Genetic diversity and population structure of the sweet leaf herb, Stevia rebaudiana B., cultivated and landraces germplasm assessed by EST-SSRs genotyping and steviol glycosides phenotyping.

BACKGROUND: Stevia rebaudiana (Asteraceae), native from Paraguay, accumulates steviol glycosides (SGs) into its leaves. These compounds exhibit acaloric intense sweet taste which answers to consumer demands for reducing daily sugar intake. Despite the developpement of S. rebaudiana cultivation all over the world, the development of new cultivars is very recent, in particular due to a colossal lack of (1) germplasm collection and breeding, (2) studies on genetic diversity and its structuring, (3) genomic tools. RESULTS: In this study, we developped 18 EST-SSR from 150,258 EST from The Compositae Genome Project of UC Davis ( http://compgenomics.ucdavis.edu/data/ ). We genotyped 145 S. rebaudiana individuals, issued from thirty-one cultivars and thirty-one landraces of various origins worldwide. Markers polymorphic information content (PIC) ranged between 0.60 and 0.84. An average of 12 alleles per locus and a high observed heterozygoty of 0.69 could be observed. The landraces revealed twice as many private alleles as cultivars. The genotypes could be clustered into 3 genetic populations. The landraces were grouped in the same cluster in which the oldest cultivars "Eirete" and "MoritaIII" type are also found. The other two clusters only include cultivated genotypes. One of them revealed an original genetic variability. SG phenotypes could not discriminate the three genetic clusters but phenotyping showed a wide range of composition in terms of bitter to sweet SGs. CONCLUSION: This is the first study of genetic diversity in Stevia rebaudiana involving 145 genotypes, including known cultivars as well as landrace populations of different origin. This study pointed out the structuration of S. rebaudiana germplasm and the resource of the landrace populations for genetic improvement, even on the trait of SG's composition.

Genome-wide association study reveals new loci involved in Arabidopsis thaliana and Turnip mosaic virus (TuMV) interactions in the field.

The genetic architecture of plant response to viruses has often been studied in model nonnatural pathosystems under controlled conditions. There is an urgent need to elucidate the genetic architecture of the response to viruses in a natural setting. A field experiment was performed in each of two years. In total, 317 Arabidopsis thaliana accessions were inoculated with its natural Turnip mosaic virus (TuMV). The accessions were phenotyped for viral accumulation, frequency of infected plants, stem length and symptoms. Genome-wide association mapping was performed. Arabidopsis thaliana exhibits extensive natural variation in its response to TuMV in the field. The underlying genetic architecture reveals a more quantitative picture than in controlled conditions. Ten genomic regions were consistently identified across the two years. RTM3 (Restricted TEV Movement 3) is a major candidate for the response to TuMV in the field. New candidate genes include Dead box helicase 1, a Tim Barrel domain protein and the eukaryotic translation initiation factor eIF3b. To our knowledge, this study is the first to report the genetic architecture of quantitative response of A. thaliana to a naturally occurring virus in a field environment, thereby highlighting relevant candidate genes involved in plant virus interactions in nature.

Family-based linkage and association mapping reveals novel genes affecting Plum pox virus infection in Arabidopsis thaliana.

Sharka is a devastating viral disease caused by the Plum pox virus (PPV) in stone fruit trees and few sources of resistance are known in its natural hosts. Since any knowledge gained from Arabidopsis on plant virus susceptibility factors is likely to be transferable to crop species, Arabidopsis's natural variation was searched for host factors essential for PPV infection. To locate regions of the genome associated with susceptibility to PPV, linkage analysis was performed on six biparental populations as well as on multiparental lines. To refine quantitative trait locus (QTL) mapping, a genome-wide association analysis was carried out using 147 Arabidopsis accessions. Evidence was found for linkage on chromosomes 1, 3 and 5 with restriction of PPV long-distance movement. The most relevant signals occurred within a region at the bottom of chromosome 3, which comprises seven RTM3-like TRAF domain-containing genes. Since the resistance mechanism analyzed here is recessive and the rtm3 knockout mutant is susceptible to PPV infection, it suggests that other gene(s) present in the small identified region encompassing RTM3 are necessary for PPV long-distance movement. In consequence, we report here the occurrence of host factor(s) that are indispensable for virus long-distance movement.

