Deep-sequencing of Solanum commersonii small RNA libraries reveals riboregulators involved in cold stress response.

Among wild species used in potato breeding, Solanum commersonii displays the highest tolerance to low temperatures under both acclimated (ACC) and non-acclimated (NACC) conditions. It is also the first wild potato relative with a known whole genome sequence. Recent studies have shown that abiotic stresses induce changes in the expression of many small non-coding RNA (sncRNA). We determined the small non-coding RNA (sncRNAome) of two clones of S. commersonii contrasting in their cold response phenotypes via smRNAseq. Differential analysis provided evidence that expression of several miRNAs changed in response to cold stress conditions. Conserved miR408a and miR408b changed their expression under NACC conditions, whereas miR156 and miR169 were differentially expressed only under ACC conditions. We also report changes in tasiRNA and secondary siRNA expression under both stress conditions. Our results reveal possible roles of sncRNA in the regulatory networks associated with tolerance to low temperatures and provide useful information for a more strategic use of genomic resources in potato breeding.

Exploring tomato Solanum pennellii introgression lines for residual biomass and enzymatic digestibility traits.

BACKGROUND: Residual biomass production for fuel conversion represents a unique opportunity to avoid concerns about compromising food supply by using dedicated feedstock crops. Developing tomato varieties suitable for both food consumption and fuel conversion requires the establishment of new selection methods. RESULTS: A tomato Solanum pennellii introgression population was assessed for fruit yield, biomass phenotypic diversity, and for saccharification potential. Introgression lines 2-5, 2-6, 6-3, 7-2, 10-2 and 12-4 showed the best combination of fruit and residual biomass production. Lignin, cellulose, hemicellulose content and saccharification rate showed a wide variation in the tested lines. Within hemicellulose, xylose value was high in IL 6-3, IL 7-2 and IL 6-2, whereas arabinose showed a low content in IL 10-2, IL 6-3 and IL 2-6. The latter line showed also the highest ethanol potential production. Alkali pre-treatment resulted in the highest values of saccharification in most of lines tested, suggesting that chemical pretreatment is an important factor for improving biomass processability. Interestingly, extreme genotypes for more than one single trait were found, allowing the identification of better genotypes. Cell wall related genes mapping in genomic regions involved into tomato biomass production and digestibility variation highlighted potential candidate genes. Molecular expression profile of few of them provided useful information about challenged pathways. CONCLUSIONS: The screening of S. pennellii introgression population resulted very useful for delving into complex traits such as biomass production and digestibility. The extreme genotypes identified could be fruitfully employed for both genetic studies and breeding.

Phenotypic and molecular characterization of plants regenerated from non-cryopreserved and cryopreserved wild Solanum lycopersicum mill. Seeds.

BACKGROUND: Before cryopreservation is routinely used, its effect on the trueness-to-type of the regenerated plant material needs to be evaluated. OBJECTIVE: In this work, we studied the effect of seed cryopreservation on the phenotypic and molecular characteristics of wild Solanum lycopersicum Mill. plants. METHODS: Thirty-five morphological traits of plants regenerated from cryopreserved seeds were compared to those measured on plants regenerated from non-cryopreserved seeds. RESULT: No statistically significant differences were observed between cryopreserved and non-cryopreserved samples, either in the first or in the second generation post-liquid nitrogen exposure. However, at the molecular level, the genetic analyses performed on the second generation plants germinated from control and cryopreserved seeds using 14 nuclear Simple Sequences Repeats (SSR) markers uncovered some changes in microsatellite length between control and cryopreserved samples. These results confirm at the botanical phenotype level the effectiveness of seed cryostorage for conservation and regeneration of true-to-type S. lycopersicum plants. CONCLUSION: Further experiments are required to clarify potential phenotypic effects of the changes observed in the DNA.

Overview of tomato (Solanum lycopersicum) candidate pathogen recognition genes reveals important Solanum R locus dynamics.

