AFLP fingerprinting of tartary buckwheat accessions (Fagopyrum tataricum) displaying rutin content variation.

In light of the economic importance of buckwheat as well as existence of enormous accessions of Fagopyrum species in the Himalayan regions of India, the characterization of tartary buckwheat for rutin content variation vis-à-vis DNA fingerprinting was undertaken so as to identify fingerprint profiles unique to high rutin content accessions. Rutin content analysis in mature seeds of 195 accessions of Fagopyrum tataricum showed a wide range of variation (6 µg/mg to 30 µg/mg D.W.) with most of the accessions (81%) containing 10-16 µg/mg of rutin followed by 14% accessions with significantly higher rutin content (17 µg/mg to 30 µg/mg) and 5% accessions with low rutin content (≤10 µg/mg). AFLP fingerprinting of 18 accessions having high (≥17 µg/mg) and low rutin content (≤10 µg/mg) with 19 EcoRI/MseI primer combinations yielded 136 polymorphic fragments out of total 907. The hierarchical and model-based cluster analyses of AFLP data strongly suggested that the 18 populations of F. tataricum were clustered into two separate groups. The high and low rutin content accessions were clustered into two separate groups based on AFLP fingerprinting. The AFLP fingerprints associated with high rutin content accessions of F. tataricum are expected to be useful for evaluation, conservation and genetic improvement of buckwheat.

Expression of flavonoid biosynthesis genes vis-à-vis rutin content variation in different growth stages of Fagopyrum species.

Buckwheat is one of the field crops with the highest concentration of rutin, an important flavonoid of medicinal value. Two species of buckwheat, Fagopyrum esculentum and Fagopyrum tataricum, are the major sources of rutin. Seeds of latter contain 40-50× higher rutin compared to the former. The physiological and molecular bases of rutin content variation between Fagopyrum species are not known. The current study investigated the differences in rutin content in seeds and in other tissues and growth stages of two Fagopyrum species, and also correlated those differences with the expression of flavonoid pathway genes. The analysis of rutin content dynamics at different growth stages, S1-S9 (from seed germination to mature seed formation) of Fagopyrum species revealed that rutin content was higher during seedling stages of F. tataricum (3.5 to 4.6-fold) compared to F. esculentum and then increased exponentially from stages S3 to S6 (different leaf maturing stages and inflorescence) of F. esculentum, whereas it fluctuated in F. tataricum. The rutin content was highest in the inflorescence stage (S6) of both species, with a relatively higher biosynthesis and accumulation during post-flowering stages of F. tataricum compared to F. esculentum. The expression of flavonoid pathway genes, through qRT-PCR, in different growth stages vis-à-vis rutin content variation showed differential expression for four genes, PAL, CHS, CHI and FLS with the amounts of transcripts relatively higher in F. tataricum compared to F. esculentum, thereby, correlating these genes with the biosynthesis and accumulation of rutin. The expression of PAL was highest, 7.69 and 8.96-fold in Stages 2 (seedling stage) and 9 (fully developed seeds) of F. tataricum compared to F. esculentum, respectively. The expression of the CHS gene correlated with the rutin content because it was highest in the flowers (S6) and fully developed seeds (S9) of both Fagopyrum species, with relatively higher transcript amounts (2.13 and 3.19-fold, respectively) in F. tataricum (IC-329457) compared to F. esculentum (IC-540858). This study provides useful information on molecular and physiological dynamics of rutin biosynthesis and accumulation in Fagopyrum species and the correlation of expression of flavonoid biosynthesis genes with the rutin content can be useful in planning for genetic improvement.

Triplex mediated delivery of a platinum complex to a specific DNA target site.

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.