Tuber-bearing Solanum Species Research Articles

A quest for the potato of the future: Characterization of wild tuber-bearing Solanum species for de novo domestication.

The potato (Solanum tuberosum) is a staple food worldwide, but modern potato cultivation relies heavily on the use of pesticides to control pests and diseases. However, many wild Solanum species are highly resistant to biotic and abiotic stresses relevant for potato production. Several of those species have been used in potato breeding to confer resistances which has only been moderately successful. Instead, we propose an alternative approach to utilize the potential of wild Solanum germplasm. Recently, de novo domestication has been suggested to create more resilient crops: Instead of introducing resistance genes into existing crops, domestication traits could be introduced into resistant wild crop relatives to create new crops. Therefore, we selected ten promising species from the 107 known wild tuber-bearing Solanum species for their resistance to biotic and abiotic stresses based on existing scientific literature for experimental characterization looking at tuberization under short and long-day conditions, tuber glycoalkaloid content, starch digestibility and performance in tissue culture. Based on this, the highly pest and disease resistant S. bulbocastanaum was chosen. Our results showed that it produced relatively large tubers, also under long-day conditions and performed exceptionally well in tissue culture.

Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato

BackgroundPotato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY.ResultsThe presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY.ConclusionsThe new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

Molecular dissection of an intronic enhancer governing cold-induced expression of the vacuolar invertase gene in potato.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as "cold-induced sweetening," is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.

Recognition of Pep-13/25 MAMPs of Phytophthora localizes to an RLK locus in Solanum microdontum.

Pattern-triggered immunity (PTI) in plants is mediated by cell surface-localized pattern recognition receptors (PRRs) upon perception of microbe-associated molecular pattern (MAMPs). MAMPs are conserved molecules across microbe species, or even kingdoms, and PRRs can confer broad-spectrum disease resistance. Pep-13/25 are well-characterized MAMPs in Phytophthora species, which are renowned devastating oomycete pathogens of potato and other plants, and for which genetic resistance is highly wanted. Pep-13/25 are derived from a 42 kDa transglutaminase GP42, but their cognate PRR has remained unknown. Here, we genetically mapped a novel surface immune receptor that recognizes Pep-25. By using effectoromics screening, we characterized the recognition spectrum of Pep-13/25 in diverse Solanaceae species. Response to Pep-13/25 was predominantly found in potato and related wild tuber-bearing Solanum species. Bulk-segregant RNA sequencing (BSR-Seq) and genetic mapping the response to Pep-25 led to a 0.081 cM region on the top of chromosome 3 in the wild potato species Solanum microdontum subsp. gigantophyllum. Some BAC clones in this region were isolated and sequenced, and we found the Pep-25 receptor locates in a complex receptor-like kinase (RLK) locus. This study is an important step toward the identification of the Pep-13/25 receptor, which can potentially lead to broad application in potato and various other hosts of Phytophthora species.

Identification of metabolites associated with boiled potato sensory attributes in freshly harvested and stored potatoes

Modern potato breeding programmes have to balance agronomic and consumer preferences, as they seek to develop high yielding and nutritious potatoes that are tolerant to abiotic/biotic stresses. The recent focus on consumer preference has generated sensory data to assess potato flavour intensity, umami, sweetness, sourness, bitterness and mealiness in a panel of 60 cultivated and four wild potato accessions. The potato tubers were assessed immediately after harvest and after a five-week cold storage period. The cultivated accessions included diploid landraces of S. tuberosum L. Andigenum groups Phureja and Stenotonum, diploid hybrids of these, and advanced tetraploid breeding lines. The wild accessions represented tuber-bearing Solanum species, implicated in potato domestication. Sensory data highlighted 20 advanced breeding lines that were relatively weak in potato flavour, sweetness and umami before storage, but increased to the intensities of the landrace and hybrids bred from them after storage. The data observed 39 metabolites which were significantly different between storage times (on average ≥3-fold higher) and associated with sweetness, umami, bitterness and sourness tastes. A comparison to potatoes with high potato flavour emphasised that lower levels of specific metabolites associated with sweetness (e.g. glucose, fructose, glycine and serine), sourness (oxalic, ascorbic and citric acid) and umami (aspartic acid) were related to a higher rating for potato flavour. This corroborates the hypothesis, that perception of sensory traits is based on a complex metabolic composition in potato tubers. These data also indicate that metabolomic analysis can play an important role in augmenting future breeding programmes considering consumer traits.

