Ricardo W. Masuelli

Crossability relationships among wild potato-species with different ploidies and Endosperm Balance Numbers (EBN)

Three wild potato species with different ploidies and Endosperm Balance Numbers (EBN) were crossed in a complete diallel design and the development of the embryo and endosperm as well as the type of seeds produced were analyzed. The compatible crosses – intraspecific intra-EBN and interspecific intra-EBN – produced more than 89% plump seeds, whereas in the incompatible crosses – intraspecific inter-EBN and interspecific inter-EBN – more than 85% of the seeds were not as well developed or were shrunken. The histological analysis revealed that inviable seeds had less developed or collapsed endosperms and thicker endotheliums than viable ones. A gradation of crossabilities was observed among species. The self-compatible species Solanum acaule had good performance as a female but not as a male parent. Among the self-incompatible species, Solanum gourlayi was the best male parent but had a poor performance as a female, whereas Solanum commersonii had an intermediate behavior. Differences in crossabilities among genotypes within species were also observed. These results can not be solely explained by the EBN hypothesis. It is, therefore, suggested that the EBN may be part of a more complex system of interspecific barriers acting at the pre- and post-zygotic levels.

Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii

BackgroundThe wild potato Solanum ruiz-lealii Brüch. (2n = 2x = 24), a species of hybrid origin, is endemic to Mendoza province, Argentina. Recurrent flower malformations, which varied among inflorescences of the same plant, were observed in a natural population. These abnormalities could be the result of genomic instabilities, nucleus-cytoplasmic incompatibility or epigenetic changes. To shed some light on their origin, nuclear and mitochondrial DNA of plants with normal and plants with both normal and malformed flowers (from here on designated as plants with normal and plants with abnormal flower phenotypes, respectively) were analyzed by AFLP and restriction analyses, respectively. Also, the wide genome methylation status and the level of methylation of a repetitive sequence were studied by MSAP and Southern blots analyses, respectively.ResultsAFLP markers and restriction patterns of mitochondrial DNA did not allow the differentiation of normal from abnormal flower phenotypes. However, methylation patterns of nuclear DNA discriminated normal and abnormal flower phenotypes into two different groups, indicating that abnormal phenotypes have a similar methylation status which, in turn, was different from the methylation patterns of normal phenotypes. The abnormal flower phenotype was obtained by treating a normal plant with 5-Azacytidine, a demethylating agent, giving support to the idea of the role of DNA methylation in the origin of flower abnormalities. In addition, the variability detected for DNA methylation was greater than the detected for nucleotide sequence.ConclusionThe epigenetic nature of the observed flower abnormalities is consistent with the results and indicates that in the diploid hybrid studied, natural variation in methylation profiles of anonymous DNA sequences could be of biological significance.

Assessment of somaclonal variation in asparagus by RAPD fingerprinting and cytogenetic analyses

Abstract RAPD and cytogenetic analyses were carried out to detect somaclonal variation in somatic embryo-derived plants from two elite genotypes (‘265’ and ‘357’) of Asparagus officinalis L. cv. Argenteuil. These two approaches were chosen for their capacity to detect, respectively, changes at the level of DNA sequence or chromosomal structure. Seventy-seven plants regenerated from three embryogenic lines (‘265-1’, ‘265-2’ and ‘357-1’) exhibited no intraclonal variation for 157 RAPD markers, even though two plants from line ‘357-1’ presented dwarf phenotypes. Cytogenetic analyses revealed that the dwarf plants were mixoploid; two other, phenotypically normal plants from line ‘357-1’ were aneuploids and presented many meiotic irregularities. The remaining regenerants from this and the other lines were genetically stable with regard to the donor plants. Molecular markers and cytogenetic analysis are complementary approaches that should allow a correct assessment of somaclonal variation in asparagus.

An endophytic bacterium isolated from roots of the halophyte Prosopis strombulifera produces ABA, IAA, gibberellins A1 and A3 and jasmonic acid in chemically-defined culture medium

This paper informs the characterization by 16SrDNA partial sequence analysis of an endophytic diazotrophic bacterium isolated from roots of the halophyte shrub Prosopis strombulifera. The bacterium produced ABA, IAA, GA1, GA3 and jasmonic acid in chemically-defined culture medium as assessed by GC-EIMS. The results emphasize the role of phytohormones produced by endophytic bacteria in the association host-beneficial microorganisms, especially under conditions of adverse environments.

Changes in micro RNA expression in a wild tuber-bearing Solanum species induced by 5-Azacytidine treatment

Phenotypic plasticity is often postulated as a principal characteristic of tuber-bearing wild Solanum species. The hypotheses to explore this observation have been developed based on the presence of genetic variation. In this context, evolutionary changes and adaptation are impossible without genetic variation. However, epigenetic effects, which include DNA methylation and microRNAs expression control, could be another source of phenotypic variation in ecologically relevant traits. To achieve a detailed mechanistic understanding of these processes, it is necessary to separate epigenetic from DNA sequence-based effects and to evaluate their relative importance on phenotypic variability. We explored the potential relevance of epigenetic effects in individuals with the same genotype. For this purpose, a clone of the wild potato Solanum ruiz-lealii, a non-model species in which natural methylation variability has been demonstrated, was selected and its DNA methylation was manipulated applying 5-Azacytidine (AzaC), a demethylating agent. The AzaC treatment induced early flowering and changes in leaf morphology. Using quantitative real-time PCR, we identified four miRNAs up-regulated in the AzaC-treated plants. One of them, miRNA172, could play a role on the early flowering phenotype. In this work, we showed that the treatment with AzaC could provide meaningful results allowing to study both the phenotypic plasticity in tuber-bearing Solanum species and the inter-relation between DNA methylation and miRNA accumulations in a wide range of species.

