1,721,002 research outputs found
A quick method for the isolation of plant DNA suitable for RAPD analysis.
No full textA simple method to isolate genomic DNA suitable for RAPD analysis from a large number of samples is described. The average DNA concentration of 360 preparations from alfalfa (Medicago sativa L.) plants belonging to six ecotypes, as measured by optical density at 260 nm, was 736 ng/micronl, and the purity, as measured by the ratio between the optical densities at 260 and 280 nm, was 2.08. The corresponding figures for 216 preparations of Kentucky bluegrass (Poa pratensis L.) plants belonging to nine progenies were 270 ng/micronl and 1.89. The assay by agarose gel electrophoresis of genomic and restricted DNA samples revealed a good DNA quality. Amplification tests showed reproducible and comparable RAPD profiles over the range of concentrations obtained by the protocol presented. Our results indicate that the optical DNA quantification can be reduced to a single measurement on a polled sample of aliquots from a random subset of preparations
Inheritance and mapping of 2n egg production in diploid alfalfa
No full textThe production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes
Alternative selectable marker genes for durum wheat genetic transformation
No full textThe production of transgenic plants relies on the delivery into the target tissues of selectable marker genes together with the useful genes, in order to allow the regeneration into a plant of the only cells that have integrated and express the foreign DNA. The most efficient selectable markers commonly employed in plant genetic transformation are bacterial genes conferring resistance to antibiotics or herbicides. However, the presence of such genes in crop plants grown in the field destined to human or animal feeding has become a matter of concern for the public opinion. Thus, the scientific community is being stimulated to test alternative selection systems based on new genes conferring resistance to chemicals other than antibiotics or herbicides. In the present work we show the application of two new alternative selection systems for durum wheat biolistic transformation. The first one is a “positive” selection based on the use of mannose as the selective agent and the Escherichia coli phosphomannose isomerase (pmi) gene as the selectable marker, conferring to the transformed plant cells the ability to grow on a medium containing mannose as the only carbon source. The second alternative marker is the hemL gene isolated from Synechococcus strain GR6, coding for a mutant form of the enzyme GSA-AT (glutamate-semialdehyde aminotransferase) which is insensitive to the phytotoxin gabaculine. The hemL gene was found very efficient in tobacco (Gough et al. 2000) and alfalfa (Rosellini et al. 2007) genetic transformation using gabaculine as selective substance. The hemL gene can be a good candidate for a safer selection system as it is present in all plant species and it is involved in one metabolic step only, so that unintended effects of its over-expression in plants are not probable. The GSA-AT cDNA from Medicago sativa was cloned and point-mutated to reproduce the gabaculine-resistance mutation of the Synechococcus hemL gene, and it has been shown to be an efficient marker in alfalfa and tobacco transformation (Rosellini et al., unpublished). In the present work both pmi and the hemL gene were compared to the conventional bar marker gene from S. Hygroscopicus conferring resistance to Bialaphos herbicide. Pmi gene showed a more efficient selectable marker than bar both when delivered in the form of traditional plasmid vector, and in the form of linear minimal expression cassettes (1.14% and 1.50% vs 0.99%). A co-transformation experiment was carried out on wheat calli by delivering plasmids pAHC20 and pAPCK-GSA carrying the bar and the mutant GSA-AT genes, respectively. After gene delivery the two marker genes were evaluated during all the selection phases, from callus regeneration to adult plant formation and compared for their transformation and selection efficiency. Transformation with pAPCK-GSA was more efficient than with the other plasmid
Self-sterility in lucerne: assessment as a practical tool for hybrid cultivar development.
No full textMETHYLATION STATE IN MEDICAGO SATIVA POLYPLOIDIZED PLANTS: AN EPIGENETIC STUDY
No full textMost plant species are either polyploidy or have experience polyploidization during their evolution: this indicates a selective advantage of polyploidization in plants. Polyploidization is known to affect gene expression in several ways, including epigenetic mechanisms. The best described epigenetic mechanism is DNA hypermethylation, or the predominant marking of CpG, CpCpG, CpHpHp, and CpNpG motifs in DNA. The sole methyl donor for all eukaryotes, S-adenosylmethionine, provides the methyl group essential for such Arabidopsis thaliana enzymes as CHROMOMETHYLASE 3 (CMT3) and
DOMAINS REARRANGED METYLASE 2 (DRM2) responsible for marking of CpNpG motifs, and METHYLTRANSFERASE 1 (MET1), the marker of CpG islands.
This study is aimed at gathering new information on the general “methylation state” in Medicago sativa, an important autopolyploid forage species affected by chromosome doubling via
sexual polyploidization. 2x and 4x progenies obtained by crossing 2x plants that produce both n and 2n eggs and pollen respectively, are used. The Medicago truncatula sequence database of CpG islands in MET, CMT, DRM and DEMET (DEMETHYLTRASFERASE) family was analyzed by two different bioinformatic approaches to assess the methylation status of virtually any group of CpG sites within CpG island;
this is a step toward understanding the methylation-affected biological processes. Methylase and demethylase gene expression changes were investigated by RT-qPCR and the
first results will be presented. These studies can be useful to understand the basis for the polyploidy advantage in agricultural crops
Genetic control of parthenogenesis in Poa pratensis L.: results from a sexual x apomictic cross.
No full textOsservazioni sulla maturazioni del vitigno “Malvasia nera” in alcune zone del Chianti Classico (Study on ripening of ‘Malvasia nera’ cultivated in several vineyards of ‘Chianti Classico’ DOC area
No full textComportamento del vitigno “Malvasia nera di Lecce” su due portinnesti in cinque zone del chianti classico (Agronomical behaviour of the cv. Malvasia on eleven sites in the area of ‘Chianti Classico’)
No full text- …
