Search for Resistant Genotypes to Cuscuta campestris Infection in Two Legume Species, Vicia sativa and Vicia ervilia.

The dodders (Cuscuta spp.) are parasitic plants that feed on the stems of their host plants. Cuscuta campestris is one of the most damaging parasitic plants for the worldwide agricultural production of broad-leaved crops. Its control is limited or non-existent, therefore resistance breeding is the best alternative both economically and environmentally. Common vetch (Vicia sativa) and bitter vetch (Vicia ervilia) are highly susceptible to C. campestris, but no resistant genotypes have been identified. Thus, the aim of this study was to identify in V. sativa and V.ervilia germplasm collections genotypes resistant to C. campestris infection for use in combating this parasitic plant. Three greenhouse screening were conducted to: (1) identify resistant responses in a collection of 154 accessions of bitter vetch and a collection of 135 accessions of common vetch genotypes against infection of C. campestris; (2) confirm the resistant response identified in common vetch accessions; and (3) characterize the effect of C. campestris infection on biomass of V. sativa resistant and susceptible accessions. Most common vetch and bitter vetch genotypes tested were susceptible to C. campestris. However, the V. sativa genotype Vs.1 exhibited high resistance. The resistant phenotype was characterized by a delay in the development of C. campestris posthaustorial growth and a darkening resembling a hypersensitive-like response at the penetration site. The resistant mechanism was effective in limiting the growth of C. campestris as the ratio of parasite/host shoot dry biomass was more significantly reduced than the rest of the accessions. To the best or our knowledge, this is the first identification of Cuscuta resistance in V. sativa genotypes.

Resistance against Orobanche crenata in Bitter Vetch (Vicia ervilia) Germplasm Based on Reduced Induction of Orobanche Germination.

Bitter vetch (Vicia ervilia (L.) Willd.) is a legume well adapted to cultivation in marginal areas, being an important source of protein for animal feed in low input cropping systems. Surprisingly, it is an underutilized crop as it could be a good alternative to increase the sustainability of extensive rainfed cropping systems. In Mediterranean rainfed cropping systems, the productivity of bitter vetch is severely reduced by the parasitic weed species Orobanche crenata (Forsk). To date, few resistant bitter vetch genotypes have been identified. O. crenata infection process initiates with the recognition of germination factors exuded by roots of susceptible hosts. In this work, the interaction of a collection of bitter vetch accessions and O. crenata has been analyzed in order to discover accessions with low germination induction activity. Through a combination of field and rhizotron experiments, two bitter vetch accessions were selected showing low germination-induction activity, which resulted in less infection. In addition, in vitro germination assays revealed that the low germination activity was due to low exudation of germination factors and not due to the exudation of germination inhibitors. The selected low germination-inducers genotypes could be the basis for a new breeding program generating locally adapted alternatives with resistance to O. crenata.

Identification of Vicia ervilia Germplasm Resistant to Orobanche crenata.

Bitter vetch (Vicia ervilia L.) is an ancient grain legume used as animal feed in the Mediterranean basin. This legume has a large economical potential because of its high yield under low inputs and good protein content, as well as resistance to cold and drought. Nevertheless, its growth and production area are affected in the presence of the broomrape weed species Orobanche crenata. Due to the small bitter vetch size, infection by as few as two or three O. crenata per vetch plant can be devastating. There are no efficient methods of selectively controlling O. crenata in this crop, for which reason the development of varieties resistant and tolerant to O. crenata infection is needed. Phytogenetic resources are valuable reserves for species survival. They represent important genetic variability and allow the possibility of finding characters of interest, such as new resistance sources. A large-scale field screening of a collection of 102 bitter vetch accessions indicated that most bitter vetch accessions were susceptible but allowed us to select 16 accessions with low levels of O. crenata infection. Next, we used a combination of field and rhizotron experiments to investigate the resistant response of selected bitter vetch genotypes in detail by studying the performance and resistance mechanisms. These experiments led to the identification of three different mechanisms that block O. crenata parasitism. A pre-attachment mechanism of low induction of O.crenata germination was identified in two bitter vetch accession Ve.055 and Ve.155. In addition, a post-attachment mechanism of resistance to O. crenata penetration was identified inthe accession Ve.125. In addition, the field-resistant accession Ve.123 showed susceptible response in rhizotron, indicating that a late mechanism acting after vascular connection, most probably related with bitter vetch of escape due to fructification precocity was acting against O. crenata development.

Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

INTRODUCTION: Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. OBJECTIVE: To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. METHODOLOGY: Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. RESULTS: In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. CONCLUSION: Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue.

Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.

The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.

Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions.

Reverse transcription quantitative real-time polymerase chain reaction is the most accurate measure of gene expression in biological systems. The data are analyzed through a process called normalization. Internal standards are essential for determining the relative gene expression in different samples. For this purpose, reference genes are selected based on their constitutive expression across samples. At present, there has not yet been any reference gene identified in any organism that is universally optimal across different tissue types or disease situations. Our goal was to test the regulation of 11 potential references for pea. These included eight commonly used and three new candidates. Twenty-six samples, including different tissues, treatments and genotypes, were addressed in this analysis. For reliable data normalization, the most suitable combination of reference genes in each experimental set was constructed with at least two out the five more stably expressed references in the whole experimental series (i.e. protein phosphatase 2A, beta-tubulin, GH720838, actin and GH720808). To validate the determined measure of gene-stability, the gene-specific variation was calculated using different normalization factors. The most non-specific variation was removed when the most stable genes were used, highlighting the importance of the adequate choice of internal controls in gene expression experiments. The set of reference genes presented here will provide useful guidelines as starting point for reference gene selection in pea studies under conditions other than those tested here.

Gene expression analysis of molecular mechanisms of defense induced in Medicago truncatula parasitized by Orobanche crenata.

The infection of Medicago truncatula Gaertn. roots with the obligate parasite Orobanche crenata Forsk. is a useful model for studying the molecular events involved in the legumes-parasite interaction. In order to gain insight into the identification of gene-regulatory elements involved in the resistance mechanism, the temporal expression pattern of ten defense-related genes was carried out using real-time quantitative reverse-transcription polymerase chain reaction assays. The induction of all of the analyzed transcripts significantly increased over a range from 2- to 321-fold higher than the control depending on the gene and time point. The transcriptional changes observed in response to O. crenata infection suggest that resistance could rely on both, the induction of general defense-related genes and more specific responses.