To investigate the genome-wide spatial arrangement of R loci, a complete catalogue of tomato (Solanum lycopersicum) and potato (Solanum tuberosum) nucleotide-binding site (NBS) NBS, receptor-like protein (RLP) and receptor-like kinase (RLK) gene repertories was generated. Candidate pathogen recognition genes were characterized with respect to structural diversity, phylogenetic relationships and chromosomal distribution. NBS genes frequently occur in clusters of related gene copies that also include RLP or RLK genes. This scenario is compatible with the existence of selective pressures optimizing coordinated transcription. A number of duplication events associated with lineage-specific evolution were discovered. These findings suggest that different evolutionary mechanisms shaped pathogen recognition gene cluster architecture to expand and to modulate the defence repertoire. Analysis of pathogen recognition gene clusters associated with documented resistance function allowed the identification of adaptive divergence events and the reconstruction of the evolution history of these loci. Differences in candidate pathogen recognition gene number and organization were found between tomato and potato. Most candidate pathogen recognition gene orthologues were distributed at less than perfectly matching positions, suggesting an ongoing lineage-specific rearrangement. Indeed, a local expansion of Toll/Interleukin-1 receptor (TIR)-NBS-leucine-rich repeat (LRR) (TNL) genes in the potato genome was evident. Taken together, these findings have implications for improved understanding of the mechanisms of molecular adaptive selection at Solanum R loci.

High-throughput genomics enhances tomato breeding efficiency.

Tomato (Solanum lycopersicum) is considered a model plant species for a group of economically important crops, such as potato, pepper, eggplant, since it exhibits a reduced genomic size (950 Mb), a short generation time, and routine transformation technologies. Moreover, it shares with the other Solanaceous plants the same haploid chromosome number and a high level of conserved genomic organization. Finally, many genomic and genetic resources are actually available for tomato, and the sequencing of its genome is in progress. These features make tomato an ideal species for theoretical studies and practical applications in the genomics field. The present review describes how structural genomics assist the selection of new varieties resistant to pathogens that cause damage to this crop. Many molecular markers highly linked to resistance genes and cloned resistance genes are available and could be used for a high-throughput screening of multiresistant varieties. Moreover, a new genomics-assisted breeding approach for improving fruit quality is presented and discussed. It relies on the identification of genetic mechanisms controlling the trait of interest through functional genomics tools. Following this approach, polymorphisms in major gene sequences responsible for variability in the expression of the trait under study are then exploited for tracking simultaneously favourable allele combinations in breeding programs using high-throughput genomic technologies. This aims at pyramiding in the genetic background of commercial cultivars alleles that increase their performances. In conclusion, tomato breeding strategies supported by advanced technologies are expected to target increased productivity and lower costs of improved genotypes even for complex traits.

Assessment of genetic variability of haploids extracted from tetraploid (2n = 4x = 48) Solanum tuberosum.

The objectives of this study were to assess the genetic variability of haploids (2n = 2x = 24) extracted from tetraploid Solanum tuberosum through 4x x 2x crosses with Solanum phureja. Molecular and phenotypic analyses were performed to fingerprint the genotypes used and to evaluate their potential use in breeding programs. AFLP analysis revealed the presence of specific bands derived from the tetraploid seed parent S. phureja, as well as ex novo originated bands. On average, 210 bands were visualized per genotype, 149 (70%) of which were common to both parental genotypes. The percentage of S. tuberosum specific bands ranged from 25.1% to 18.6%, with an average of 22%. The fraction of genome coming from S. phureja ranged from 1.9% to 6.5%, with an average value of 4%. The percentage of ex novo bands varied from 1.9% to 9.0%. The presence of S. phureja DNA is very interesting because it indicated that S. phureja pollinator is involved in the mechanism of haploid formation. The characterization for resistance to Erwinia carotovora subsp. carotovora and potato virus X (PVX) provided evidence that haploids may express traits that are lacking in the tetraploids they come from, which can be useful for both genetic studies and breeding purposes. It is noteworthy that genotypes combining resistance to both diseases and good pollen stainability were identified. Other possible breeding implications owing to the presence of S. phureja genome in the haploids analyzed are discussed.

