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Wheat (Triticum aestivum L.) is an important winter and spring crop in South Africa with average dryland yields of 2.5 t/ha, while irrigated wheat yields 5 t/ha. Diuraphis noxia (Kurdjumov) commonly referred to as the Russian wheat aphid was recorded in 1978 to South Africa and has become a major pest in the wheat industry. It can cause over 80% yield loss in the summer rainfall region if not controlled. There are five described RWA biotypes in SA, namely RWASA1 to RWASA5. We investigated the genetic variation of 24 specimens which are representative of four South African RWA biotypes using three molecular techniques, AFLP, cDNA-AFLP and microsatellites. With AFLP and cDNA-AFLP, we detected variation among specimens within a biotype. The cDNA-AFLP results increased the amount of the variation detected compared to the genomic AFLP results, but it was not sufficient to be linked to a specific biotype. With microsatellites, we were able to detect variation among biotypes and distinguish between them. We were unable to identify markers unique to a biotype. We found evidence that the genetic diversity may be linked to geographic location of the RWA, but further investigation is required to confirm this., Agricultural Research Council-Small Grain, National Research Foundation (NRF)

Seedlings of Phillyrea latifolia L., a Mediterranean shrub, were exposed for 90 days to 110 nl l –1 ozone (O3). Comparison of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) patterns for treated and control plants allowed the identification and cloning of 88 differential sequences induced by O3. The differential expression of 67 cloned sequenceswas further confirmed byRT-PCR. The functions of 36 cloned sequences, corresponding to seven of the twelve gene functional classes of Arabidopsis, were presumed on the basis of their homology with characterized gene sequences. Ozone induction of genes homologous to 24 of the clones has been reported in other plant species, whereas the induction of the 12 remaining sequences has not been observed before. Ozone activation of these newly identified genes could be a result of the chronic exposure to low O3 concentration, because in most previous studies, acute treatments, involving high O3 dosages, were applied. Possible roles of the cloned sequences in the response of P. latifolia to O3 and other causes of oxidative stress are discussed.

Seedlings of Phillyrea latifolia L., a Mediterranean shrub, were exposed for 90 days to 110 nl l –1 ozone (O3). Comparison of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) patterns for treated and control plants allowed the identification and cloning of 88 differential sequences induced by O3. The differential expression of 67 cloned sequenceswas further confirmed byRT-PCR. The functions of 36 cloned sequences, corresponding to seven of the twelve gene functional classes of Arabidopsis, were presumed on the basis of their homology with characterized gene sequences. Ozone induction of genes homologous to 24 of the clones has been reported in other plant species, whereas the induction of the 12 remaining sequences has not been observed before. Ozone activation of these newly identified genes could be a result of the chronic exposure to low O3 concentration, because in most previous studies, acute treatments, involving high O3 dosages, were applied. Possible roles of the cloned sequences in the response of P. latifolia to O3 and other causes of oxidative stress are discussed.

Background: Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. Results: cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was confirmed vi

Salinity is an important environmental factor limiting growth and productivity of plants, and affects almost every aspect of the plant physiology and biochemistry. The objective of this study was to apply cDNA-AFLP and to identify differentially expressed genes in response to NaCl stress vs. no-stress in Populus simonii × Populus nigra in order to develop genetic resources for genetic improvement. Selective amplification with 64 primer combinations allowed the visualization of 4407 transcript-derived fragments (TDFs), and 2027 were differentially expressed. Overall, 107 TDFs were re-sequenced successfully, and 86 unique sequences were identified in 10 functional categories based on their putative functions. A subset of these genes was selected for real-time PCR validation, which confirmed the differential expression patterns in the leaf tissues under NaCl stress vs. no stress. Differential expressed genes will be studied further for association with salt or drought-tolerance in P. simonii×P. nigra. This study suggests that cDNA-AFLP is a useful tool to serve as an initial step for characterizing transcriptional changes induced by NaCl salinity stress in P. simonii×P. nigra and provides resources for further study and application in genetic improvement and breeding. All unique sequences have been deposited in the Genbank as accession numbers GW672587–GW672672 for public use.

