Influence of altitude and enhanced ultraviolet-B radiation on tuber production, seed viability, leaf pigments and morphology in the wild potato species Solanum kurtzianum Bitter & Wittm collected from an elevational gradient.

Climate change could lead to an upward shift in plant distribution, exposing populations to higher levels of ultraviolet (UV)-B radiation. In the framework of an in situ strategy for conserving potato wild relatives, we evaluated the effect of high UV-B levels on natural population of Solanum kurtzianum. The hypothesis is that plants from naturally higher altitudes are more adapted to increased UV-B radiation. Two populations from low and high altitudes were field supplemented using UV-B-lamps (+UV-B) or excluded from it with plastic filters. Additionally, to assess in which extent the plant responses to these artificial experimental conditions are reproducible in natural conditions, three genotypes were cultivated in two mountain experimental gardens (EG) at different elevations. +UV-B treatment induced changes in leaf morphology and increases in phenolic compounds in both populations, indicating plant adaptation, since chlorophylls and reproductive structures were not negatively affected. These results indicate that this environmental factor may not limit the displacement of populations towards sites with higher UV-B levels. Meanwhile, in higher-altitude EG a tubers yield reduction, mainly through a decreased tuber number and a bigger accumulation of phenolic compounds than in +UV-B treatment were observed, suggesting that UV-B is not the only factor involved in plants adaptation to high altitude environments.

Short-term hybridisation activates Tnt1 and Tto1 Copia retrotransposons in wild tuber-bearing Solanum species.

Interspecific hybridisation in tuber-bearing species of Solanum is a common phenomenon and represents an important source of variability, crucial for adaptation and speciation of potato species. In this regard, the effects of interspecific hybridisation on retrotransposon families present in the genomes, and their consequent effects on generation of genetic variability in wild tuber-bearing Solanum species, are poorly characterised. The aim of this study was to analyse the activity of retrotransposons in inter- and intraspecific hybrids between S. kurtzianum and S. microdontum, obtained by controlled crosses, and the effects on morphological, genetic and epigenetic variability. For genetic and epigenetic analysis, S-SAP (sequence-specific amplification polymorphism) and TMD (transposon methylation display) techniques were used, respectively, with specific primers for Tnt1 and Tto1 retrotransposon families (Order LTR, Superfamily Copia). The results indicate that at morphological level, interspecific hybrid genotypes differ from their parental species, whereas derived intraspecific hybrids do not. In both cases, we observed significant reductions in pollen grain viability, and a negative correlation with Tnt1 mobility. Both retrotransposons, Tto1 and Tnt1, were mobilised in the genotypes analysed, with mobility ranging from 0 to 7.8%. Furthermore, at the epigenetic level, demethylation was detected in the vicinity of Tnt1 and Tto1 in the hybrids compared with the parental genotypes. These patterns were positively correlated with the activity of the retrotransposons. The results suggest a possible mechanism through which hybridisation events generate genetic variability in tuber-bearing species of Solanum through retrotranposon activation.

Reproductive ecology and genetic variability in natural populations of the wild potato, Solanum kurtzianum.

The cultivated potato (Solanum tuberosum ssp. tuberosum) has more than 200 related wild species distributed along the Andes, adapted to a wide range of geographical and ecological areas. Since the last century, several collection expeditions were carried out to incorporate genetic variability into the potato germplasm around the world. However, little is known about the reproductive ecology and genetic population structure of natural potato population from field studies. The aim of this work is to study, in the field, the genetic variability and reproductive strategies of populations of one of the most widely distributed potato species in Argentina, Solanum kurtzianum, growing in Mendoza province. AFLP markers showed that the genetic variability is mainly present among plants within populations, indicating that in the sampled populations, sexual reproduction is more relevant than clonal multiplication (by tubers). Additional evidence was obtained evaluating the genetic diversity in populations with a distribution in patches, where several genotypes were always detected. From a field study performed in the Villavicencio Natural Reserve, we found that the average number of plump seeds per fruit was 94.3, identified and calculated the foraging distance of four insect pollinators, and demonstrated the seed dispersal by storm water channels. We argue that the breeding system, the two modes of reproduction and the ecological interaction described here may have a prominent role in determining the genetic structure of S. kurtzianum populations, and discuss the importance of field studies on population genetics, reproductive biology and ecology to design collections and conservation strategies.

Molecular bases of the postzygotic barriers in interspecific crosses between the wild potato species Solanum acaule and Solanum commersonii.

To investigate the molecular bases of postzygotic hybridization barriers in tuber-bearing Solanums, the wild species Solanum commersonii Dunal ex Poir. (cmm, 2n = 2x = 24, 1EBN) and Solanum acaule Bitter (acl, 2n = 4x = 48, 2EBN) were crossed in intra- and interspecific genotypic combinations, and the transcriptome of immature seeds was analyzed by using the cDNA-AFLP technique. From a total of 423 analyzed cDNA fragments, 107 (25.3%) were differentially regulated in the compatible (acl × acl and cmm × cmm) versus incompatible (acl × cmm) crosses. DNA sequence data were obtained from 21 fragments and RT-PCR analyses were carried out with five fragments to validate the cDNA-AFLP differential pattern. Sequence analysis suggested a possible role for the differentially expressed sequences in cytokinesis, cell cycle, secondary and hormonal metabolism, biodegradation, and transport. In situ hybridization experiments with fragments encoding an ubiquitin-fold modifier 1 precursor and a possible vesicle transport protein revealed expression of these genes in the embryo and endosperm. The results suggest that the collapse of the embryo and endosperm in incompatible crosses may be related to alterations in cell cycle and cytokinesis.

