Transcription analysis of apple fruit development using cDNA microarrays

The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and maturation thus potentially involved in fruit quality traits. The expression profile of 1,536 transcripts was analysed by microarray hybridisation. A total of 177 genes resulted to be differentially expressed in at least one of the developmental stages considered. Gene ontology annotation was employed to univocally describe gene function, while cluster analysis allowed grouping genes according to their expression profile. An overview of the transcriptional changes and of the metabolic pathways involved in fruit development was obtained. As expected, August and September are the two months where the largest number of differentially expressed genes was observed. In particular, 85 genes resulted to be up-regulated in September. Even though most of the differentially expressed genes are involved in primary metabolism, several other interesting functions were detected and will be presented

Molecular markers linked to the apple scab resistance gene Vbj derived from Malus baccata jackii

Breeding for scab-resistant apple cultivars by pyramiding several resistance genes in the same genetic background is a promising way to control apple scab caused by the fungus Venturia inaequalis. To achieve this goal, DNA markers linked to the genes of interest are required in order to select seedlings with the desired resistance allele combinations. For several apple scab resistance genes, molecular markers are already available; but until now, none existed for the apple scab resistance gene Vbj originating from the crab apple Malus baccata jackii. Using bulk segregant analysis, three RAPD markers linked to Vbj were first identified. These markers were transformed into more reliable sequence-characterised amplified region (SCAR) markers that proved to be co-dominant. In addition, three SSR markers and one SCAR were identified by comparing homologous linkage groups of existing genetic maps. Discarding plants showing genotype-phenotype incongruence (GPI plants) plants, a linkage map was calculated. Vbj mapped between the markers CH05e03 (SSR) and T6-SCAR, at 0.6cM from CH05e03 and at 3.9cM from T6-SCAR. Without the removal of the GPI plants, Vbj was placed 15cM away from the closest markers. Problems and pitfalls due to GPI plants and the consequences for mapping the resistance gene accurately are discussed. Finally, the usefulness of co-dominant markers for pedigree analysis is also demonstrate

Inheritance studies of apple scab resistance and identification of Rvi14, a new major gene that acts together with other broad-spectrum QTL

Scab, caused by the fungal pathogen Venturia inaequalis, is the most common disease of cultivated apple (Malus domestica). The fungal races 6 and 7 have now overcome the major resistance gene Vf, which is widely used in apple breeding programmes. New breeding strategies to achieve durable resistance are thus necessary. The aim of this study was to determine the genetic basis of quantitative resistance of the apple cultivar ‘Du¨lmener Rosenapfel’, known to be scab resistant under different environmental conditions. An F1 progeny derived from the cross between the susceptible cultivar ‘Gala’ and ‘Du¨lmener Rosenapfel’ was tested in a greenhouse with a multi-isolate inoculum of V. inaequalis. Rvi14, a new major gene that conditions a chlorotic-type reaction, was mapped on linkage group (LG) 6 in a genomic region not known to be involved in disease resistance. A further three quantitative trait loci (QTL) for resistance were identified. One co-localized with Rvi14 on LG6, whereas the remaining two were detected on LG11 and LG17, in genomic regions already reported to carry broad-spectrum QTL in other genetic backgrounds. Since a selective genotyping approach was used to detect QTL, an expectation-maximization (EM) computation was used to estimate the corrected QTL contributions to phenotypic variation and was validated by entire progeny genotyping

Microsatellite markers spanning the apple ( Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database ( http://www.hidras.unimi.it ) to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15c

Development and test of 21 multiplex PCRs composed of SSRs spanning most of the apple genome

A series of 21 multiplex (MP) polymerase chain reactions containing simple sequence repeat (SSR) markers spanning most of the apple genome has been developed. Eighty-eight SSR markers, well distributed over all 17 linkage groups (LGs), have been selected. Eighty-four of them were included in 21 different MPs while four could not be included in any MPs. The 21 MPs were then used to genotype approximately 2,000 DNA samples from the European High-quality Disease-Resistant Apples for a Sustainable agriculture project. Two SSRs (CH01d03 and NZAL08) were discarded at an early stage as they did not produce stable amplifications in the MPs, while the scoring of the multilocus (ML) SSR Hi07d11 and CN44794 was too complex for large-scale genotyping. The testing of the remaining 80 SSRs over a large number of different genotypes allowed: (1) a better estimation of their level of polymorphism; as well as of (2) the size range of the alleles amplified; (3) the identification of additional unmapped loci of some ML SSRs; (4) the development of methods to assign alleles to the different loci of ML SSRs and (5) conditions at which an SSR previously described as ML would amplify alleles of a single locus to be determined. These data resulted in the selection of 75 SSRs out of the 80 that are well suited and recommended for large genotyping project

A possible endophytic symbiont of Androsace brevis (Hegetschw.) Cesati (Primulaceae)

