Analysis of genetic and epigenetic variation in in vitro propagated potato somatic hybrid by AFLP and MSAP marker

Background: Genetic and epigenetic changes (DNA methylation) were examined in the tissue-culture propagated interspecific potato somatic hybrids between dihaploid Solanum tuberosum and S. pinnatisectum. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) were applied to detect the genetic and epigenetic changes, respectively in the somatic hybrids mother plants (1st cycle) and their regenerants (30th cycles sub-cultured). Results: To detect genetic changes, eight AFLP primer combinations yielded a total of 329 scorable bands of which 49 bands were polymorphic in both mother plants and regenerants. None of the scorable bands were observed in term of loss of original band of mother plant or gain of novel band in their regenerants. AFLP profiles and their cluster analysis based on the Jaccard’s similarity coefficient revealed 100% genetic similarity among the mother plant and their regenerants. On the other hand, to analyze epigenetic changes, eight MSAP primer pair combinations detected a few DNA methylation patterns in the mother plants (0 to 3.4%) and their regenerants (3.2 to 8.5%). Out of total 2320 MSAP sites in the mother plants, 2287 (98.6%) unmethylated, 21 (0.9%) fully methylated and 12 (0.5%) hemi-methylated, and out of total 2494 MSAP sites in their regenerants, 2357 (94.5%) unmethylated, 79 (3.1%) fully methylated and 58 (2.3%) hemi-methylated sites were amplified. Conclusion: The study concluded that no genetic variations were observed among the somatic hybrids mother plants and their regenerants by eight AFLP markers. However, minimum epigenetic variations among the samples were detected ranged from 0 to 3.4% (mother plants) and 3.2 to 8.5% (regenerants) during the tissue culture process.

Genetic diversity among native varieties and commercial cultivars of Solanum tuberosum ssp. tuberosum L. present in Chile

Background: The object of this work was to determine the genetic and allelic diversity of Solanum species present in Chile, assessing allelic distribution among native varieties and commercial cultivars of Solanum tuberosum ssp. tuberosum L., using microsatellite markers. Results: A high level of allelic richness was found in the potatoes studied. The seven microsatellite markers used presented a total of 64 allelic variants among native varieties and commercial cultivars of Solanum tuberosum ssp. tuberosum. The SSR loci generated an average of 9.16 alleles/locus. The group with the highest PIC was that of native varieties collected in the Chiloe archipelago. The high PIC values found indicate that the native varieties from Chiloe have a low level of interrelation, representing wide genetic diversity. Conclusions: The markers with the highest number of alleles in native varieties corresponded to loci STG 0016 and LEMALX. Commercial cultivars do not present the same genetic variability as native varieties, and the allelic richness of commercial cultivars is lower than that of native varieties of S. tuberosum ssp. tuberosum. Most of the native varieties were clustered in accordance with their geographical location, while commercial cultivars, were clustered in accordance with their breeding programs in Chile and Europe, with the exception of Shepody.

Virulence function of pectobacterium atrosepticum secretion system mutants on evaluation of some solanum tuberosum resistance genes

P.atrosepticum is a commercially important pathogen. It causes blackleg in the field and soft rot of tubers after the harvest. This effect is due to secretion of depolymerases and other virulence factors by several mechanisms including T3SS The effect of bacterial T3SS on Solanumtuberosum [S. tuberosum] varieties and its relationship with S. tuberosum resistance gene expression were studied. A P. atrosepticum HrpW gene was cloned, sequenced and constructed a phylogenic tree with some phytopathogen. The virulence properties of P. atrosepticum strains were investigated and then S. tuberosum varieties were tested for their sensitivity against P. atrosepticum. PR-5 and HIN genes copy-number for infiltrated S. tuberosum tubers were assessed. The results show that infiltrated tubers of P. atrosepticum T3SS mutants were significantly more macerated than the wild type ones. PR-5 and HIN expression amounts were depended on bacterial T3SS function.

Isolation and characterization of the tissue and development-specific potato snakin-1 promoter inducible by temperature and wounding

Snakin-1 (StSN1) is a broad-spectrum antimicrobial peptide isolated from Solanum tuberosum. Homologous proteins have been identified in a wide range of species but there is no apparent consensus in the roles they play. A 1394 bp fragment of the 5’upstream region of StSN1 gene, designated PStSN1, was isolated from the potato genome and sequenced. Bioinformatics analyses revealed a total of 55 potential regulatory motifs related to tissue-specificity, stress, defence and hormone responsiveness, among others. PStSN1 spatial and temporal activity was studied in transgenic Arabidopsis plants expressing a reporter gene under this promoter control (PStSN1::GUS). Histochemical staining revealed PStSN1::GUS expression in the root vasculature, cotyledons, young leaves and floral organs. Moreover, GUS staining was detected in young developmental stages gradually decreasing as the plant aged. Stress treatments on transgenic plants showed that PStSN1 activity was induced by high/low temperature and wounding. The characterization of PStSN1 in a model plant establishes a framework for the understanding of its possible biological functions and provides a potential tool for plant modification through genetic engineering.