RTM3, which controls long-distance movement of potyviruses, is a member of a new plant gene family encoding a meprin and TRAF homology domain-containing protein.

Restriction of long-distance movement of several potyviruses in Arabidopsis (Arabidopsis thaliana) is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2, and RTM3. RTM1 encodes a protein belonging to the jacalin family, and RTM2 encodes a protein that has similarities to small heat shock proteins. In this article, we describe the positional cloning of RTM3, which encodes a protein belonging to an undescribed protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its amino-terminal region and a coiled-coil domain at its carboxy-terminal end. Involvement in the RTM resistance system is the first biological function experimentally identified for a member of this new gene family in plants. Our analyses showed that the coiled-coil domain is not only highly conserved between RTM3-homologous MATH-containing proteins but also in proteins lacking a MATH domain. The cluster organization of the RTM3 homologs in the Arabidopsis genome suggests the role of duplication events in shaping the evolutionary history of this gene family, including the possibility of deletion or duplication of one or the other domain. Protein-protein interaction experiments revealed RTM3 self-interaction as well as an RTM1-RTM3 interaction. However, no interaction has been detected involving RTM2 or the potyviral coat protein previously shown to be the determinant necessary to overcome the RTM resistance. Taken together, these observations strongly suggest the RTM proteins might form a multiprotein complex in the resistance mechanism to block the long-distance movement of potyviruses.

Metabolic Profile Discriminates and Predicts Arabidopsis Susceptibility to Virus under Field Conditions.

As obligatory parasites, plant viruses alter host cellular metabolism. There is a lack of information on the variability of virus-induced metabolic responses among genetically diverse plants in a natural context with daily changing conditions. To decipher the metabolic landscape of plant-virus interactions in a natural setting, twenty-six and ten accessions of Arabidopsis thaliana were inoculated with Turnip mosaic virus (TuMV), in two field experiments over 2 years. The accessions were measured for viral accumulation, above-ground biomass, targeted and untargeted metabolic profiles. The phenotypes of the accessions ranged from susceptibility to resistance. Susceptible and resistant accessions were shown to have different metabolic routes after inoculation. Susceptible genotypes accumulate primary and secondary metabolites upon infection, at the cost of hindered growth. Twenty-one metabolic signatures significantly accumulated in resistant accessions whereas they maintained their growth as mock-inoculated plants without biomass penalty. Metabolic content was demonstrated to discriminate and be highly predictive of the susceptibility of inoculated Arabidopsis. This study is the first to describe the metabolic landscape of plant-virus interactions in a natural setting and its predictive link to susceptibility. It provides new insights on plant-virus interactions. In this undomesticated species and in ecologically realistic conditions, growth and resistance are in a permanent conversation.

Mapping with a few plants: using selective mapping for microsatellite saturation of the Prunus reference map.

The concept of selective (or bin) mapping is used here for the first time, using as an example the Prunus reference map constructed with an almond x peach F2 population. On the basis of this map, a set of six plants that jointly defined 65 possible different genotypes for the codominant markers mapped on it was selected. Sixty-three of these joint genotypes corresponded to a single chromosomal region (a bin) of the Prunus genome, and the two remaining corresponded to two bins each. The 67 bins defined by these six plants had a 7.8-cM average length and a maximum individual length of 24.7 cM. Using a unit of analysis composed of these six plants, their F1 hybrid parent, and one of the parents of the hybrid, we mapped 264 microsatellite (or simple-sequence repeat, SSR) markers from 401 different microsatellite primer pairs. Bin mapping proved to be a fast and economic strategy that could be used for further map saturation, the addition of valuable markers (such as those based on microsatellites or ESTs), and giving a wider scope to, and a more efficient use of, reference mapping populations.

Development and characterization of 96 microsatellite markers suitable for QTL mapping and accession control in an Arabidopsis core collection.