The human gastrointestinal (GI) tract provides home to a complex microbial community, collectively termed microbiota. Although major efforts have been made to describe the diversity and stability of the microbiota, functional studies have been largely restricted to intestinal isolates and include few community studies. The aim of this study was to explore the in situ gene expression of the fecal microbiota and to evaluate the RNA fingerprinting method cDNA-AFLP (cDNA amplified fragment length polymorphism) for this purpose. To this end, cDNA-AFLP analysis of enriched mRNA revealed that two healthy subjects showed highly divergent expression profiles with considerable fluctuations in time. Subsequent excision and sequence determination of bands from the mRNA-enriched profiles resulted in 122 identifiable sequences (transcripts and rRNAs). The classification of retrieved transcripts into functional clusters based on COG (cluster of orthologous genes) annotation showed that most assigned transcripts belonged to the metabolism cluster (26% of all sequences), underlining that even at the very end of the intestinal tract the microbiota is still very active. This study furthermore revealed that cDNA-AFLP is a useful tool to compare gene expression profiles in time in complex microbial communities

Plant parasitic nematodes infect roots and trigger the formation of specialized feeding sites by substantial reprogramming of the developmental process of root cells. In this article, we describe the dynamic changes in the tomato root transcriptome during early interactions with the potato cyst nematode Globodera rostochiensis. Using amplified fragment length polymorphism-based mRNA fingerprinting (cDNA-AFLP), we monitored 17 600 transcript-derived fragments (TDFs) in infected and uninfected tomato roots, 1¿14 days after inoculation with nematode larvae. Six hundred and twenty-four TDFs (3.5%) showed significant differential expression on nematode infection. We employed GenEST, a computer program which links gene expression profiles generated by cDNA-AFLP and databases of cDNA sequences, to identify 135 tomato sequences. These sequences were grouped into eight functional categories based on the presence of genes involved in hormone regulation, plant pathogen defence response, cell cycle and cytoskeleton regulation, cell wall modification, cellular signalling, transcriptional regulation, primary metabolism and allocation. The presence of unclassified genes was also taken into consideration. This article describes the responsiveness of numerous tomato genes hitherto uncharacterized during infection with endoparasitic cyst nematodes. The analysis of transcriptome profiles allowed the sequential order of expression to be dissected for many groups of genes and the genes to be connected with the biological processes involved in compatible interactions between the plant and nematode

Background: After 10-year-use of AFLP (Amplified Fragment Length Polymorphism) technology for DNA fingerprinting and mRNA profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. AFLP marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cDNA-AFLP for transcriptome profiling and differentially expressed gene cloning. The evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. This may be achieved by means of the Gene Ontology (GO), consisting in three structured vocabularies (i.e. ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. In this paper, the functional annotation of about 8,000 AFLP-derived ESTs retrieved in the NCBI databases was carried out by using GO terminology. Results: Descriptive statistics on the type, size and nature of gene sequences obtained by means of AFLP technology were calculated. The gene products associated with mRNA transcripts were then classified according to the three main GO vocabularies. A comparison of the functional content of cDNA-AFLP records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ESTs of Arabidopsis and rice, respectively. On the whole, the statistical parameters adopted for the in silico AFLP-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable GO results. Such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species. Conclusion: Reliable GO annotations of AFLP-derived sequences can be gathered through the optimization of the experimental step

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis. © 2007 Springer-Verlag., SCOPUS: ar.j, info:eu-repo/semantics/published

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis. © 2007 Springer-Verlag., SCOPUS: ar.j, info:eu-repo/semantics/published

A cDNA-AFLP experiment was designed to identify and clone nucleotide sequences induced during seed germination in Arabidopsis thaliana. Sequences corresponding to known genes involved in processes important for germination, such as mitochondrial biogenesis, protein synthesis and cell cycle progression, were isolated. Other sequences correspond to Arabidopsis BAC clones in regions where genes have not been annotated. Notably, a number of the sequences cloned did not correspond to available sequences in the databases from the Arabidopsis genome, but instead present significant similarity with DNA from other organisms, for example fish species; among them, some may encode transposons. A number of the sequences isolated showed no significant similarity with any sequences in the public databases. Oligonucleotides derived from these new sequences were used to amplify genomic DNA of Arabidopsis. Expression analysis of representative sequences is presented. This work suggests that, during germination, there may be a massive transposon mobilization that may be useful in the annotation of new genome sequences and identification of regulatory mechanisms.