Examination of the hybrid origin of the wild potato Solanum ruiz-lealii Brücher

The common potato, tetraploid Solanum tuberosum spp. tuberosum L. (tbr), has a narrow genetic base but a large number of related tuber-bearing species that harbor genetic diversity for agronomic characters. The taxonomic status of Solanum ruiz-lealii Brücher (rzl), a diploid species endemic to Mendoza province, Argentina, is controversial. It has been described as a new species of non-hybrid origin and as a natural hybrid between S. kurtzianum Bitt. & Wittm. (ktz) and S. chacoense Bitt. (chc). The hypothesis of the hybrid origin of rzl is examined systematically for the first time by phenetic analyses of morphological and molecular (SSR markers) data, and cytological analyses of interspecific hybrids. The morphological, cytological and molecular data are congruent, and suggest that rzl is not a recent natural hybrid between the ktz and the chc populations studied but has probably originated by divergence of chc, or by hybridization between chc and another taxon.

Pollen-mediated gene flow from a commercial potato cultivar to the wild relative S. chacoense Bitter under experimental field conditions in Argentina

The common potato, Solanum tuberosum ssp. tuberosum (tbr, 2n = 4x = 48; 4EBN), has many closely related wild tuber-bearing species. Around 28 to 35 of them spontaneously grow in Argentina overlapping, in some areas, with the crop and/or experimental transgenic potatoes. Although it is well proven that hybridization barriers in potatoes can be incomplete, information on gene flow between cultivated and wild germplasm is scarce. Thus, a gene flow field experiment with a circular array was set up in Balcarce, Argentina, in 2009, and evaluated over two seasons. The tetraploid tbr cultivar Huinkul MAG and one compatible cloned genotype of the related wild potato S. chacoense Bitter (chc, 2n = 2x = 24; 2EBN), which produced 2n eggs, were used, respectively, as pollen donor and receptor. Berries with hybrid seeds - as revealed by ploidy and RAPD profiles - were obtained in one season, at 30 m from the pollen donor. These results reinforce others previously obtained with the same pollen donor and a male sterile tbr cultivar in a similar array, pointing out to the need of increasing isolation distances in areas of overlap between cultivated and wild potato germplasm to prevent or minimize undesirable pollen-mediated gene flow.

Evolution of nematode-resistant Mi-1 gene homologs in three species of Solanum

Plants have evolved several defense mechanisms, including resistance genes. Resistance to the root-knot nematode Meloidogyneincognita has been found in wild plant species. The molecular basis for this resistance has been best studied in the wild tomato Solanum peruvianum and it is based on a single dominant gene, Mi-1.2, which is found in a cluster of seven genes. This nematode attacks fiercely several crops, including potatoes. The genomic arrangement, number of copies, function and evolution of Mi-1 homologs in potatoes remain unknown. In this study, we analyzed partial genome sequences of the cultivated potato species S. tuberosum and S. phureja and identified 59 Mi-1 homologs. Mi-1 homologs in S. tuberosum seem to be arranged in clusters and located on chromosome 6 of the potato genome. Previous studies have suggested that Mi-1 genes in tomato evolved rapidly by frequent sequence exchanges among gene copies within the same cluster, losing orthologous relationships. In contrast, Mi-1 homologs from cultivated potato species (S. tuberosum and S. phureja) seem to have evolved by a birth-and-death process, in which genes evolve mostly by mutations and interallelic recombinations in addition to sequence exchanges.

Comparison of two detection systems to reveal AFLP markers in plants

Since their development, AFLP (amplified fragment length polymorphism) markers have been used for a wide variety of analyses and, up to this day, are considered highly informative, robust, and reproducible molecular markers. Originally, the visualization of the amplified fragments was done in polyacrylamide gels, followed by silver staining or by developing in an X-ray plate, when radioactivity is used. In the last 14 years, capillary electrophoresis of fluorescently labeled fragments has been gradually replacing gel-based systems. However, the latter continue to be better for isolating and cloning AFLP fragments. In this report, we compare the results obtained by capillary electrophoresis with those from silver staining. We found that if fluorescence-labeled amplification products are loaded in a polyacrylamide gel, duplicated bands (doublets) are seen. This phenomenon is probably due to a delay in the migration of the strand that carries the fluorophore. Therefore, we recommend a minimum separation of 4...

Genetic and morphological variability in populations of the wild diploid potato species Solanum maglia and Solanum kurtzianum from Argentina

Solanum maglia Schltdl., a wild potato species that has its widest geographical distribution in Chile, is restricted in Argentina to Quebrada La Cumbre (quebrada = gorge), Mendoza province, where no other potato species has been re- ported. During two collecting expeditions carried out in 2006 and 2007 in this gorge, tubers of two potato populations sep- arated by 500 m (area 1 and 2) were sampled. The morphological and genetic diversity of these two newly sampled populations and of five accessions from the same gorge classified as S. maglia, a plant of Solanum kurtzianum collected outside this gorge, and a Chilean plant of S. maglia were examined by comparing 24 morphological characters and electro- phoretic patterns for simple sequence repeat and amplified fragment length polymorphism markers, respectively. Based on the current taxonomic concept, the results support the classification of plants from area 1 as S. kurtzianum and those from area 2 as S. maglia, except for one plant from the former area, which shared electrophoretic bands from both species. This is the first report on the sympatry of populations of the two species in Quebrada La Cumbre. The importance of perform- ing in situ population studies in the same sites over years is discussed.