Genetic And Epigenetic Variation Induced By Tissue Culture In Rice

In the first part of this study, we addressed the issue of somaclonal variation in rice cv. Nipponbare by firstly investigating the genomic variations using two molecular markers, RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat), followed by sequencing of selected bands that manifested genomic variations, and pairwise sequence analysis taking advantage of the whole genome sequence of the rice cultivar. Finally, transpositional activity of the active MITE, mPing, was analyzed by locus-specific PCR amplification. Data showed that two-year-old calli and their regenerated plants, as analyzed with 24 RAPD and 20 ISSR primers, manifested moderate levels of genomic variations (20.83% and 17.04% respectively). To test whether DNA methylation plays a role in somaclonal variation, the calli were treated with 5-azacytidine that reduces cytosine methylation by blocking activity of DNA methyltransferase. Though dwarfism occurred in regenerants from treated calli (a hallmark of the drug treatment), there is only a slight increase in the frequency of somaclonal variation detected in the treated calli and their regenerated plants, relative to untreated controls. mPing also remained immobile in both treated and untreated calli. Nevertheless, dendrograms constructed according to the Jacard`s coefficient calculated by UPGMA of the ISSR bands revealed that the 5-azacytidine treated and untreated somaclones were grouped into two distinct clusters, suggesting a possible directed effect of the treatment on the genomic changes, depending on the marker used. Sequence analysis indicated a low level of variation (0.31%), with single base substitutions being predominant.In the second, three indica rice cultivars, V14, V27, and R09, widely cultivated in Burundi, were used to investigate changes in cytosine DNA methylation induced by tissue culture by means of methylation-sensitive amplified polymorphism (MSAP) method. Five regenerated plants, 2 callus cell lines, together with the mother donor plant for each cultivar were subjected to MSAP analysis with 17 of primer combinations. Changes in DNA methylation during tissue culture occurred in all three cultivars, but the frequencies were genotype-dependent. Frequencies of decrease in DNA methylation sites among cultivars were in this order: V27, V14, and R09, V27 showing more demethylation sites. Four patterns of DNA methylation changes among somaclones compared to their parents for each cultivar were produced. B2, D1, C1, D3, and A1 patterns frequently appeared in V14 with a total of 229 sites whereas D1, A1, C1, and B1 were frequent in V27 with a total of 241 sites, and D1, C1, and D2 patterns were more frequent in R09 variety with a total of 203 sites, showing also a decrease in DNA methylation in the same order as above. BlastN and BlastX results showed that on the 23 isolated sites, 19 sequenced fragments showed no significant similarity while 4 remaining did not affect cellular genes and retrotransposons, but instead targeted sequences coding for hypothetical proteins and putative transposon. To assess the behavior of mPing elements following changes in DNA methylation, two selected genotypic somaclonal lines with their respective rice parents were used in the Transposon Display analysis and showed high frequencies of insertions of mPing elements than deletions among cultivars in this order:...