Androsace brevis is a narrow endemic plant living on windy ridges and peaks in a restricted area of Southern Alps in Lombardy and Switzerland, preferring acid soils with low nitrogen content and flowering immediately after the snowmelt. The species is proposed as a model species to study the effects of climate change on the web of interactions in mountain ecosystems (microbiota and pollinators). During a preliminary work aimed at developing molecular markers for A. brevis, a significant amount of prokaryotic DNA, not compatible with an environmental contamination, was detected. It seems to be a symbiotic relationship. The bacterial genome was de novo assembled and identified as belonging to the Beijerinkiaceae family, Rhizobiales order. Nor the genus neither the species could be identified: nothing similar has been described in the NCBI database so far. To evaluate the diffusion of the bacterium, specific PCR primers were designed and tested on a large number of A. brevis individuals, from eight natural populations. The presence of the bacterium was confirmed in all samples. Beijerinckiaceae family includes bacteria living in the phyllosphere, often methylotrophs or methanotrophs sharing nitrogen fixation capability (allowing to thrive in habitats where nitrogen sources are scarce, such as A. brevis growth-substrate) and capable to promote plant growth. In the present work, we tested different selective growth media to isolate the bacterium. The bioinformatic analysis of the functional domains predicted in the bacterial genome suggested a possible symbiotic relationship with the plant, experimentally supported by preliminary observations confirming the presence of endophytic bacteria inside plant tissues (leaves). Based on the above, the isolation and identification of this microorganism could help to clarify the very peculiar ecology of this alpine plant and to reduce the current lack of knowledge about high-altitude plant-bacteria interactions

Survival of Listeria monocytogenes in uncooked Italian dry sausage (salami).

This study was undertaken to supplement existing information on the survival of Listeria monocytogenes in Italian salami. The fact that Italian salami is frequently consumed by a large number of people poses some serious health implications. Some raw materials have been found to be microbiologically contaminated, for their production occurs without any thermic treatment, and these are in circulation throughout Italy all year round. We selected the product for its microbiological, technological, and commercial characteristics. We analyzed 1,020 samples taken during the autumn and winter 2002 and spring and summer 2003 periods and immediately before selling. The samples were collected from 17 plants with an annual production of between 1 and 2,000 metric tons and with a distribution of products in over 80% of Italy in geographic terms. To detect and enumerate L. monocytogenes, we followed International Organization for Standardization (ISO) 11290 part 1 and 2: 1996 (modified using chromogenic medium Agar Listeria according to Ottarviani and Agosti [ALOA]). L. monocytogenes was found in 22.7% of samples, but the contamination level was less than 10 CFU/g. Contamination prevalence ranged from 1.6 to 58.3% and was lower than 10% in 5 of the 17 plants checked. The most frequently isolated serotypes were 1/2c, 1/2a, 1/2b, and 4b. Additional studies are necessary to establish if the exposure to a small number of L. monocytogenes cells through the consumption of salami represents a significant health risk and, in light of the future introduction of the SANCO/4198/2001 revision 21 "Commission Regulation on Microbiological Criteria for Foodstuffs," is a necessary investigation

A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

<p>Abstract</p> <p>Background</p> <p>Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression.</p> <p>Results</p> <p>We report that peptides extracted from wheat bud chromatin induce growth inhibition, G₂arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G₂cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression.</p> <p>Conclusion</p> <p>The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G₂checkpoint pathway activation.</p

Loss of Arc renders the visual cortex impervious to the effects of sensory experience or deprivation

A myriad of mechanisms have been suggested to account for the full richness of visual cortical plasticity. We found that visual cortex lacking Arc is impervious to the effects of deprivation or experience. Using intrinsic signal imaging and chronic visually evoked potential recordings, we found that Arc−/− mice did not exhibit depression of deprived-eye responses or a shift in ocular dominance after brief monocular deprivation. Extended deprivation also failed to elicit a shift in ocular dominance or open-eye potentiation. Moreover, Arc−/− mice lacked stimulus-selective response potentiation. Although Arc−/− mice exhibited normal visual acuity, baseline ocular dominance was abnormal and resembled that observed after dark-rearing. These data suggest that Arc is required for the experience-dependent processes that normally establish and modify synaptic connections in visual cortex.Howard Hughes Medical InstituteNational Science Foundation (U.S.

Are nutrition and physical activity associated with gut microbiota? A pilot study on a sample of healthy young adults

BACKGROUND: The literature shows that gut microbiota composition is related with health, and a lot of individual and outer factors may determine its variability. In particular, nutrition and exercise seem to influence the presence in the gut of the two major bacterial phyla of Firmicutes and Bacteroidetes. STUDY DESIGN: An ongoing cross-sectional investigation is aimed to explore these associations in humans. METHODS: Healthy Caucasian young adults were asked to provide a fecal sample in order to analyze their gut microbiome considering their Body Mass Index (BMI), adherence to Mediterranean diet and Physical Activity (PA) level. RESULTS: A total of 59 participants (49.1% males, mean age 23.1 ± 3.14 years) were enrolled so far. Firmicutes (61.6±14.6) and Bacteroidetes (30.7 ± 13.3) showed the highest relative abundance in fecal samples. The Pearson's analysis showed a significant negative correlation between PA and Firmicutes (r =-0.270, p = 0.03). Linear regression confirmed a significant decrease of this phylum with the increase of PA (R2 = 0.07, p = 0.03). CONCLUSIONS: These preliminary results suggest the association between physical activity and gut microbiota composition in healthy humans