Relative gene expression levels of transgene gshI (γ-glutamylcysteine synthetase cloned from E. coli) were analyzed by qRT-PCR in two transgenic poplar (Populus × canescens) clones (11ggs and 6lgl) and wild type (WT). An extremely high expression level of transgene gshI was observed in the 6lgl clone (13.5-fold) compared to 11ggs (1.0) sample...s, which level was doubled (1.8-fold) in the DHAC (5.6-dihydro-5'-azacytidine hydrochloride) treated (at 10-4 M for 7 days) 6lgl samples but not in the 11ggs clone (0.4-fold). Contrary to this result, relative copy number of transgene gshI in the 6lgl clone was found to be less 60% less (1.0) then in the 11ggs samples (1.6). Relative expression levels of proper poplar gene gsh1-poplar showed significantly higher responsiveness to DHAC treatment than transgene gshI with the highest expression level in the untransformed (WT) poplar
clone (19.7-fold) compared to transformed 6lgl (8.7-fold) and 11ggs (2.5-fold) clones. For internal controls constitutively expressed housekeeping genes a-tubulin were applied. For data analysis, the 2−ΔΔCt method was used. DHAC applied in long-term cultures (27 days) at low concentrations (10-8 – 10-6 M) showed morphogenetic activity by initiating de novo root development of leaf discs.
The evolution of water melon (Citrullus lanatus) microsatellites from the 15th century (Debrecen); 13th (Buda); and 18th century, (Pannonhalma) were analyzed. Microsatellite (nSSR, nuclear simple sequence repeat) and cpDNA profiles of the aDNA (ancient DNA) of seed remains were compared to modern water melon cultivars and landraces. Sixteen pri...mer pairs were applied. Sequence analysis at the (CT)26 and cpDNA trnV loci revealed a (CT)3 and Adenin deletions, respectively, form the current water melon cultivar compared to the medieval sample. Cila-1), a new LTR retrotansposon has been described. For morphological reconstruction, a dendrogram produced by SPSS11 based on the presence versus absence of 24 phenotypic characters were also analyzed.
Genetic variation of somatic clones (1 to 35) of black poplar (Populus nigra) developed from two anther-donor trees N-SL and N-309 was determined by five SSR primer pairs. Twenty SSR alleles were detected, the number of alleles per marker ranged from 1 to 6, with an average of 3.3 including WPMS-2 (5 alleles), WPMS-4 (6 alleles), WPMS-6 (2 alle...les), WPMS-20 (6 alleles) and PTR-4 (1 allele) detected by ALF (automatic laser fluorometer). A
dendrogram produced by SPSS11 based on the presence versus absence of SSR alleles discriminated the groups of somatic clones of N-SL from somatic clones of N-309. The polymorphic markers of WPMS-2 (5 alleles), WPMS-4 (6 alleles) and WPMS-20 (6 alleles) revealed clonal variation in 1 clone (37) out of the 6 from the N-309 tree, and three subgroups out of the 29 somatic clones from the N-SL tree (17 and 24), (2 and 14) and (10 and 15). The remaining 23 of the 29 N-SL somatic clones with uniform genetic similarity suggests a good degree of genetic stability in black poplar. Nevertheless, the new SSR-clones may provide useful new genetic resources for poplar breeding.
Seed remains of medieval millet, recovered from a 15th century layer (King’s Palace, Budapest, Hungary), showed reddish yellow grain color after rehydrating on tissue culture medium that was close to grain color of modern cultivar Omszkoje. aDNA of medieval c. millet was extracted successfully, analyzed and compared to modern common millets b...y ISSR, SSR, CAPS and mtDNA. Analyses of fragments and sequences revealed
polymorphism at seven ISSR loci (22 alleles) and at the 5S-18S rDNA locus of mtDNA. CAPS analysis of the 5S-18S rDNA fragment revealed no SNPs in the restriction sites of six endonucleases TaqI, BsuRI, HinfI, MboI, AluI and RsaI. Sequence alignments of the restriction fragments RsaI also revealed
consensus sequence in the medieval sample compared to a modern variety. Morphological characterization of twenty common millet (Panicum miliaceum L., 2n=4×=36) cultivars and landraces revealed four distinct clusters which were apparently consistent with the grain colors of black, black and brown, red, yellow, and white. In the comparative AFLP, SSR and mtDNA analysis modern millet cv. ‘Topáz’ was used. AFLP analysis revealed that extensive DNA degradation had occurred in the 4th CENT. ancient millet resulting in only 2 (1.2%) AFLP fragments (98.8% degradation),
compared to the 15th CENT. medieval millet with 158 (40%) fragments (60% degradation) and modern millet cv. ‘Topáz’ with 264 fragments (100%). Eight AFLP fragments were sequenced after reamplification and cloning. Microsatellite (SSR) analysis at the nuclear gln4, sh1, rps28 and rps15 loci of the medieval DNA revealed one SNP (single nucleotide polymorphism) at the 29th position (A to G) of rps28 locus compared to modern millet.
Mitochondrial (mtDNA) fragment (MboI) amplified at the 5S-18S-rDNA locus in the medieval millet showed no molecular changes compared to modern millet. The results underline the significance of survived aDNA extraction and analysis of excavated seeds for comparative analysis and molecular reconstruction of ancient and extinct plant genotypes. An attempted phenotype reconstruction indicated that medieval common millet showed the closest morphological similarity to modern millet cultivar Omszkoje.
ITS (internal tanscribed spacer) profiles of the aDNA (ancient DNA) of seed remains extracted from an extinct sample recovered from the 15th century (Budapest, Hungary) were compared to 31 modern melon cultivars and landraces. An aseptic incubation followed by ITS analysis was used to exclude the exogenously and endogenously contaminated (Asper...gillus) medieval seeds and to detect SNPs in ITS1-5.8S-ITS2 region of rDNA (ribosomal DNA). SNPs were observed at the 94–95 bp (GC to either RC, RS or AG) of ITS1; and at 414 bp (A-to-T substitution), 470 bp (T to Y or C), 610 bp (A to R or G) of ITS2. The results facilitate the final aim of molecular and morphological reconstructions of ancient melon tpyes.
Morphological diversity of melon (Cucumis melo); phenotype reconstruction of a medieval sample. Morphological diversity among 47 melon (Cucumis melo) cultivars and landraces from Hungarian germplasm collection (ABI, Tápiószele) were analyzed with an ultimate aim to characterize morphologically cv. Hógolyó, which showed the closest genetic s...imilarity to a medieval melon recovered from the 15th century. Cultivars based on fruit morphology were grouped into the three main types of melon as reticulatus, cantalupensis and inodorus. Cluster analysis (by SPSS-11) based on 23 morphological (quantitative and qualitative) traits recorded revealed an extreme diversity among accessions, nevertheless cultivars were clustered into main melon clusters with only two exceptions of inodorus type cv. Zimovka J. and Afghanistan. Cultivars Sweet ananas and Ezüst ananász; and two Hungarian landraces Kisteleki and Nagycserkeszi showed close similarity. Cultivars Hógolyó and Túrkeve of inodorus type
were also grouped in one cluster, which provide insight into the morphological reconstruction of the medieval melon recovered from the 15th century. These results also indicate that old Hungarian landraces could be re-introduced into breeding programs for broadening genetic base of melon.