Genetic and gene expression analysis of flowering time regulation by light quality in lentil.

BACKGROUND AND AIMS: Flowering time is important due to its roles in plant adaptation to different environments and subsequent formation of crop yield. Changes in light quality affect a range of developmental processes including flowering time, but little is known about light quality-induced flowering time control in lentil. This study aims to investigate the genetic basis for differences in flowering response to light quality in lentil. METHODS: We explored variation in flowering time caused by changes in red/far-red-related light quality environments of a lentil interspecific recombinant inbred line (RIL) population developed from a cross between Lens culinaris cv. Lupa and L. orientalis accession BGE 016880. A genetic linkage map was constructed and then used for identifying quantitative trait loci (QTLs) associated with flowering time regulation under different light quality environments. Differential gene expression analysis through transcriptomic study and RT-qPCR were used to identify potential candidate genes. KEY RESULTS: QTL mapping located 13 QTLs controlling flower time under different light quality environments, with phenotypic variance explained ranging from 1.7 to 62.9 %. Transcriptomic profiling and gene expression analysis for both parents of this interspecific RIL population identified flowering-related genes showing environment-specific differential expression (flowering DEGs). One of these, a member of the florigen gene family FTa1 (LcFTa1), was located close to three major QTLs. Furthermore, gene expression results suggested that two other florigen genes (LcFTb1 and LcFTb2), MADS-box transcription factors such as LcAGL6/13d, LcSVPb, LcSOC1b and LcFULb, as well as bHLH transcription factor LcPIF6 and Gibberellin 20 oxidase LcGA20oxC,G may also be involved in the light quality response. CONCLUSIONS: Our results show that a major component of flowering time sensitivity to light quality is tightly linked to LcFTa1 and associated with changes in its expression. This work provides a foundation for crop improvement of lentil with better adaptation to variable light environments.

Indication of CPAP without a sleep study in patients with high pretest probability of obstructive sleep apnea.

OBJECTIVE: To evaluate the performance of clinical criteria (CC) for diagnosis and initiation of empirical treatment with continuous positive airway pressure (CPAP) in patients with suspected obstructive sleep apnea (OSA) compared with the treatment decision based on sleep studies (polysomnography or respiratory polygraphy), guidelines, and experience of participating physicians. METHODS: This was a simulated intention-to-treat study in a retrospective (G1) and prospective (G2) cohort. Four observers (two per group) called CC1 and CC2 reviewed the sleep questionnaires and indicated CPAP if the patients presented snoring, frequent apneas (≥ 3-4/week), body mass index (BMI) > 25 kg/m2, sleepiness (Epworth > 11), or tiredness (at least 3-4 times per week) and some comorbidity (hypertension, coronary/cerebrovascular event, diabetes). Ten independent observers formed two groups of five (FD1 and FD2) and were blinded to each other's opinion. These observers in FD1 and FD2 decided CPAP treatment based on guidelines of the Spanish Society of Pulmonology and Thoracic Surgery (SEPAR) or guidelines of the American Academy of Sleep Medicine (AASM) and factored in their own opinion. Sensitivity (S), specificity (Sp), and positive/negative likelihood ratios (LR+/-) were calculated with the test method: CC1/2, and the reference method: majority decision of FD1/2. RESULTS: A total of 653 patients (264 women, 40%) were studied. Median age was 54 years, BMI 28 kg/m2, and apnea hypopnea index (AHI) 16.5 events/h. S ranged from 21 to 25% (p 0.60), Sp 96.1 to 97.6% (p 0.39), and LR+ of clinical criteria 6.4 to 8.9 (p 0.52). CONCLUSION: CPAP indication without a previous sleep study showed a low sensitivity (≅ 22%) but a specificity greater than 95% in patients with high pretest probability for OSA (snoring, report of frequent apneas, BMI > 25 kg/m2 and sleepiness or tiredness plus comorbidity).

Construction of a high-density interspecific (Lens culinaris x L. odemensis) genetic map based on functional markers for mapping morphological and agronomical traits, and QTLs affecting resistance to Ascochyta in lentil.

Usage of high-throughput sequencing approaches allow for the generation and characterization of reference transcriptome datasets that support gene-based marker discovery, which in turn can be used to build genetic maps among other purposes. We have obtained a transcriptome assembly including 49,453 genes for the lentil (Lens culinaris Medik.) cultivar Alpo using RNAseq methodology. This transcriptome was used as reference to obtain 6,306 quality polymorphic markers (SNPs and short indels) analyzing genotype data from a RIL population at F7 generation derived from the interspecific cross between L. culinaris cv. Alpo and L. odemensis accession ILWL235. L. odemensis is a wild species included in the secondary gene pool and can be used as a source for gene introgression in lentil breeding programs. Marker data were used to construct the first genetic interspecific map between these two species. This linkage map has been used to precisely identify regions of the CDC-Redberry lentil draft genome in which the candidate genes for some qualitative traits (seed coat spotting pattern, flower color, and stem pigmentation) could be located. The genome regions corresponding to a significant single quantitative trait locus (QTL) controlling "time to flowering" located in chromosome 6 and three QTLs regulating seed size and positioned in chromosomes 1 and 5 (two QTLs) were also identified. Significant QTLs for Ascochyta blight resistance in lentil were mapped to chromosome 6 in the genome region or close to it where QTLs for Ascochyta blight resistance have previously been reported.