Identification and Characterization of Potato Zebra Chip Resistance Among Wild Solanum Species

Potato zebra chip (ZC) disease, associated with the uncultured phloem-limited bacterium, Candidatus Liberibacter solanacearum (CLso), is transmitted by the potato psyllid Bactericera cockerelli. Potato ZC disease poses a significant threat to potato production worldwide. Current management practices mainly rely on the control of the psyllid to limit the spread of CLso. The present study investigated new sources of ZC resistance among wild Solanum species. A taxonomically diverse collection of tuber-bearing Solanum species was screened; one ZC-resistant accession and three ZC-tolerant accessions were identified among the 52 screened accessions. Further characterization of the resistant accession showed that the resistance was primarily associated with antibiosis effects due to differences in leaf trichome density and morphology of the wild accession, which could limit the psyllid feeding and oviposition. This germplasm offers a good resource for further understanding ZC and psyllid resistance mechanisms, contributing to potato breeding efforts to develop ZC resistance cultivars. Alternatively, it could be used as a potential trap crop to manage psyllid and control ZC disease.

South American species <i>Solanum alandiae</i> Card. and <i>S. okadae</i> Hawkes et Hjerting as potential sources of genes for potato late blight resistance

For several decades, wild species of Solanum L. section Petota Dumort. have been involved in potato cultivar breeding for robust resistance to pests and diseases. Potato late blight (LB) is caused by oomycete Phytophthora infestans (Mont.) de Bary, and the genes for race-specific resistance to P. infestans (Rpi genes) have been introgressed into cultivated potatoes by remote crosses and trans- or cisgenesis, first from S. demissum Buk. and, more recently, from other wild species, such as S. bulbocastanum Dun., S. stoloniferum Schlechtd. et Bché, and S. venturii Hawkes et Hjerting (according to the nomenclature by Hawkes, 1990). Most wild species already involved in breeding for LB resistance came from North and Central Americas: series Bulbocastana (Rydb.) Hawkes, Demissa Buk. and Longipedicellata Buk., and some Rpi genes of these species have been already characterized in much detail. Rpi genes of South American species, including the series Tuberosa (Rydb.) Hawkes, have not been sufficiently investigated. Among the latter, this study focuses on the Rpi genes of S. alandiae Card. and S. okadae Hawkes et Hjerting. Four accessions of S. alandiae, one accession of S. okadae and 11 clones of interspecific potato hybrids comprising S. alandiae germplasm from the VIR collection were PCR-screened using specific SCAR (Sequence Characterized Amplified Region) markers for eight Rpi genes. SCAR amplicons of five Rpi genes registered in this study were validated by comparing their sequences with those of prototype genes deposited in the NCBI Genbank. Among the structural homologues of Rpi genes found in S. alandiae and S. okadae, of special interest are homologues of CC-NB-LRR resistance genes with broad specificity towards P. infestans races, in particular R2=Rpi-blb3, R8, R9a, Rpi-vnt1 and Rpi-blb2 (94–99, 94–99, 86–89, 92–98 and 91% identity with the prototype genes, respectively). Our data may help to better understand the process of Rpi gene divergence along with the evolution of tuberbearing Solanum species, particularly in the series Tuberosa.

Genetic Diversity of Solanum pinnatisectum Dun. and Solanum chacoense Bitt. by Resistance to Potato Virus Y and Results of DNA Analysis

The potato Solanum tuberosum L. has the greatest diversity of cultivated and wild relatives, whose genetic nature has been studied insufficiently. The aim of the present study was to examine the resistance to PVY in S. chacoense Bitt. and S. pinnatisectum Dun. and to search for DNA markers linked to resistance genes. For the first time, representative populations of two diploid tuber-bearing Solanum species were evaluated by the resistance to PVY and the presence of DNA markers linked to the Ry (extreme resistance) or Ny (hypersensitivity) genes localized on the long arm of chromosome 9. Nine accessions of S. chacoense and six accessions of S. pinnatisectum represented by 168 and 170 genotypes, respectively, from the VIR collection were assessed by resistance to artificial infection with PVY. The differences in segregation of two wild potato species into phenotypic classes with response to PVY infection have been established. The diversity of visible reactions of S. chacoense plants after PVY infection differs from the uniform type of symptoms observed in S. pinnatisectum. DNA analysis was performed in 170 S. chacoense genotypes and 44 S. pinnatisectum genotypes using the Ry186, S1d11, and CT220 markers linked to genes determining viral resistance in potato varieties, breeding clones, or species of the genus Solanum L. Most genotypes of S. chacoense and S. pinnatisectum have amplified CT220 marker linked to the Nxphu gene and the Sw-5 gene in the IvP35 line of the species S. phureja Juz. et Buk. and tomato, respectively. The STS Ry186 marker linked to the Rychc gene in Japanese varieties created on the basis of S. chacoense was identified in a small number of S. chacoense genotypes; however, no association between the DNA marker and resistance phenotype was detected. The CAPS S1d11/AcsI marker, which distinguishes between the PVY resistant and susceptible genotypes of both wild potato species, was developed. The plants of two wild-growing tuber-bearing species of Solanum L. were first examined for the presence of the GBSS gene-specific PCR marker (the gene encoding granules-bound starch synthase). A significant part of the genotypes (15% in S. pinnatisectum and 25% in S. chacoense) did not reveal the genetic marker regulating amylose biosynthesis in the forming starch granules. No significant differences in the ability to form tubers depending on the genotypes with or without GBSS marker, indicating the possible presence of an alternative allelic variant of the GBSSI gene in S. chacoense and S. pinnatisectum species, were detected.