Selection for aneuploid potato hybrids combining a low wild genome content and resistance traits from Solanum commersonii.

A breeding scheme based on the production of progenies with odd ploidy was followed to introduce useful genes from the wild Solanum commersonii (cmm) into S. tuberosum (tbr) genome. Hybrids from 5 x x 4 x crosses were characterized for traits of interest, and selection was assisted by amplified fragment length polymorphism (AFLP) analysis. As expected, most of the hybrids were aneuploids, with a trend towards a low degree of aneuploidy. Despite the fact that aneuploidy has often been associated with a reduction in male and female fertility, most of the hybrids were fertile following crosses with tbr, making it possible to produce viable offspring. A screening for resistance traits deriving from cmm was also carried out. With respect to freezing resistance, the killing temperatures of cold-acclimated genotypes were distributed between the wild and cultivated parental values, with some hybrids displaying an acclimation capacity higher than 3 degrees C. A wide variability was also found for tuber soft rot resistance, and hybrids with high levels of resistance were identified. Selection of hybrids was based on a two-stage scheme that consisted of conventional phenotypic selection followed by an estimation of the wild genome content still present in order to identify hybrids combining noteworthy traits with a low wild genome content. Previously selected cmm-specific AFLPs were used to monitor the degree of wild genome content still present in each hybrid. The percentage of cmm-specific markers ranged from 59% to 91%, with an average value of 75%. AFLP analysis was employed to assist in the selection of valuable hybrids for further breeding efforts.

Substitutes for genome differentiation in tuber-bearing Solanum: interspecific pollen-pistil incompatibility, nuclear-cytoplasmic male sterility, and endosperm.

The cultivated potato, Solanum tuberosum L. (2n=4x=48), has a very large number of related wild and cultivated tuber-bearing species widely distributed in the Americas. These species, grouped in 16 taxonomic series, range from the diploid to the hexaploid level. Polyploid species are either disomic or polysomic, and sexual polyploidization via genetically controlled 2 n gametes has played a major role in their evolution. Species are separated in nature by geographical and ecological barriers. However, there are several examples of sympatric species that share the same niches but do not readily cross (i.e., the diploids S. commersonii and S. chacoense in certain areas of Argentina). External barriers alone are, therefore, not sufficient to explain species integrity. In addition, there is no strong evidence indicating that genome differentiation is important in the group. In this review we present evidence supporting the assertion that interspecific pollen-pistil incompatibility, nuclear-cytoplasmic male sterility, and the endosperm are major forces that strengthen the external hybridization barriers allowing, at the same time and under specific circumstances, a certain amount of gene exchange without jeopardizing the integrity of the species.

Glycoalkaloids and acclimation capacity of hybrids between Solanum tuberosum and the incongruent hardy species Solanum commersonii.

F(1) and backcross hybrids between sexually incompatible species Solanum commersonii and Solanum tuberosum were characterized for glycoalkaloid content and capacity to cold acclimate. Glycoalkaloid (GA) analysis revealed that F(1) triploids and BC(1) pentaploids contained the glycoalkaloids of both parents. In BC(2) (near) tetraploids the situation was different, in that some hybrids produced the GAs of both parents, whereas others contained only the GAs of S. tuberosum. This suggested that the GAs from S. commersonii may be lost rapidly, and that they may have a simple genetic control. The total tuber GA content of BC(1) and BC(2) groups averaged quite acceptable levels (165.9 mg/kg in BC(1) and 192.8 mg/kg in BC(2)), with six genotypes having a GA content <200 mg/kg fresh weight. The F(1) triploid hybrids expressed a capacity to cold acclimate similar to S. commersonii, whereas BC(1) and BC(2) genotypes generally displayed an acclimation capacity higher than the sensitive parent but lower than S. commersonii. However, one BC(1) and two BC(2) genotypes with an acclimation capacity as high as S. commersonii were identified. The polar lipid fatty acid composition in S. commersonii and its hybrid derivatives showed that, following acclimation, there was a significant increase in 18:3. Correlation analysis between the capacity to cold acclimate and the increase in 18:3 was significant, suggesting that the increase in 18:3 can be used as a biochemical marker for the assisted selection of cold-acclimating genotypes in segregating populations.