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.(AU)

Genomic instability in Solanum tuberosum x Solanum kurtzianum interspecific hybrids.

The use of interspecific crosses in breeding is an important strategy in improving the genetic base of the modern cultivated potato, Solanum tuberosum L. Until now, it has normally been interspecific Solanum hybrids that have been morphologically and cytologically characterized. However, little is known about the genomic changes that may occur in the hybrid nucleus owing to the combination of genomes of different origin. We have observed novel AFLP bands in Solanum tuberosum x Solanum kurtzianum diploid hybrids; 40 novel fragments were detected out of 138 AFLP fragments analyzed. No cytological abnormalities were observed in the hybrids; however, we found DNA methylation changes that could be the cause of the observed genomic instabilities. Of 277 MSAP fragments analyzed, 14% showed methylation patterns that differed between the parental species and the hybrids. We also observed frequent methylation changes in the BC1 progeny. Variation patterns among F1 and BC1 plants suggest that some methylation changes occurred at random. The changes observed may have implications for potato breeding as an additional source of variability.

The importance of Arabidopsis seed mutants in the elucidation of the molecular basis of Endosperm Balance Number in tuber-bearing Solanum species.

The Endosperm Balance Number (EBN) is an important concept for potato breeding and has evolutionary importance in tuber-bearing Solanum species. The EBN is part of the post-zygotic hybridization barriers in the group and represents a reproductive isolating mechanism. Few genes have been proposed to be involved in its genetic control; until now, however, neither specific genes nor its molecular basis have been well established. Histological observations of embryo and endosperm development in inter-EBN crosses in tuber-bearing Solanum revealed phenotypes similar to those recently described in Arabidopsis seed mutants. The common feature between them is that the endosperm nuclei become greatly enlarged and that embryos are arrested at the globular stage. The proteins encoded by the Arabidopsis TITAN genes are related to chromosome dynamics and cell division. Based on the sequence of titan mutants, genes in potato species related to cell cycle and microtubule assembly were isolated. In this article a perspective model is proposed to explore the utility of Arabidopsis mutants associated with cell cycle control as a tool to elucidate the molecular basis of EBN in potato. Further research focused on the expression pattern of these genes in intra- and inter-EBN crosses in potato species will be performed.

Mitotic-cycle time and the development of embryo and endosperm in compatible and incompatible crosses in tuber-bearing Solanum species.

To understand the relationship between early seed development and the EBN (endosperm balance number) hypothesis, the embryo and endosperm growth rates in crosses among Solanum spp. with the same and different EBNs were analyzed. For the embryo, the differences in the mean cell-doubling time (MCDT) between the compatible species 2x Solanum gourlayi and Solanum acaule was 3.9 h, whereas the incompatible species Solanum commersonii had MCDT differences of 10 and 13.9 h with 2x S. gourlayi and S. acaule, respectively. The embryo growth rates of the 2EBN species S. acaule and S. gourlayi were almost twice as fast as that of the 1EBN species S. commersonii. Nuclei of variable sizes were observed in the endosperms resulting from incompatible crosses. The author discusses the possibility that the collapse of the endosperm in inter-EBN crosses could be caused by differences in the MCDT between the parents that produce hybrid endosperms with high levels of DNA synthesis and transcription activity, resulting in increased nuclear size. A model is proposed to explain the formation of enlarged endosperm nuclei in incompatible crosses in the genus Solanum.

Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis.

A system of randomly amplified polymorphic DNA (RAPD) markers was developed to facilitate the transfer of S. bulbocastanum (blb) genes into the S. tuberosum (tbr) genome by hybridization and backcrossing. DNA from tbr, blb and the hexaploid hybrid was used as a template for polymerase chain reaction (PCR) amplification. Polymorphic RAPD products, originating from 10-mer primers, specific for blb were cloned and sequenced at their ends to allow the synthesis of 18-mer primers. The 18-mer primers allowed a more reproducible assay than the corresponding RAPDs. Of eight 18-mer primer pairs, four amplified the expected products specific for blb. However, the stringency of the primer annealing conditions needed to be carefully optimized to avoid amplification of the homeologous tbr product, suggesting that the original RAPD polymorphisms were due to single base-pair changes rather than deletions or insertions. Two primers used for amplification of backcross 2 progeny segregated in a 1∶1 (presence:absence) ratio; the other two were unexpectedly absent. The most likely explanation for the loss of these markers is irregular meiosis in the original hexaploid hybrid and subsequent elimination of chromosomes. Cytological analysis of the meiosis in the hybrid demonstrated widespread irregular pairing and the presence of lagging univalents. In addition, the first backcross individual used as the parent for the second backcross had 54 chromosomes instead of the predicted 60. In conclusion, our results demonstrate that PCR technology can be used for the efficient isolation of taxon-specific markers in Solanum. Furthermore, by the use of these markers we detected the loss of chromosomes that was subsequently shown by cytological analysis to be caused by irregular meiosis of the somatic hybrid.