Polymorphisms and Evolution of Solanum bulbocastanum Genes for Broad-Spectrum Resistance to Phytophthora infestans

For two decades, Solanum bulbocastanum Dun. has been challenging potato geneticists with its durable resistance to Phytophthora infestans Mont. de Bary, the causal agent of late blight, the most devastating potato disease. In this study, highly specific SCAR (sequence characterized amplified region) markers for three S. bulbocastanum genes of broad-spectrum resistance to P. infestans,RB/Rpi-blb1,Rpi-blb2 and Rpi-blb3, were employed to screen a vast collection of wild tuber-bearing species from several series of Solanum L. section Petota Dumort. and also potato hybrids and varieties. In S. bulbocastanum and S. stoloniferum Schlechtd. et Bche, the sequences of the markers Rpi-blb1-820 and Rpi-sto1-890, which recognize the LRR and CC regions respectively differed inconsiderably in the orthologous genes RB/Rpi-blb1 and Rpi-sto1. These two markers were tightly linked in diverse wild Solanum genotypes. These markers as well as RB-226, another marker recognizing the LRR region of the Rpi-blb1 gene, were obviously related to high resistance to P. infestans. One more marker recognizing the CC region of the gene, RB-629, was found in much wider range of Solanum genotypes; it was not related to resistance to P. infestans and is therefore considered non-specific. In addition to Rpi-blb1 = Rpi-sto1, the Rpi-blb2 and Rpi-blb3 genes were registered in both S. bulbocastanum and S. stoloniferum accessions. In most cases, three bulbocastanum genes were not linked together. The markers specific for Rpi-blb1 = Rpi-sto1 genes were found in the Solanum series Bulbocastana (Rydb.) Hawkes (genome designation BB) and Longipedicellata Buk. (genome designation AABB) and were absent from five Pinnatisecta Rydb. species also comprising genome B. The marker for Rpi-blb2 was found in the series Bulbocastana and Longipedicellata and also in three accessions of Pinnatisecta, while the Rpi-blb3 gene, the orthologue of widely spread R2 gene for resistance to P. infestans, was present in all three Solanum series. In contrast with Rpi-blb1 = Rpi-sto1, two other functional RB homologues, Rpi-bt1 in S. bulbocastanum and RBver in S. verrucosum Schlechtd. (genome AA), apparently diverged earlier in the evolution of tuber-bearing Solanum species. Our evidence on functional Rpi-blb1 = Rpi-sto1 genes in three Petota series substantiates the assumption that the B genome of tetraploid Longipedicellata evolved from diploid Bulbocastana rather than from diploid Pinnatisecta progenitors. Similar to wild Solanum species, the markers Rpi-blb1-820 and Rpi-sto1-890 were tightly linked in advanced potato hybrids and established varieties bred from S. stoloniferum as the conventional source of bulbocastanum genes for late blight resistance, but did not always match the markers for the Rpi-blb2 and Rpi-blb3 genes and the marker for the Rysto gene as an independent indicator of stoloniferum germplasm.

Potato mosaic viruses which infect plants of tuber-bearing Solanum spp. growing in the VIR field gene bank