CPAP indication based on clinical data and oximetry for patients with suspicion of obstructive sleep apnea: A multicenter trial.

BACKGROUND AND OBJECTIVE: The usefulness of pulse oximetry for the management of obstructive sleep apnea is controversial. The aim of this study was to assess the accuracy for indication of Continuous Positive Airway Pressure (CPAP) treatment in patients with suspected obstructive sleep apnea (OSA) based on clinical and oximetry data as compared to polysomnography (PSG). METHODS: This multicenter observational study involved seven sleep laboratories. Patients with suspicion of OSA who completed a standardized sleep questionnaire and a diagnostic PSG were enrolled. Eight observers logged on to a website independently and blindly. Seven observers only accessed the clinical data, curve and pulse oximetry results (Os-SO2-test method), while the eighth observer had full access to all indicators of PSG (O-PSG-reference method). Once observers assessed the information available on the website, they had to choose between three CPAP treatment options (yes/no/do not know) based on their knowledge and criteria. RESULTS: 411 subjects (228 men), median age 54 years, were available for evaluation. Os-SO2 had lower sensitivity (S), greater specificity (Sp) and positive likelihood ratio (PLR) to prescribe CPAP in patients more symptomatic (Epworth Sleepiness Scale-ESS > 10 or comorbidities) than those with fewer symptoms (ESS < 11 without comorbidities) (S 45-75% versus 45-91%, p 0.028); Sp 93.8-100% versus 68.5-96.6%, p 0.004; PLR > 10 versus 2.9-17, p<0.01). CONCLUSIONS: Due to its low false positive rate, a strategy based on pulse oximetry and clinical data was a consistent tool to indicate CPAP treatment in most symptomatic patients with a suspicion of OSA.

A genome-wide identification and comparative analysis of the lentil MLO genes.

Powdery mildew is a widespread fungal plant disease that can cause significant losses in many crops. Some MLO genes (Mildew resistance locus O) have proved to confer a durable resistance to powdery mildew in several species. Resistance granted by the MLO gene family members has prompted an increasing interest in characterizing these genes and implementing their use in plant breeding. Lentil (Lens culinaris Medik.) is a widely grown food legume almost exclusively consumed as dry seed with an average world production of 4.5 million tons. Powdery mildew causes severe losses on certain lentil cultivars under particular environmental conditions. Data mining of the lentil CDC Redberry draft genome allowed to identify up to 15 gene sequences with homology to known MLO genes, designated as LcMLOs. Further characterization of these gene sequences and their deduced protein sequences demonstrated conformity with key MLO protein characteristics such as the presence of transmembrane and calmodulin binding domains, as well as that of other conserved motifs. Phylogenetic and other comparative analyses revealed that LcMLO1 and LcMLO3 are the most likely gene orthologs related to powdery mildew response in other species, sharing a high similarity with other known resistance genes of dicot species, such as pea PsMLO1 and Medicago truncatula MtMLO1 and MtMLO3. Sets of primers were designed as tools to PCR amplify the genomic sequences of LcMLO1 and LcMLO3, also to screen lentil germplasm in search of resistance mutants. Primers were used to obtain the complete sequences of these two genes in all of the six wild lentil relatives. Respective to each gene, all Lens sequences shared a high similarity. Likewise, we used these primers to screen a working collection of 58 cultivated and 23 wild lentil accessions in search of length polymorphisms present in these two genes. All these data widen the insights on this gene family and can be useful for breeding programs in lentil and close related species.

Deep Super-SAGE transcriptomic analysis of cold acclimation in lentil (Lens culinaris Medik.).

