature, and to investigate their distribution in the wheat genome. Molecular markers that correspond to candidate genes for plant responses to pathogens were also projected onto the map, particularly considering NBS-LRR and receptor-like protein kinases. More than 80 independent QTL and 51 resistance genes from 62 different mapping populations were projected onto the consensus map using the Biomercator statistical software. Twenty-four MQTL that comprised 2-6 initial QTL that had widely varying confidence intervals were found on 15 chromosomes. The co-location of the resistance QTL and genes was investigated. Moreover, from analysis of the sequences of DArT markers, 28 DArT clones mapped on wheat chromosomes have been shown to be associated with the NBS-LRR genes and positioned in the same regions as the MQTL for powdery mildew resistance. CONCLUSIONS: The results from the present study provide a detailed analysis of the genetic basis of resistance to powdery mildew in wheat. The study of the Creso × Pedroso durum-wheat population has revealed some QTL that had not been previously identified. Furthermore, the analysis of the co-localization of resistance loci and functional markers provides a large list of candidate genes and opens up a new perspective for the fine mapping and isolation of resistance genes, and for the marker-assisted improvement of resistance in wheat.

Description of durum wheat linkage map and comparative sequence analysis of wheat mapped DArT markers with rice and Brachypodium genomes.

BACKGROUND: The importance of wheat to the world economy, together with progresses in high-throughput next-generation DNA sequencing, have accelerated initiatives of genetic research for wheat improvement. The availability of high density linkage maps is crucial to identify genotype-phenotype associations, but also for anchoring BAC contigs to genetic maps, a strategy followed for sequencing the wheat genome. RESULTS: Here we report a genetic linkage map in a durum wheat segregating population and the study of mapped DArT markers. The linkage map consists of 126 gSSR, 31 EST-SSR and 351 DArT markers distributed in 24 linkage groups for a total length of 1,272 cM. Through bioinformatic approaches we have analysed 327 DArT clones to reveal their redundancy, syntenic and functional aspects. The DNA sequences of 174 DArT markers were assembled into a non-redundant set of 60 marker clusters. This explained the generation of clusters in very small chromosome regions across genomes. Of these DArT markers, 61 showed highly significant (Expectation < E-10) BLAST similarity to gene sequences in public databases of model species such as Brachypodium and rice. Based on sequence alignments, the analysis revealed a mosaic gene conservation, with 54 and 72 genes present in rice and Brachypodium species, respectively. CONCLUSIONS: In the present manuscript we provide a detailed DArT markers characterization and the basis for future efforts in durum wheat map comparing.

Validation of a Spanish version of the psychological inflexibility in pain scale (PIPS) and an evaluation of its relation with acceptance of pain and mindfulness in sample of persons with fibromyalgia.

BACKGROUND: Psychological flexibility has been suggested as a fundamental process in health. The Psychological Inflexibility in Pain Scale (PIPS) is one of the scales employed for assessing psychological inflexibility in pain patients. The aim of this study was to validate the Spanish version of the PIPS and secondly, to compare it to two other psychological constructs, the acceptance of pain and mindfulness scales. METHODS: The PIPS was translated into Spanish by two bilingual linguistic experts, and then, back-translated into English to assess for equivalence. The final Spanish version was administered along with the Pain Visual Analogue Scale, Fibromyalgia Impact Questionnaire, Hospital Anxiety Depression Scale, Pain Catastrophizing Scale, Chronic Pain Acceptance Questionnaire and the Mindful Attention Awareness Scale, to 250 Spanish patients with fibromyalgia. Face validity, construct validity, reliability (internal consistency and test-retest) and convergent validity were tested. Also a multiple regression analysis was carried out.The usual guidelines have been followed for cross-cultural adaptations. RESULTS: Data were very similar to the ones obtained in the original PIPS version. The construct validity confirmed the original two-components solution which explained 61.6% of the variance. The Spanish PIPS had good test-retest reliability (intraclass correlation coefficient 0.97) and internal consistency reliability (Cronbach's alpha: 0.90). The Spanish PIPS' score correlated significantly with worse global functioning (r = 0.55), anxiety (r = 0.54), depression (r = 0.66), pain catastrophizing (r = 0.62), pain acceptance (r = -0.72) and mindfulness (r = -0.47), as well as correlating modestly with pain intensity (r = 0.12). The multiple regression analyses showed that psychological inflexibility, acceptance and mindfulness are not overlapped. CONCLUSIONS: The Spanish PIPS scale appears to be a valid and reliable instrument for the evaluation of psychological inflexibility among a sample of fibromyalgia patients. These results ensure the use of this scale in research as well as in clinical practice. Psychological inflexibility measures processes different from other related components such as acceptance and mindfulness.

Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study.

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

QTL mapping for kernel-related traits in a durum wheat x T. dicoccum segregating population.

Durum wheat breeding relies on grain yield improvement to meet its upcoming demand while coping with climate change. Kernel size and shape are the determinants of thousand kernel weight (TKW), which is a key component of grain yield, and the understanding of the genetic control behind these traits supports the progress in yield potential. The present study aimed to dissect the genetic network responsible for kernel size components (length, width, perimeter, and area) and kernel shape traits (width-to-length ratio and formcoefficient) as well as their relationships with kernel weight, plant height, and heading date in durum wheat. Quantitative Trait Locus (QTL) mapping was performed on a segregating population of 110 recombinant inbred lines, derived from a cross between the domesticated emmer wheat accession MG5323 and the durum wheat cv. Latino, evaluated in four different environments. A total of 24 QTLs stable across environments were found and further grouped in nine clusters on chromosomes 2A, 2B, 3A, 3B, 4B, 6B, and 7A. Among them, a QTL cluster on chromosome 4B was associated with kernel size traits and TKW, where the parental MG5323 contributed the favorable alleles, highlighting its potential to improve durum wheat germplasm. The physical positions of the clusters, defined by the projection on the T. durum reference genome, overlapped with already known genes (i.e., BIG GRAIN PROTEIN 1 on chromosome 4B). These results might provide genome-based guidance for the efficient exploitation of emmer wheat diversity in wheat breeding, possibly through yield-related molecular markers.

