Gábor Gyulai

Genetic variation of melon (C. melo) compared to an extinct landrace from the Middle Ages (Hungary) I. rDNA, SSR and SNP analysis of 47 cultivars

SummaryMicrosatellite 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.

Morphological and molecular analysis of common millet (P. miliaceum) cultivars compared to an aDNA sample from the 15th century (Hungary)

SummaryMorphological 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 (Kings 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.

AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus x canescens L.) for copper in vitro

Abstract Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRIDMseI digestion. Four fragments generated by EcoAGTDMseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. \ canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10-1 to 10-5 ᴍ) incubated for 21 days on aseptic tissue culture media WPM containing 1 μᴍ Cu. Zn2+ caused phytotoxicity only at high concentrations (10-1 to 10-2 ᴍ). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.

Seed remains of common millet from the 4th (Mongolia) and 15th (Hungary) centuries: AFLP, SSR and mtDNA sequence recoveries

Gyulai, G., Humphreys, M. O., Lagler, R., Szabo, Z., Toth, Z., Bittsanszky, A., Gyulai, F., Heszky, L. (2006). Seed remains of common millet from the 4th (Mongolia) and 15th (Hungary) centuries: AFLP, SSR, and mtDNA sequence recoveries. Seed Science Research, 16, (3), 179-191. Keywords: ancient DNA, excavated seeds, Panicum miliaceum Sponsorship: Hungarian Scientific Research Group OTKA-M-04 563, Organization for Economic Co-operation and Development grant OECD-JA00018642, the USA–Hungarian Fulbright Commission, and a Sze´chenyi Fellowship (SzO- 4/2003).

RT-PCR analysis and stress response capacity of transgenic gshi-poplar clones (Populus x canescens) in response to paraquat exposure

Abstract Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) 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 × 10-9 to 4.0 × 10-6 m) for 21 days. Significant differences at sublethal (4.0 × 10-7 m) and bleaching (4.0 × 10-6 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 Fmax), 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 × 10-7 m) samples of the transgenic poplar line (cyt-ECS).