The INRAE Centre for Vegetable Germplasm: Geographically and Phenotypically Diverse Collections and Their Use in Genetics and Plant Breeding.

The French National Research Institute for Agriculture, Food and the Environment (INRAE) conserves and distributes five vegetable collections as seeds: the aubergine* (in this article the word aubergine refers to eggplant), pepper, tomato, melon and lettuce collections, together with their wild or cultivated relatives, are conserved in Avignon, France. Accessions from the collections have geographically diverse origins, are generally well-described and fixed for traits of agronomic or scientific interest and have available passport data. In addition to currently conserving over 10,000 accessions (between 900 and 3000 accessions per crop), the centre maintains scientific collections such as core collections and bi- or multi-parental populations, which have also been genotyped with SNP markers. Each collection has its own merits and highlights, which are discussed in this review: the aubergine collection is a rich source of crop wild relatives of Solanum; the pepper, melon and lettuce collections have been screened for resistance to plant pathogens, including viruses, fungi, oomycetes and insects; and the tomato collection has been at the heart of genome-wide association studies for fruit quality traits and environmental stress tolerance.

Effector prediction and characterization in the oomycete pathogen Bremia lactucae reveal host-recognized WY domain proteins that lack the canonical RXLR motif.

Pathogens that infect plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen Bremia lactucae by searching the predicted proteome for the WY domain, a structural fold found in effectors that has been implicated in immune suppression as well as effector recognition by host resistance proteins. We predicted 55 WY domain containing proteins in the genome of B. lactucae and found substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis of 21 effectors containing WY domains revealed 11 that elicited cell death on wild accessions and domesticated lettuce lines containing resistance genes, indicative of recognition of these effectors by the host immune system. Only two of the 11 recognized effectors contained the canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens.

Genetic analysis of cadmium accumulation in lettuce (Lactuca sativa).

This work characterized mechanisms controlling cadmium (Cd) tolerance and accumulation in lettuce at both the physiological and genetic levels. These traits were evaluated in 18 Lactuca accessions representing a large genetic diversity. Cd tolerance and accumulation in roots and shoots as well as Cd translocation from roots to the shoot varied independently, and with a significant range of variation. Analyses of F1 progenies of crosses between cultivars with contrasted phenotypes showed that high tolerance to Cd, low Cd accumulation and low Cd root-shoot translocation were recessive traits. Results of analyses of F2 progenies of different crosses suggest that root Cd concentration and root-shoot Cd translocation were under a complex genetic determinism involving at least two loci. This work thus revealed that limiting both Cd accumulation and Cd root-shoot translocation in lettuce is possible and depends on recessive loci. Differences in the ability to accumulate Cd in roots in the long term could not be linked to differences in short-term 109Cd uptake into, or efflux from, roots. In contrast, the cultivar with the highest root-shoot Cd translocation was the same in the long term and in the short term, which suggests that this trait relies on processes that are implemented quickly (i.e. in less than three days) after the start of Cd exposure.

Successful gene tagging in lettuce using the Tnt1 retrotransposon from tobacco.

The tobacco (Nicotiana tabacum) element Tnt1 is one of the few identified active retrotransposons in plants. These elements possess unique properties that make them ideal genetic tools for gene tagging. Here, we demonstrate the feasibility of gene tagging using the retrotransposon Tnt1 in lettuce (Lactuca sativa), which is the largest genome tested for retrotransposon mutagenesis so far. Of 10 different transgenic bushes carrying a complete Tnt1 containing T-DNA, eight contained multiple transposed copies of Tnt1. The number of transposed copies of the element per plant was particularly high, the smallest number being 28. Tnt1 transposition in lettuce can be induced by a very simple in vitro culture protocol. Tnt1 insertions were stable in the progeny of the primary transformants and could be segregated genetically. Characterization of the sequences flanking some insertion sites revealed that Tnt1 often inserted into genes. The progeny of some primary transformants showed phenotypic alterations due to recessive mutations. One of these mutations was due to Tnt1 insertion in the gibberellin 3beta-hydroxylase gene. Taken together, these results indicate that Tnt1 is a powerful tool for insertion mutagenesis especially in plants with a large genome.

Systematic silencing of a tobacco nitrate reductase transgene in lettuce (Lactuca sativa L.).

A population of 50 independent transgenic lettuces transformed with a nitrate reductase coding sequence under the control of the 35S promoter was studied. None of them showed significantly lower nitrate levels when compared with the untransformed plants, despite the presence of nitrate reductase (NR) activity that derives from the transgene in at least four of the transformants. No repercussion on total NR activity (endogenous+transgenic) was detected in these plants. Nevertheless, 28% of the transformants showed phenotypes characteristic of a general silencing of the NR genes as already described in tobacco and potato, i.e. bleaching of the leaves leading to the death of the plant. By northern blots, it was shown that the transgene was silenced in these chlorotic plants and also in the plants that did not show symptoms of chlorosis. Thus a silencing process specifically directed against the NR mRNA derived from the transgene occurred very early in the development of all the plants studied, whatever homologous endogenous NR mRNA is present in the plant. In some cases this transgene-specific silencing was shown subsequently to extend to the homologous endogenous NR mRNA. These results suggest that, in lettuce, the level of nitrate reductase mRNA is under tight expression control and this is able specifically to target transgenic transcripts by a post-transcriptional gene silencing (PTGS) mechanism during the first stage of development of the plantlet.

A simple and efficient method for testing Lettuce mosaic virus resistance in in vitro cultivated lettuce.

The potential of a new in vitro inoculation and propagation method developed for Lettuce mosaic virus (LMV) on lettuce (Lactuca sativa L.) was evaluated by studying LMV infection on in vitro cultivated seedlings or on newly regenerated plantlets. Lettuce cultivars differing by their LMV-resistance status were inoculated with various natural LMV isolates as well as with Green Fluorescent Protein (GFP)-tagged recombinant virus isolates. A good correlation was observed between the known resistance status of the cultivars and the results obtained by in vitro screening. The results show that this resistance assay can be greatly improved by the use of GFP-tagged virus isolates. The main advantages of this method are reduced space requirements and an improved environmental safety, especially for the handling of recombinant virus, of quarantine virus or of transgenic plants.

SNP-based codominant markers for a recessive gene conferring resistance to corky root rot (Rhizomonas suberifaciens) in lettuce (Lactuca sativa).

The analysis of F2 progeny and derived F3 families of Lactuca sativa segregating for resistance to corky root rot caused by Rhizomonas suberifaciens permitted the identification of restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism (SNP) markers linked to the recessive resistance gene cor. PCR-based markers were identified by bulked segregant analysis (BSA). Allele-specific primers were generally designed with the 3 terminal base coinciding with an SNP, matching one of the alleles and mismatching the other, and with an additional subterminal 3 base mismatching both alleles. Codominant, robust, and inexpensive molecular markers were obtained that used standardized PCR conditions. Some of the markers could be analyzed in multiple Lactuca mapping populations that did not segregate for disease resistance allowing the cor locus to be located on several maps. The consistent low density of markers around cor in these maps suggests that cor may be in an area with an elevated rate of recombination. Evaluation of these markers in a large sample of cultivars and landraces identified pairs of flanking polymorphic markers that can be used for marker-assisted selection of corky root resistance.

The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus.

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.