BACKGROUND: Frost is one of the main abiotic stresses limiting plant distribution and crop production. To cope with the stress, plants evolved adaptations known as cold acclimation or chilling tolerance to maximize frost tolerance. Cold acclimation is a progressive acquisition of freezing tolerance by plants subjected to low non-freezing temperatures which subsequently allows them to survive exposure to frost. Lentil is a cool season grain legume that is challenged by winter frost in some areas of its cultivation. RESULTS: To better understand the genetic base of frost tolerance differential gene expression in response to cold acclimation was investigated. Recombinant inbred lines (RILs) from the cross Precoz x WA8649041 were first classified as cold tolerant or cold susceptible according to their response to temperatures between -3 to -15 °C. Then, RILs from both extremes of the response curve were cold acclimated and the leaf transcriptomes of two bulks each of eight frost tolerant and seven cold susceptible RILs were investigated by Deep Super-SAGE transcriptome profiling. Thus, four RNA bulks were analysed: the acclimated susceptible, the acclimated tolerant and the respective controls (non-acclimated susceptible and non-acclimated tolerant). Approximately 16.5 million 26 nucleotide long Super-SAGE tags were sequenced in the four sets (between ~3 and 5.4 millions). In total, 133,077 different unitags, each representing a particular transcript isoform, were identified in these four sets. Tags which showed a significantly different abundance in any of the bulks (fold change ≥4.0 and a significant p-value <0.001) were selected and used to identify the corresponding lentil gene sequence. Three hundred of such lentil sequences were identified. Most of their known homologs coded for glycine-rich, cold and drought-regulated proteins, dormancy-associated proteins, proline-rich proteins (PRPs) and other membrane proteins. These were generally but not exclusively over-expressed in the acclimated tolerant lines. CONCLUSIONS: This set of candidate genes implicated in the response to frost in lentil represents an useful base for deeper and more detailed investigations into this important agronomic trait in future.

Obtaining retrotransposon sequences, analysis of their genomic distribution and use of retrotransposon-derived genetic markers in lentil (Lens culinaris Medik.).

Retrotransposons with long terminal repeats (LTR-RTs) are widespread mobile elements in eukaryotic genomes. We obtained a total of 81 partial LTR-RT sequences from lentil corresponding to internal retrotransposon components and LTRs. Sequences were obtained by PCR from genomic DNA. Approximately 37% of the LTR-RT internal sequences presented premature stop codons, pointing out that these elements must be non-autonomous. LTR sequences were obtained using the iPBS technique which amplifies sequences between LTR-RTs. A total of 193 retrotransposon-derived genetic markers, mainly iPBS, were used to obtain a genetic linkage map from 94 F7 inbred recombinant lines derived from the cross between the cultivar Lupa and the wild ancestor L. culinaris subsp. orientalis. The genetic map included 136 markers located in eight linkage groups. Clusters of tightly linked retrotransposon-derived markers were detected in linkage groups LG1, LG2, and LG6, hence denoting a non-random genomic distribution. Phylogenetic analyses identified the LTR-RT families in which internal and LTR sequences are included. Ty3-gypsy elements were more frequent than Ty1-copia, mainly due to the high Ogre element frequency in lentil, as also occurs in other species of the tribe Vicieae. LTR and internal sequences were used to analyze in silico their distribution among the contigs of the lentil draft genome. Up to 8.8% of the lentil contigs evidenced the presence of at least one LTR-RT similar sequence. A statistical analysis suggested a non-random distribution of these elements within of the lentil genome. In most cases (between 97% and 72%, depending on the LTR-RT type) none of the internal sequences flanked by the LTR sequence pair was detected, suggesting that defective and non-autonomous LTR-RTs are very frequent in lentil. Results support that LTR-RTs are abundant and widespread throughout of the lentil genome and that they are a suitable source of genetic markers useful to carry out further genetic analyses.

Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse.

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

Analysis of the argK-tox gene cluster in nontoxigenic strains of Pseudomonas syringae pv. phaseolicola.

The analysis of 46 isolates obtained directly from different and distant common bean fields from the northwestern part of Spain revealed that they do not produce phaseolotoxin. The isolates were classified as race 5, and their analysis revealed that they do not carry the argK-tox gene cluster involved in the biosynthesis of the phaseolotoxin.

The genetic structure of tetraploid Avena: a comparison of isozyme and RAPD markers.

Isozymes were the first widely used molecular markers in plant population analysis. They yielded valuable information on the amount and the structure of genetic variability. DNA technology has provided new types of markers based on DNA sequence, which make it possible to study polymorphisms in a much greater proportion of the genome. This is the reason why the use of isozymes is less popular nowadays. This effect would be justified if all markers provided the same type of information on polymorphism and genetic relationships among populations; otherwise, it would be necessary to use different markers to obtain the complete picture of the genetic structure of populations and species. In this study, we compared data of isozyme and RAPD markers in the populations of two tetraploid species of wild oats: Avena barbata populations collected in Argentina, and Avena murphyi populations collected in Spain and Morocco. The samples were evaluated for 9 isozymatic systems and 10 primers. The structure of genetic variability was studied using Nei's method, and the relationships between populations were estimated using Hedrick and Jaccard's similarities for isozymes and RAPDs, respectively. As expected, RAPDs were more polymorphic than isozymes, but the information obtained from both markers was weakly correlated. The various reasons for this observation are discussed, but our conclusion is that in order to study the structure of genetic variability, several types of markers should be used.