Lagler, R., Gyulai, G., Humphreys, M. O., Szabo, Z., Horvaith, L., Bittsanszky, A., Kiss, J., Holly, L., Heszky, L. (2005). Morphological and molecular analysis of common millet (P. miliaceum) cultivars compared to an aDNA sample from the 15th century (Hungary). Euphytica, 146, (1-2), 77-85 Keywords: ancientDNA - CAP - mtDNA - ISSR - SNPsMorphological characterization of 20 common millet (Panicum miliaceum L., 2n = 4x = 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. 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 commom millet was extracted successfully, analyzed and compared to modern common millets by ISSR, SSR, CAP and mtDNA. Analyses of fragments and sequences revealed polymorphism at seven ISSR loci (15 alleles) and at the 5S-18S rDNA locus of mtDNA. CAP 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. An attempted phenotype reconstruction indicated that medieval common millet showed the closest morphological similarity to modern millet cultivar Omszkoje.Peer reviewe

Microsatellite profiles of 47 melon cultivars and landraces were analyzed and compared to the aDNA (ancient DNA) of seed remains from an extinct sample recovered from the 15th century (Budapest, Hungary). An aseptic incubation followed by ITS (internal transcribed spacer) analysis was used to exclude the exogenously and endogenously contaminated medieval seeds and to detect SNPs (single nucleotide polymorphism) 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) and 633 bp (A-to-G transition) of ITS2. For comparative microsatellite analysis SSRs (simple sequence repeats) detected by ALF (automated laser fluorometer) was used. Eight of the 20 SSR primer pairs amplified 40 microsatellite alleles in identical fragment ranges. A total of 485 alleles were detected in the 47 melon cultivars. The number of alleles per marker ranged from 2 to 7 with an average of 5.7 including CMCT44 (2 alleles), CMAG59 (5 alleles), CMGA104 (5 alleles), CMCT134 (4 alleles), CMTA134 (6 alleles), CMCTT144 (7 alleles), CMTC168 (6 alleles) and CMCT170 (5 alleles). Sequence analysis of the microsatellite alleles showed different fragment lengths depending on changes in the number of unit of core sequences. Dendrogram produced by SPSS11 based on the presence versus absence of SSR alleles revealed that medieval melon had the closest genetic similarity to a registered melon cultivar Hógolyó selected from an old Hungarian melon landrace. These results also indicated that cloned DNA sequences recovered from aDNA of medieval melon can be of use for molecular breeding of modern melon cultivars via gene transfer.

Stress response capacity (Fv/Fm at 690 nm and F690/F735 at F max ) of untransformed hybrid poplar, Populus ¥ canescens (P. tremula ¥ P. alba), and two transgenic lines overexpressing γ-ECS (γ-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 ¥ 10 Ð9 to 4.0 ¥ 10 Ð6 m) for 21 days. Significant differences at sublethal (4.0 ¥ 10 Ð7 m) and bleaching (4.0 ¥ 10 Ð6 m) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at F max ), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR of the cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression of the constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities of gene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression of the paraquat treated (4.0 ¥ 10 Ð7 m) samples of the transgenic poplar line (cyt-ECS).

Seed remains of common millet (Panicum miliaceum L.) were excavated from sites of AD 4th-century Darhan (Mongolia), and AD 15th-century Budapest (Hungary). Because the 15th-century medieval grains looked so intact, a germination test was carried out under aseptic conditions, which resulted in swelling of the grains but no cell proliferation or germination. Ancient DNA (aDNA) was extracted from the aseptic grains; analysed for amplified fragment length polymorphisms (AFLP), simple sequence repeats (SSR) and mitochondrial DNA (mtDNA); and compared with the modern millet cultivar 'Topaz'. AFLP analysis revealed that extensive DNA degradation had occurred in the 4th-century ancient millet, resulting in only 2 (1.2%) AFLP fragments (98.8% degradation) amplified by MseCAA -EcoAGT, compared to the 15th-century medieval millet, with 158 (40%) fragments (60% degradation), and modern millet cultivar 'Topaz' with 264 fragments (100%). EcoAGT-Mse-CAA was found to be the most effective selectiveprimer combination for the analysis of medieval and modern millet. Eight AFLP fragments were sequenced after re-amplification and cloning. Microsatellite (SSR) analysis at the nuclear gln4, sh1, rps28 and rps15 loci revealed one SNP (single nucleotide polymorphism) at the 29th position (A ! G) of rps28 locus, compared to modern millet. An mtDNA fragment (Mbo I), amplified at the 18S -5S ribosomal DNA (rDNA) locus in the medieval millet, showed no molecular changes compared to modern millet. The results underline the significance of aDNA extraction and analysis of excavated seeds for comparative analysis and molecular reconstruction of ancient and extinct plant genotypes.

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 similarity 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 typewere 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.