In dog thyrocytes in primary culture, thyrotropin (TSH), through cAMP, positively controls proliferation and differentiation. As until now, the key events and the genes involved in the action of TSH remain largely uncharacterized, our goal was to identify new differentially expressed genes in TSH-induced thyroid proliferation. Using cDNA-AFLP, we visualized 105 different transcripts showing significant differential expression during the stimulation of dog thyrocytes with TSH for different times, in the presence of insulin. Northern blot and RT-PCR analyses confirmed the pattern expression of 5 clones encoding known proteins: thrombospondine 1, TNFr1, RhoE, RalB, and annexin A2. These regulations provide molecular counterparts of in vivo physiological effects of TSH: angiogenesis (decreased thrombospondin 1), decreased apoptosis (decreased TNFR1) and actin filament disruption, macropinocytosis and thyroid hormone secretion (decreased RhoE)., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Vascular secondary growth results from the activity of the vascular cambium, which produces secondary phloem and secondary xylem. By means of cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis along aspen stems, several potential regulatory genes involved in the progressive transition from primary to secondary growth were identified. A total of 83 unique transcript-derived fragments (TDFs) was found to be differentiated between the top and the bottom of the stem. An independent RT-PCR expression analysis validated the cDNA-AFLP profiles for 19 of the TDFs. Among these, seven correspond to new genes encoding putative regulatory proteins. Emphasis was laid upon two genes encoding, respectively, an AP2/ERF-like transcription factor (PtaERF1) and a RING finger protein (PtaRHE1); their differential expression was further confirmed by reverse northern analysis. In situ RT-PCR revealed that PtaERF1 was expressed in phloem tissue and that PtaRHE1 had a pronounced expression in ray initials and their derivatives within the cambial zone. These results suggest that these genes have a potential role in vascular tissue development and/or functioning., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Vascular secondary growth results from the activity of the vascular cambium, which produces secondary phloem and secondary xylem. By means of cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis along aspen stems, several potential regulatory genes involved in the progressive transition from primary to secondary growth were identified. A total of 83 unique transcript-derived fragments (TDFs) was found to be differentiated between the top and the bottom of the stem. An independent RT-PCR expression analysis validated the cDNA-AFLP profiles for 19 of the TDFs. Among these, seven correspond to new genes encoding putative regulatory proteins. Emphasis was laid upon two genes encoding, respectively, an AP2/ERF-like transcription factor (PtaERF1) and a RING finger protein (PtaRHE1); their differential expression was further confirmed by reverse northern analysis. In situ RT-PCR revealed that PtaERF1 was expressed in phloem tissue and that PtaRHE1 had a pronounced expression in ray initials and their derivatives within the cambial zone. These results suggest that these genes have a potential role in vascular tissue development and/or functioning., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

In dog thyrocytes in primary culture, thyrotropin (TSH), through cAMP, positively controls proliferation and differentiation. As until now, the key events and the genes involved in the action of TSH remain largely uncharacterized, our goal was to identify new differentially expressed genes in TSH-induced thyroid proliferation. Using cDNA-AFLP, we visualized 105 different transcripts showing significant differential expression during the stimulation of dog thyrocytes with TSH for different times, in the presence of insulin. Northern blot and RT-PCR analyses confirmed the pattern expression of 5 clones encoding known proteins: thrombospondine 1, TNFr1, RhoE, RalB, and annexin A2. These regulations provide molecular counterparts of in vivo physiological effects of TSH: angiogenesis (decreased thrombospondin 1), decreased apoptosis (decreased TNFR1) and actin filament disruption, macropinocytosis and thyroid hormone secretion (decreased RhoE)., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

'Gala' and 'Fuji' are two high-quality apple (Malus domestica Borkh) cultivars. Their fruits mature and tree-ripen over a long period of time, and are resistant to pre-harvest drop. "Tree factor," a putative inhibitor of system 2 ethylene production is hypothesized to account for differences in ethylene production between attached and detached apple fruits. Three years of field data revealed two distinct patterns of maturation and ripening behavior in these two cultivars. 'Gala,' an early cultivar, demonstrated a typical positive "tree factor." Studies of the ripening pattern of 'Fuji' apple, which is a late-maturing cultivar, did not. 'Fuji' data were confounded by cold weather in the late fall. The natural progression of tree-ripening did not lead to the high concentrations of internal ethylene routinely measured in stored fruits. The stimulation of ethylene found in picked 'Gala' fruits ripened in the orchard might be explained by wounding stress coupled with a loss of nutrients and the water stress. Our alternative explanation for "tree factor" is the effect of continued termination of the phloem and xylem connection. The strength of the "tree factor" declined as 'Gala' fruit maturity progressed. Therefore, the "tree factor" tends to be more obvious in fruits with shorter growing period that mature during warm weather. To investigate differential gene expression that accompanies maturation and tree ripening, we used cDNA-AFLP (Amplified Fragment Length Polymorphism) to identify changes in transcript profiling during tree-ripening, and in the ripening of harvested fruits. Two hundred differentially-expressed transcript-derived fragments were isolated from 'Gala.' Ripening-related genes including those known to function in the key processes of defense and stress, cell wall degradation, pigment production and aroma biosynthesis were identified. Clones similar to housekeeping genes involved in protein biosynthesis and degradation, intracellular trafficking and sorti

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.