Wyniki wyszukiwania

1

Real-time PCR for the detection of precise transgene copy number in durum wheat

100%

Gadaleta A., Giancaspro A., Cardone M., Blanco A.

Cellular and Molecular Biology Letters

|

2011

|

tom 16

|

nr 4

Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs. This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

2

Genetic mapping of polyphenol oxidase in tetraploid wheat

100%

Simeone R., Pasqualone A., Clodoveo M. L., Blanco A.

Cellular and Molecular Biology Letters

|

2002

|

tom 07

|

nr 2B

Pasta colour is one of the main factors inﬂuencing pasta quality. It is the product of a desirable yellow component, an undesirable brown component and, under some drying conditions, a red component. The brown colour depends on enzymatic and chemical factors. Polyphenol oxidase (PPO; E.C. 1.14.18.1) is one of the enzymatic factors. It is mainly localised in the peripheral part of the wheat kernel, and is involved in the oxidation of endogenous wheat phenolic compounds resulting in the production of highly coloured products. Therefore, a knowledge of the genetic control of PPO activity could enable the developing of better strategies in breeding programs to reduce pasta darkening. The aim of this study was to map the gene(s) affecting PPO activity using a set of recombinant inbred (RI) lines, derived from a cross between Triticum turgidum L. var. durum cultivar Messapia and the accession MG4343 of Triticum turgidum L. var. dicoccoides. After performing linkage analysis, the gene for high PPO activity was mapped on the long arm of the chromosome 2A and its characteristic was found highly associated to the RFLP marker Xutv1427-2A, with a value of LOD equal to 29.84. The identification of molecular markers linked to loci controlling the PPO activity may potentially accelerate wheat breeding since the selection of plants can be carried out by genotype rather than phenotype.

3

Heterochromatin organization in metaphase chromosomes and interphase nuclei of Dasypyrum breviaristatum [Lindb f] Frederiksen

100%

Pignone D., Galasso I., Blanco A., Cremonini R.

Acta Societatis Botanicorum Poloniae

|

2000

|

tom 69

|

nr 1

4

An evaluation of a new approach to the regeneration of Helichrysum italicum [Roth] G. Don, and the molecular characterization of the variation among sets of differently derived regenerants

76%

Perrini R., Alba V., Ruta C., Morone-Fortunato I., Blanco A., Montemurro C.

Cellular and Molecular Biology Letters

|

2009

|

tom 14

|

nr 3

377-394

A protocol for the induction of regeneration from leaves of Helichrysum italicum was established. Calli were found to form on the basal medium only when it was supplemented with thidiazuron (TDZ) alone or in combination with naphthalene acetic acid (NAA), with a percentage ranking of at least 80%. The hormone-free medium showed the highest percentage of shoot regeneration (62%) even though no callus formed. AFLP markers were employed to verify tissue culture-induced variation in the regenerated plantlets obtained by direct shoot regeneration or the indirect shoot regeneration process (callus formation). Seven out of the eleven AFLP primer pairs yielded polymorphic patterns. The average number of fragments per primer pair was 64.1. Singletons were represented by 12 (2.7%) fragments. Student’s T-test was performed both on the average number of shared fragments and on the nucleotide diversity, and no significant statistical difference was observed between the two regeneration treatments.

5

The cytogenetics and molecular characteristics of a translocated chromosome 1AS.1AL-1DL with a Glu-D1 locus in durum wheat

76%

Blanco A., Cenci A., Simeone R., Gadaleta A., Pignone D., Galasso I.

Cellular and Molecular Biology Letters

|

2002

|

tom 07

|

nr 2A

Wheat quality depends directly on the grain protein content and protein composition. High and low molecular weight glutenin subunits play an important role in determining the visco-elastic properties of gluten. In an attempt to improve the breadmaking quality of hexaploid triticale, a fragment of wheat chromosome 1D, containing the Glu-D1 allele encoding the 5+10 subunits, was translocated to the long arm of chromosome 1A by Lukaszewski and Curtis [1], The 1A.1D translocation chromosome was transferred to tetraploid wheat [2], making the Glu-D1 locus available for the improvement of durum wheat. The goal of this study was to evaluate using cytogenetics and molecular approaches the amount of chromatin introgressed in durum wheat. Fluorescence in situ hybridization with total genomic DNA (GISH) of Aegilops squarrosa L. indicated that the translocated chromosome 1A.1D had a terminal 1DL segment of about 35-40% of the recombinant arm length. Several pairs of microsatellite primers from chromosome 1A and 1D were used to genetically characterize the recombinant chromosome. The mapping data indicated that a 1AL segment, at least 150 cM long, was substituted by a 1DL segment with a minimal length of 72 cM, and that the translocation breakpoint was near the 1A centromeric region. The genetic and physical data highlight a substantial discrepancy between the recombinational and physical map distances. We are using a targeted strategy via the Ph pairing manipulation system to generate smali intercalary 1D chromosome segments in a durum wheat background.

6

Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study

52%

Cifarelli R. A., D`onofrio O., Grillo R., Mango T., Cellini F., Piarulli L., Simeone R., Giancaspro A., Colasuonno P., Blanco A., Gadaleta A.

Cellular and Molecular Biology Letters

|

2013

|

tom 18

|

nr 2

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

7

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum [L.] Thell.

51%

Blanco A., Simeone R., Cenci A., Gadaleta A., Tanzarella O. A., Porceddu E., Salvi S., Tuberosa R., Figliuolo G., Spagnoletti P., Roder M. S., Korzun V.

Cellular and Molecular Biology Letters

|

2004

|

tom 09

|

nr 3

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the highdensity Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.

Ograniczanie wyników

Real-time PCR for the detection of precise transgene copy number in durum wheat

Genetic mapping of polyphenol oxidase in tetraploid wheat

Heterochromatin organization in metaphase chromosomes and interphase nuclei of Dasypyrum breviaristatum [Lindb f] Frederiksen

An evaluation of a new approach to the regeneration of Helichrysum italicum [Roth] G. Don, and the molecular characterization of the variation among sets of differently derived regenerants

The cytogenetics and molecular characteristics of a translocated chromosome 1AS.1AL-1DL with a Glu-D1 locus in durum wheat

Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum [L.] Thell.