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.

Transferability of molecular markers from major legumes to Lathyrus spp. for their application in mapping and diversity studies.

Lathyrus cicera L. (chickling pea) and L. sativus L. (grass pea) have great potential among grain legumes due to their adaptability to inauspicious environments, high protein content and resistance to serious diseases. Nevertheless, due to its past underused, further activities are required to exploit this potential and to capitalise on the advances in molecular biology that enable improved Lathyrus spp. breeding programmes. In this study we evaluated the transferability of molecular markers developed for closely related legume species to Lathyrus spp. (Medicago truncatula, pea, lentil, faba bean and lupin) and tested the application of those new molecular tools on Lathyrus mapping and diversity studies. Genomic and expressed sequence tag microsatellite, intron-targeted amplified polymorphic, resistance gene analogue and defence-related gene markers were tested. In total 128 (27.7 %) and 132 (28.6 %) molecular markers were successfully cross-amplified, respectively in L. cicera and L. sativus. In total, the efficiency of transferability from genomic microsatellites was 5 %, and from gene-based markers, 55 %. For L. cicera, three cleaved amplified polymorphic sequence markers and one derived cleaved amplified polymorphic sequence marker based on the cross-amplified markers were also developed. Nine of those molecular markers were suitable for mapping in a L. cicera recombinant inbred line population. From the 17 molecular markers tested for diversity analysis, six (35 %) in L. cicera and seven (41 %) in L. sativus were polymorphic and discriminate well all the L. sativus accessions. Additionally, L. cicera accessions were clearly distinguished from L. sativus accessions. This work revealed a high number of transferable molecular markers to be used in current genomic studies in Lathyrus spp. Although their usefulness was higher on diversity studies, they represent the first steps for future comparative mapping involving these species.

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.

Simultaneous determination of nine anticoagulant rodenticides in soil and water by LC-ESI-MS.

A new and sensitive analytical method is presented to determine nine anticoagulant rodenticide (chlorophacinone, bromadiolone, pindone, diphacinone, warfarin, coumatetralyl, brodifacoum, floucomafen, and difenacoum) residues in water and soil samples by LC-ESI-MS. Rodenticides were extracted from soil using a methanol and ammonium formate 30 mM mixture, while ethyl acetate was employed in the water samples. A Gemini 5 µm C18 column was employed, and a mobile phase comprising a mixture of ammonium formate 30 mM and di-n-butylamine 30 mM in water (pH 3.5), ammonium formate 30 mM and di-n-butylamine 20 mM in water (pH 4.4), ammonium formate 30 mM in water (pH 6.5), and methanol in a gradient elution mode was selected. The method was fully validated and it was found to be selective and precise in terms of linearity and accuracy. Extraction recoveries ranged from 90 to 104% for the compounds studied, while the detection and quantification limits were between 0.09 and 2.2 µg/kg in soil or 0.08 and 1.7 µg/L in water. The method was applied to simultaneously measure these compounds in water and soil samples.

Analysis of anticoagulant rodenticide residues in Microtus arvalis tissues by liquid chromatography with diode array, fluorescence and mass spectrometry detection.

We describe here a fast and selective analytical method to determine the levels of four anticoagulant rodenticides (chlorophacinone, bromadiolone, brodifacoum and difenacoum) in animal tissues by liquid chromatography (LC) using different detection methods: fluorescence (FLD), diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS). Rodenticides were extracted from freeze-dried and homogenized tissue samples (liver, intestine and muscle) that had been obtained from the common vole (Microtus arvalis). These samples were diluted in 5 mL of methanol, the solution was shaken and centrifuged, and the supernatant was removed and evaporated to dryness. The residue was reconstituted in 1 mL of methanol (liver samples) or 1 mL of the mobile phase (muscle and intestine samples), and injected onto an LC-DAD-FLD-MS system coupled to electrospray ionization (ESI) in negative mode. After conducting an LC optimization study, we selected a Gemini 5 µm C18 column, a mobile phase composed of a mixture of 30 mM ammonium formate in water and methanol (26:74, v/v), and we used an isocratic elution mode. The method was fully validated and shown to be selective, precise, accurate, and linear in the range from ~5µg/kg (ESI-MS) or ~50 µg/kg (DAD-FLD) to 10,000 µg/kg, depending on the compound analyzed. Recoveries ranged from 82% to 103%, while the limits of detection and quantification ranged from 9-89 µg/kg (FLD-DAD) and 0.6-4.6 µg/kg (ESI-MS). This method was successfully used to simultaneously measure the aforementioned compounds in M. arvalis tissues.

Pea detection in food and feed samples by a real-time PCR method based on a specific legumin gene that allows diversity analysis.

Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.