Cytological and breeding behavior of pentaploids derived from 3x x 4x crosses in potato.

Cytology and breeding behavior of Solanum commersonii - S. tuberosum hybrids derived from 3 x x 4 x crosses was examined. The chromosome number of hybrids ranged from hypo-pentaploid (2 n=5 x - 8=52), to hyper-pentaploid (2 n=5 x + 7=67), with the euploid pentaploid 2 n=5 x=60 class predominant. The high variability in chromosome number of the 3 x x 4 x hybrids was attributed to the fact that meiotic restitution during megasporogenesis of the 3 x female may have involved poles with various chromosome numbers, resulting in 2 n eggs with 24-48 chromosomes. Microsporogenesis analyses provided evidence that chromosome pairing between S. commersonii and S. tuberosum genomes occurred. In addition, chromosome distribution at anaphase I and anaphase II revealed an average chromosome number of 29.5 and 29.1 per pole, respectively. To further study the extent of transmission of extra genome chromosomes from pentaploids, 5 x x 4 x and 4 x x 5 x crosses were performed, and the chromosome number of resulting progeny was determined. Ploidy ranged from 2 n=4 x=48 to 2 n=5 x=60 following 5 x x 4 x crosses, and from 2 n=4 x + 1=49 to 2 n=5 x=60 following 4 x x 5 x crosses. These results provided indirect evidence that the pentaploid hybrids produced viable aneuploid gametes with a chromosome number ranging from 24 to 36. They also demonstrated that gametes with large numbers of extra chromosomes can be functional, resulting in sporophytes between the 4 x and 5 x ploidy level. Fertility parameters of crosses involving various (aneuploid) pentaploid genotypes were not influenced by chromosome number, suggesting a buffering effect of polyploidy on aneuploidy. The possibility of successfully using (aneuploid) pentaploid genotypes for further breeding efforts is discussed.

Evaluation and use of plant biodiversity for food and pharmaceuticals.

Many epidemiological studies have shown the importance of fruit and vegetables in the human diet so as to prevent the onset of cardiovascular disease and several forms of cancer. The use for food and pharmaceuticals of two of the most widely grown and genetically well-known species in the world, the tomato and the potato, is reviewed. Tomatoes are important sources of vitamin C, potassium, folic acid and carotenoids such as lycopene and beta-carotene. It has been demonstrated that lycopene has anti-oxidant properties and interferes with the growth of cancerous cells. At the Department of Agronomy and Plant Genetics in Portici, interesting results have been obtained with the constitution of stable tomato hybrids having a high content of lycopene and vitamin C. Many of the parental lines used in constituting the hybrids come from interspecific crosses. Potato is also very important in the human diet for its content of high quality proteins, mineral salts and vitamins and it has many medicinal properties. The use of diploid wild species to transfer traits such as high content of vitamin C, mineral salts and high quality proteins into the cultivated potato through ploidy manipulation is discussed.

Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.).

Diploid (2n = 2x = 24) Solanum species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n = 4x = 48, 4EBN) and haploid (2n = 2x = 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F1 hybrids between an in vitro-doubled clone of S. commersonii (2n = 4x = 48, 2EBN) and diploid 2EBN clones were successfully used in 3x x 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid/near pentaploid BC1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.