Potato crop is particularly affected by virus diseases, and potato virus Y (PVY) currently considered the most important pathogen distributed worldwide as a diversity of strains. Wild and cultivated tuber-bearing species of the genus Solanum L., stored in the VIR collection, are used as the initial material in creation domestic potato varieties (Solanum tuberosum L.) resistant to virus diseases. The preservation and rational utilization of the potato collection is based on regular phytosanitary monitoring, including quarantine objects, foremost PSTVd (potato spindle tuber viroid). The aim of the work is to examine plants of tuber-bearing Solanum species in the field gene bank of VIR for the presence of PSTVd and PVX (potato virus X), PVS (potato virus S), PVM (potato virus M) and PVY, which are the most common viruses on potatoes in the North-West District of Russia. We examined clonal plants of 137 genotypes representing 31 species of the section Petota of the genus Solanum L. A diagnostic was carried out using ELISA, RT-PCR and indicator plants. No PSTVd was found in the studied plants, but a plural infestation by mosaic viruses was detected, more than half of the tested clones are infected with two or more viruses. In the studied samples, only 17 genotypes (12 %) are not infected by PVX, PVS, PVM and PVY according to the ELISA test. There are statistically significant differences in the virus infestation of Solanum species with different origins, according to Pearson’s chi-squared test. Among the studied genotypes of wild relatives of potatoes, the proportion of those affected by PVY was significantly higher in the South American than in the North American species (χ2 = 4.56, p = 0.03); the proportion of genotypes affected by PVХ was significantly higher in the North American species (χ2 = 8.81, p = 0.003), the critical value was χ2 = 3.841. PVY strains were identified by multiplex RT-PCR in 37 genotypes of Solanum spp. We found that 27 genotypes are infected by a common PVYO strain, two genotypes are infected by PVYNW (A) and PVYNW (B) strains, respectively, seven genotypes are infected by a mixture of PVYO +PVYNW (A) strains, and one is infected by a mixture of PVYO +PVYNTN-NW (SYRI)+SYRIII strains. The recombinant strains of PVY are detected in the North-West District of Russia for the first time. Coherency of the results of PVY strains detection by various (immunological, molecular and biological) methods is discussed.

Two different R gene loci co-evolved with Avr2 of Phytophthora infestans and confer distinct resistance specificities in potato.

Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease in potato. For sustainable management of this economically important disease, resistance breeding relies on the availability of resistance (R) genes. Such R genes against P. infestans have evolved in wild tuber-bearing Solanum species from North, Central and South America, upon co-evolution with cognate avirulence (Avr) genes. Here, we report how effectoromics screens with Avr2 of P. infestans revealed defense responses in diverse Solanum species that are native to Mexico and Peru. We found that the response to AVR2 in the Mexican Solanum species is mediated by R genes of the R2 family that resides on a major late blight locus on chromosome IV. In contrast, the response to AVR2 in Peruvian Solanum species is mediated by Rpi-mcq1, which resides on chromosome IX and does not belong to the R2 family. The data indicate that AVR2 recognition has evolved independently on two genetic loci in Mexican and Peruvian Solanum species, respectively. Detached leaf tests on potato cultivar ‘Désirée’ transformed with R genes from either the R2 or the Rpi-mcq1 locus revealed an overlapping, but distinct resistance profile to a panel of 18 diverse P. infestans isolates. The achieved insights in the molecular R – Avr gene interaction can lead to more educated exploitation of R genes and maximize the potential of generating more broad-spectrum, and potentially more durable control of the late blight disease in potato.

37. ばれいしょ近緑種の日長反応に関する研究 : (1) 同質倍数体および合成複2倍体の実生個体における日長反応(I 年次講演会要旨)

37. ばれいしょ近緑種の日長反応に関する研究 : (1) 同質倍数体および合成複2倍体の実生個体における日長反応(I 年次講演会要旨)

Variation in tuber mineral concentrations among accessions of Solanum species held in the Commonwealth Potato Collection

The variation in tuber mineral concentrations amongst accessions of wild tuber-bearing Solanum species in the Commonwealth Potato Collection (CPC) was evaluated under greenhouse conditions. Selected CPC accessions, representing the eco-geographical distribution of wild potatoes, were grown to maturity in peat-based compost under controlled conditions. Tubers from five plants of each accession were harvested, bulked and their mineral composition analysed. Among the germplasm investigated, there was a greater range in tuber concentrations of some elements of nutritional significance to both plants and animals, such as Ca, Fe and Zn (6.7-fold, 3.6-fold, and 4.5-fold, respectively) than others, such as K, P and S (all less than threefold). Significant positive correlations were found between mean altitude of the species’ range and tuber P, K, Cu and Mg concentrations. The amount of diversity observed in the CPC collection indicates the existence of wide differences in tuber mineral accumulation among different potato accessions. This might be useful in breeding for nutritional improvement of potato tubers.

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.

Agroinfiltration and PVX Agroinfection in Potato and <em>Nicotiana benthamiana</em>

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.

Agroinfiltration and PVX Agroinfection in Potato and <em>Nicotiana benthamiana</em>

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.