Genetic Mapping of Flavonoid Grain Pigments in Durum Wheat.

Pigmented cereal grains with high levels of flavonoid compounds have attracted the attention of nutritional science backing the development of functional foods with claimed health benefits. In this study, we report results on the genetic factors controlling grain pigmentation in durum wheat using a segregant population of recombinant inbred lines (RILs) derived from a cross between an Ethiopian purple grain accession and an Italian amber grain cultivar. The RIL population was genotyped by the wheat 25K SNP array and phenotyped for total anthocyanin content (TAC), grain color, and the L*, a*, and b* color index of wholemeal flour, based on four field trials. The mapping population showed a wide variation for the five traits in the different environments, a significant genotype x environment interaction, and high heritability. A total of 5942 SNP markers were used for constructing the genetic linkage map, with an SNP density ranging from 1.4 to 2.9 markers/cM. Two quantitative trait loci (QTL) were identified for TAC mapping on chromosome arms 2AL and 7BS in the same genomic regions of two detected QTL for purple grain. The interaction between the two QTL was indicative of an inheritance pattern of two loci having complementary effects. Moreover, two QTL for red grain color were detected on chromosome arms 3AL and 3BL. The projection of the four QTL genomic regions on the durum wheat Svevo reference genome disclosed the occurrence of the candidate genes Pp-A3, Pp-B1, R-A1, and R-B1 involved in flavonoid biosynthetic pathways and encoding of transcription factors bHLH (Myc-1) and MYB (Mpc1, Myb10), previously reported in common wheat. The present study provides a set of molecular markers associated with grain pigments useful for the selection of essential alleles for flavonoid synthesis in durum wheat breeding programs and enhancement of the health-promoting quality of derived foods.

Adverse Clinical Outcomes and Associated Predictors in Rivaroxaban-Treated Atrial Fibrillation Patients With Renal Impairment.

Renal impairment confers worse prognosis in patients with atrial fibrillation (AF) but there is scarce evidence about the influence of direct-acting oral anticoagulants in routine clinical practice. Herein, we compared clinical outcomes between patients with AF with and without renal impairment on rivaroxaban and investigated predictors for clinical outcomes in patients with AF with renal impairment. This was a multicenter study including patients with AF on rivaroxaban for at least 6 months. During 2.5 years follow-up, ischemic strokes (IS)/transient ischemic attacks (TIA)/systemic embolisms (SE)/myocardial infarctions (MI), major bleeding, and major adverse cardiovascular events (MACE) were recorded. Creatinine clearance (CrCl) was estimated using the Cockroft-Gault equation, renal impairment was defined as a CrCl <60 ml/min, and 1,433 patients (34.8% with CrCl <60 ml/min) were included. Patients with CrCl <60 ml/min showed higher event rates for major bleeding (1.87%/year vs 0.62%/year; p = 0.003) and MACE (1.97%/year vs 0.62%/year; p = 0.002) but similar event rates for IS/TIA/SE/MI (0.66%/year vs 0.67%/year; p = 0.955). In patients with renal impairment, CHA2DS2-VASc was associated with higher risk of IS/TIA/SE/MI; HAS-BLED and any dependency level were associated with higher risk of major bleeding; and male gender and heart failure were associated with higher risk of MACE. Antiplatelets were independently associated with increased risk of IS/TIA/SE/MI and MACE. In conclusion, in patients with AF on rivaroxaban, the incidence of IS/TIA/SE/MI did not increase in those with renal impairment, suggesting that rivaroxaban may be an effective option in this subgroup. In patients with AF, male gender, heart failure, dependency, antiplatelets, CHA2DS2-VASc, and HAS-BLED were associated with increased risk of adverse outcomes.

Avelumab in Combination With Cetuximab and Chemotherapy as First-Line Treatment for Patients With Advanced Squamous NSCLC.

Introduction: We present the results of a phase 2a trial of first-line avelumab (anti-programmed death-ligand 1 antibody) plus cetuximab (anti-EGFR antibody) in patients with advanced squamous NSCLC. Methods: Patients with recurrent or metastatic squamous NSCLC received avelumab 800 mg (d 1 and 8), cetuximab 250 mg/m2 (d 1) and 500 mg/m2 (d 8), cisplatin 75 mg/m2 (d 1), and gemcitabine 1250 mg/m2 (d 1 and 8) for four 3-week cycles, followed by avelumab 800 mg and cetuximab 500 mg/m2 every 2 weeks. The primary end point was the best overall response; the secondary end points were progression-free survival, duration of response, overall survival, and safety. Efficacy analyses were reported from an updated data cutoff. Results: A total of 43 patients were enrolled. The median follow-up was 6.6 months for the primary analyses and 9.2 months for the efficacy analyses. In the efficacy analyses, 15 patients had a confirmed partial response (objective response rate, 34.9% [95% confidence interval: 21.0%-50.9%]), and the median duration of response was 7.1 months (95% confidence interval: 4.2-12.5 mo). The median progression-free survival and overall survival were 6.1 months and 10.0 months, respectively. In the safety analyses (primary analysis), 38 patients (88.4%) had a treatment-related adverse event, of whom 24 (55.8%) had a grade 3 or higher treatment-related adverse event. Conclusions: The combination of avelumab + cetuximab and chemotherapy showed antitumor activity and tolerable safety; however, the ORR was not improved compared with those reported for current standards of care (NCT03717155).