Expressed sequence enrichment for candidate gene analysis of citrus tristeza virus resistance.

Several studies have reported markers linked to a putative resistance gene from Poncirus trifoliata ( Ctv-R) located at linkage group 4 that confers resistance against one of the most important citrus pathogens, citrus tristeza virus (CTV). To be successful in both marker-assisted selection and transformation experiments, its accurate mapping is needed. Several factors may affect its localization, among them two are considered here: the definition of resistance and the genetic background of progeny. Two progenies derived from P. trifoliata, by self-pollination and by crossing with sour orange ( Citrus aurantium), a citrus rootstock well-adapted to arid and semi-arid areas, were used for linkage group-4 marker enrichment. Two new methodologies were used to enrich this region with expressed sequences. The enrichment of group 4 resulted in the fusion of several C. aurantium linkage groups. The new one A(7+3+4) is now saturated with 48 markers including expressed sequences. Surprisingly, sour orange was as resistant to the CTV isolate tested as was P. trifoliata, and three hybrids that carry Ctv-R, as deduced from its flanking markers, are susceptible to CTV. The new linkage maps were used to map Ctv-R under the hypothesis of monogenic inheritance. Its position on linkage group 4 of P. trifoliata differs from the location previously reported in other progenies. The genetic analysis of virus-plant interaction in the family derived from C. aurantium after a CTV chronic infection showed the segregation of five types of interaction, which is not compatible with the hypothesis of a single gene controlling resistance. Two major issues are discussed: another type of genetic analysis of CTV resistance is needed to avoid the assumption of monogenic inheritance, and transferring Ctv-R from P. trifoliata to sour orange might not avoid the CTV decline of sweet orange trees.

The diversification of Citrus clementina Hort. ex Tan., a vegetatively propagated crop species.

Clementines, due to their high quality, are one of the most important cultivated citrus mandarins. As in the case of sweet orange and satsuma mandarins, genetic variability within this species is minimal when analyzed by molecular markers, because the existing varieties have not been obtained through hybridization, but through the selection of spontaneous mutations affecting traits of agronomic interest. This would explain, at least in part, the greater diversity for agronomic traits when compared to the variability for molecular markers. Another possible (nonexclusive) reason is that the types of molecular marker used are not focused on the kind of molecular change mainly involved in the origination of new clementine cultivars; i.e., are all sources of variation equally involved in the diversification of these plants? To answer this question, different kinds of markers based on primers of random sequence, simple sequence repeats, and retrotransposon sequences that may reveal point mutations, and somatic recombination and transposon activity, respectively, were used to compare the level of variability among 24 clementine varieties. Their ISSR, RAPD, and AFLP analysis provided only two polymorphic bands, distinguishing just two varieties. No variability was found by SSRs, i.e., no new allele arising through somatic recombination was detected. Instead, the amplification of sequences adjacent to retrotransposons yielded a higher number of polymorphisms (14.6 vs 2.4% for the previous mentioned marker types). Two geographical distant groups, one from North Africa and the other from Spain, have evolved in agreement with polymorphisms based on IRAP markers anchored to, at least, two different Copia-like retrotransposon sequences. Therefore, this study suggests that the DNA of this type of mobile elements is evolving faster than the DNA of other markers in this clonal lineage.

Salt tolerance in Lycopersicon species. III. Detection of quantitative trait loci by means of molecular markers.

A segregating population derived from a cross between L. esculentum cv Madrigal and a line of L. pimpinellifolium was used to identify genetic markers linked to QTLs involved in salinity tolerance in terms of yield, under a conductivity of 15 dS/m (171.1 mM NaCl). Six markers resulted, associated with QTLs affecting average fruit weight, fruit number and total weight under salinity. One of them, Aco-1, behaves reversely to the expectation from parental means; this and other features make it a promising target to obtain salt-tolerant tomatoes. Epistatic interactions were also found, thus affecting the criteria for marker-assisted selection. Although only 41% of the loci assayed were polymorphic, a high efficiency in identifying QTLs was achieved, since 43% of the marker loci are linked to QTLs for the trait under study.

Genetic variability in Lycopersicon species and their genetic relationships.

Genetic diversity has to be described and measured in order to establish breeding strategies and manage genetic resources. It is also fundamental to develop a comparative intraspecific study before attempting to discuss and conclude any phylogenetic relationship. The genetic variability of Lycopersicon species was studied using starch gel electrophoresis of 11 enzymatic systems in a hierarchical fashion. The species with the greatest genetic variability are L. chilense, L. peruvianum and L. pennellii, mainly due to the within-line component. L. chmielewskii, L. parviflorum and L. pimpinellifolium show an intermediate total variability and their between-component clearly predominates over the within-component. The least variable species are L. cheesmanii and L. esculentum. Cluster analysis resulted in three main groups: one formed by the cultigen, L. pimpinellifolium, L. cheesmanii and L. peruvianum;another by two species with self-incompatibility systems, L. pennelli and L. chilense; and another by two autogamous species L. chmielewskii and L. parviflorum. With respect to L. esculentum the farthest related species is Solanum rickii and the closest, L. pimpinellifolium.

Salt tolerance in Lycopersicon species. I. Character definition and changes in gene expression.

Salt tolerance defined in terms of fruit yield under different NaCl concentrations (171.1 and 325.1 mM) is analyzed in 11 lines belonging to: Lycopersicon esculentum, L. cheesmanii, L. chmielewski, L. peruvianum and L. pimpinellifolium. Four L. pimpinellifolium lines and two L. cheesmanii lines tolerated the 171.1mM treatment; the latter species even tolerates 325.1 mM of NaCl. Changes in gene expression induced by salt treatment were also investigated by studying anther and leaf zymograms for L. esculentum and one salt-tolerant L. pimpinellifolium line, and leaf proteinograms for all lines. Changes in leaf PRX and MDH enzymatic systems were detected, mainly in the salt-sensitive genotype (L. esculentum). Four saltrelated peptides from 14 500 to 40 000 daltons were found. A polyclonal antibody raised against one of these peptides (number 2), also binds another peptide, named 2', of much higher molecular weight, present both in control and salt-tolerant L. cheesmanii lines at the end of 171.1 mM treatment. The xero-halophyte shrub Atriplex halimus also showed a likely 2'-homologous peptide with this treatment, while its counterpart C3 species A. triangularis did not.

Salt tolerance in Lycopersicon species. II. Genetic effects and a search for associated traits.

Eleven quantitative traits, mostly related to tomato plant growth and fruit set, and their association with salt tolerance in terms of fruit yield under a 171.1 mM NaCl treatment have been investigated in 206 progeny derived from an interspecific hybrid, L. esculentum x L. pimpinellifolium, by self-pollination. None of the traits were highly correlated phenotypically to salt tolerance; however, the immunologically-detected presence of peptide 2' was significantly associated with high total fruit weight (TW) and number (FN) under saline treatment. Broad-sense heritability was estimated for these two salt-tolerance components as 53.44 and 72.59 %, respectively. Non-additive gene effects, which have to be considered in a breeding program for salt tolerance, have been detected in TW, FN and in average fruit weight (FW). Given that different types of gene action have been found depending on the presence or absence of a high NaCl concentration in the nutrient solution, a different set of genes, or genes, differently regulated, must be involved in the expression of TW, FN and other fruit-related characters depending on this environmental condition.