Role of Genetic Modification in Developing Climate Smart Agriculture to Ensure Sustained Food Security

I was a contemporary of James Watson and Francis Crick at the University of Cambridge, UK, during 1950–52. I was aware of the fact that they were working on the molecular structure of DNA in association with Maurice Wilkins and Rosalind Franklin since I used to visit the Cavendish Laboratory to attend lectures by Prof Max Perutz. Their publication on the double helix structure of the DNA molecule appeared in Nature early in 1953 [2, 7, 8]. I was then at the Genetics Laboratory of the University of Wisconsin, Madison, USA. Since then, I have been following the explosive progress of the science of molecular genetics, opening up uncommon opportunities for transferring genes across sexual barriers. I was deeply interested in this development, since my work in the early 1950s related to the transfer of genes in tuber-bearing Solanum species for characters like frost tolerance and resistance to the golden nematode, Heterodera rostochiensis. The donor for frost tolerance was the species S. acaule from the Lake Titicaca region of Peru-Bolivia, while the donor for golden nematode resistance was S. polyadenium. Achieving crosses between these species and S. tuberosum was extremely difficult, since the foreign pollen were getting inhibited in the stigma and style. I had to remove the stigma and replace it with an artificial medium which promoted pollen germination [4]. This technique later came to be known as the ‘Swaminathan artificial stigma method’. All this would not have been necessary if the recombinant DNA technology had existed then. Thus, the Watson–Crick–Wilkins– Franklin discovery paved the way for fulfilling the dream of plant and animal breeders and microbiologists with respect to producing novel genetic combinations of applied interest. The award of the Nobel Prize to my colleague at the University of Wisconsin Dr. Joshua Lederberg, for his work on microbial transformation further stimulated my interest in this fast growing area of science. The US National Academy of Sciences invited me in 1982 to deliver a lecture on ‘Biotechnology Research and Third World Agriculture’, where I emphasised the need for Third World countries, to master all recent developments in Genetics [5]. In 1980, when I joined the Union Planning Commission at the invitation of the then Prime Minister of India Mrs. Indira Gandhi, I got a National Biotechnology Board set up to achieve synergy and coordination among the work in progress in molecular genetics and genetic engineering under the umbrella of different scientific organisations like ICAR, CSIR, ICMR, Department of Atomic Energy and UGC. I served as the first chair of the National Biotechnology Board. Later, it was converted into a Department of Biotechnology during the tenure of Prime Minister Rajiv Gandhi, with Dr. S. Ramachandran serving as its first Secretary. During the last 30 years, the Government of India has invested a considerable amount of money in creating the infrastructure essential for advanced research in the broad

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.

Genome-wide analysis of plastome sequence variation and development of plastidial CAPS markers in common potato and related Solanum species

The plastome sequence of the European cultivated potato, Solanum tuberosum subsp. tuberosum (tbr, GenBank accession no. DQ386163), was compared with that of S. bulbocastanum, a wild potato relative (blb, GenBank accession no. DQ347958), in order to characterize the degree and type of variability in different genomic regions, and develop molecular markers relevant to genetics, breeding and biotechnology of potato. One hundred forty-two and 251 PICs (Potentially Informative Characters) were found in coding and non-coding sequences (NCSs), respectively. Further, while variation in coding regions was almost exclusively due to nucleotide substitutions, 25% of PICs in NCSs of tbr and blb were due to indels, most of them mononucleotide or longer tandem repeats (micro and minisatellites). Four intergenic regions were selected for further analyses in other 16 tuber-bearing Solanum species. The rps16-trnQ UUG gene spacer was found to be the most variable, forty-six PICs in this region distinguishing 18 haplotypes. Analysis of haplotype relationships, based on variability in the four intergenic regions, confirmed that the most primitive species from Central America were the most distant to S. tuberosum. Finally, polymorphic sites in the same regions were used to develop a set of CAPS (Cleaved Amplified Polymorphic Sequences) markers for species/cytoplasm identification in Solanum spp.

Cultivated and wild Solanum species as potential sources for health-promoting quality traits

In this study, several genotypes of cultivated (Solanum tuberosum subsp. andigena, S. phureja) and wild, tuber-bearing Solanum species (S. chacoense, S. pinnatisectum) were examined for concentrations of antioxidants, soluble phenols and proteins in their tuber tissue. The potato genotypes differed considerably in all these traits. Amounts of antioxidants ranged from 0.06 to 4.22 μg/mg fresh weight (fw) when the ascorbic acid equivalent was measured and from 0.08 to 3.98 μg/mg fw for the trolox equivalent. The wild species S. pinnatisectum exhibited on average higher levels of both types of antioxidants than the other Solanum species, and it also had the highest quantities of soluble phenols and proteins in its tuber tissue. Among the species, S. phureja ranked on the lowest level of antioxidant potential.