BACKGROUND: To identify plant genes involved in various key traits, QTL mapping is a powerful approach. This approach is based on the use of mapped molecular markers to identify genomic regions controlling quantitative traits followed by a fine mapping and eventually positional cloning of candidate genes. Mapping technologies using SNP markers are still rather expensive and not feasible in every laboratory. In contrast, microsatellite (also called SSR for Simple Sequence Repeat) markers are technologically less demanding and less costly for any laboratory interested in genetic mapping. RESULTS: In this study, we present the development and the characterization of a panel of 96 highly polymorphic SSR markers along the Arabidopsis thaliana genome allowing QTL mapping among accessions of the Versailles 24 core collection that covers a high percentage of the A. thaliana genetic diversity. These markers can be used for any QTL mapping analysis involving any of these accessions. We optimized the use of these markers in order to reveal polymorphism using standard PCR conditions and agarose gel electrophoresis. In addition, we showed that the use of only three of these markers allows differentiating all 24 accessions which makes this set of markers a powerful tool to control accession identity or any cross between any of these accessions. CONCLUSION: The set of SSR markers developed in this study provides a simple and efficient tool for any laboratory focusing on QTL mapping in A. thaliana and a simple means to control seed stock or crosses between accessions.

The RTM resistance to potyviruses in Arabidopsis thaliana: natural variation of the RTM genes and evidence for the implication of additional genes.

BACKGROUND: The non conventional RTM (Restricted Tobacco etch virus Movement) resistance which restricts long distance movement of some plant viruses in Arabidopsis thaliana is still poorly understood. Though at least three RTM genes have been identified, their precise role(s) in the process as well as whether other genes are involved needs to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the natural variation of the RTM genes was analysed at the amino acid level in relation with their functionality to restrict the long distance movement of Lettuce mosaic potyvirus (LMV). We identified non-functional RTM alleles in LMV-susceptible Arabidopsis accessions as well as some of the mutations leading to the non-functionality of the RTM proteins. Our data also indicate that more than 40% of the resistant accessions to LMV are controlled by the RTM genes. In addition, two new RTM loci were genetically identified. CONCLUSIONS/SIGNIFICANCE: Our results show that the RTM resistance seems to be a complex biological process which would involves at least five different proteins. The next challenges will be to understand how the different RTM protein domains are involved in the resistance mechanism and to characterise the new RTM genes for a better understanding of the blocking of the long distance transport of plant viruses.

A member of a new plant gene family encoding a meprin and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses.

Restriction of long distance movement of several potyviruses in Arabidopsis thaliana is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2 and RTM3 and acts as a non conventional resistance. RTM1 encodes a protein belonging to the jacalin family and RTM2 encodes a protein which has similarities to small heat shock proteins. The recent cloning of RTM3 which encodes a protein belonging to an unknown protein family of 29 members which has a meprin and TRAF homology (MATH) domain in its N-terminal region and a coiled-coil (CC) domain at its C-terminal end is an important breakthrough for a better understanding of this resistance process. Not only the third gene involved in this resistance has been identified and has allowed revealing a new gene family in plant but the discovery that the RTM3 protein interacts directly with RTM1 strongly suggests that the RTM proteins form a multimeric complex. However, these data also highlight striking similarities of the RTM resistance with the well known R-gene mediated resistance.

Comparative mapping and marker-assisted selection in Rosaceae fruit crops.

The development of saturated linkage maps using transferable markers, restriction fragment length polymorphisms, and micro-satellites has provided a foundation for fruit tree genetics and breeding. A Prunus reference map with 562 such markers is available, and a further set of 13 maps constructed with a subset of these markers has allowed genome comparison among seven Prunus diploid (x = 8) species (almond, peach, apricot, cherry, Prunus ferganensis, Prunus davidiana, and Prunus cerasifera); marker colinearity was the rule with all of them. Preliminary results of the comparison between apple and Prunus maps suggest a high level of synteny between these two genera. Conserved genomic regions have also been detected between Prunus and Arabidopsis. By using the data from different linkage maps anchored with the reference Prunus map, it has been possible to establish, in a general map, the position of 28 major genes affecting agronomic characters found in different species. Markers tightly linked to the major genes responsible for the expression of important traits (disease/pest resistances, fruit/nut quality, self-incompatibility, etc.) have been developed in apple and Prunus and are currently in use for marker-assisted selection in breeding programs. Quantitative character dissection using linkage maps and candidate gene approaches has already started. Genomic tools such as the Prunus physical map, large EST collections in both Prunus and Malus, and the establishment of the map position of high numbers of ESTs are required for a better understanding of the Rosaceae genome and to foster additional research and applications on fruit tree genetics.