Comparación de dos kits de RT-PCR en la detección de ARNm de dos genes endogenos de papa (Solanum tuberosum spp. Andigena) / Comparison of two kits of RT-PCR in RNAm Detection of two endegenous genes of potato (Solanum tuberosum spp. Andigena)

RT-PCR es una tecnica en la que usando ARN mensajero como molde, se obtiene complementario o cADN por transcripcion inversa, y luego se amplifica uno de los cADN por PCR, mediante el uso de primers especificos. Esta tecnica permite realizar estudios de expresion, a nivel de ARN mensajero. Con el proposito de implementar la tecnica en papa (Solanum tuberosum spp. Andigena), se utilizaron

A recombination point is conserved in the mitochondrial genome of higher plant species and located downstream from the cox2 pseudogene in Solanum tuberosum L

The potato (Solanum tuberosum L.) mitochondrial cox3/sdh4/pseudo-cox2 gene cluster has previously been identified by heterologous hybridization using a Marchantia polymorpha sdh4 probe. In our present study we used Southern blotting using sdh4 and cox2 probes to show that the sdh4 and cox2 genes are clustered in the mitochondria of potato, soybean and pea. Northern blotting revealed cotranscription of sdh4 and cox2 in potato but not in cauliflower, indicating that these genes are not clustered in cauliflower. A putative recombination point was detected downstream of the cox2 pseudogene (pseudo-cox2) in potato mitochondrial DNA (mtDNA). This sequence corresponds to a 32 bp sequence which appears to be well-conserved and is adjacent to the terminals of some mitochondrial genes in Citrullus lanatus, Beta vulgaris and Arabidopsis thaliana and is probably involved in the genic rearrangements. It is possible the potato mtDNA pseudo-cox2 gene was generated by recombination during evolution in the same way as that of several other mitochondrial genes and remains as an inactive partial copy of the functional cox2 which was also detected in potato mtDNA.

Filocrono em diferentes densidades de plantas de batata / Phyllochron at different plant densities in potato

O conceito do filocrono, ou seja, o intervalo de tempo necessário para a emissão de duas folhas consecutivas, pode ser usado para simular o aparecimento de folhas em plantas. O número de folhas, além de afetar o índice de área foliar da planta, é considerado uma excelente medida de tempo fisiológico. O objetivo do trabalho foi estimar o filocrono em batata cultivada num sistema hidropônico com substrato em diferentes densidades de plantas. O experimento foi conduzido em ambiente protegido, no Departamento de Fitotecnia da UFSM, RS, no período de 03/09/2004 a 26/10/2004. O delineamento experimental foi o inteiramente casualizado, com três repetições. Os tratamentos foram os espaçamentos de 5x5, 10x10, 15x15 e 20x20cm entre covas. Os valores de filocrono para cada densidade foram relacionados com o número de covas m-2 e de hastes m-2. Os valores de filocrono estimados variaram em função da densidade de plantas. As maiores densidades promoveram aumentos no filocrono, indicando que a densidade de plantas afeta a velocidade de surgimento de folhas em batata, e esta resposta deve ser contemplada em modelos de simulação do número de folhas nesta cultura.

Possible role of calcium dependent protein phosphorylation in the modulation of wound induced HRGP gene activation in potatoes after gamma irradiation.

Hydroxyproline rich glycoprotein (HRGP) gene is induced in both control and gamma irradiated potato tubers after wounding. The enhanced RNA synthesis in response to wounding correlated well with the accumulation of both HRGP gene transcripts and protein. Initially, the level of HRGP gene expression in gamma irradiated potatoes in response to wounding was 30% more than the corresponding controls. After post irradiation storage of 3-5 weeks, HRGP gene expression in response to wounding was significantly lower than the unirradiated samples. This low level of HRGP gene expression in irradiated potatoes was partially retrieved by 5 mM Ca2+ treatment. Prior treatment with trifluoperazine, a calcium channel blocker resulted in 35% reduction in wound induced HRGP gene expression in control potatoes, further providing evidence for the involvement of Ca2+ dependency for HRGP gene activation. A comparative study on in vivo protein phosphorylation induced by wounding in control and irradiated potatoes exhibited significant differences. A good correlation was observed in the modulation of phosphorylation and HRGP gene expression by Ca2+ in irradiated potatoes. Wound induced signal transduction system and subsequent Ca2+ dependent protein phosphorylation for the activation of HRGP gene is affected in potatoes after gamma irradiation, thus impairing the wound healing process adversely.

Newly synthesised mRNA as a probe for identification of wound responsive genes from potatoes.

A method for the selective isolation of wound induced RNA from potato tuber has been standardised by labeling with 4-thiouridine and subsequent affinity chromatography on Affigel 501 phenyl-mercury Sepharose column. About 30% of the total RNA isolated from wounded potato tissue was found to be synthesized de novo. The relative abundance of wound induced RNAs was confirmed by using two well identified wound inducible gene probes of hydroxyproline-rich glycoproteins (HRGP) and proteinase inhibitor I. The selectively separated wound induced mRNA was used to make cDNA probes. By screening a--gt10 cDNA library from wounded potato tissue with these cDNA probes, several wound responsive genes were identified. Wound inducible expression of these genes was further confirmed by Northern hybridization and their tissue specific expressions were examined. Thus, a simple method for the direct identification of wound responsive genes from potato was developed.