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AbstractTomato (Solanum lycopersicum) is one of the highest-value vegetable crops worldwide, but tomato planting can be easily affected by leaf mold. Tomato leaf mold disease is caused by Cladosporium fulvum, and plants carrying the Cf genes have effective resistance to C. fulvum. To identify leaf mold resistant varieties and screen for specific cDNA amplified fragment length polymorphism (cDNA-AFLP) molecular markers, tomato plants harbouring genes Cf-5, Cf-11, Cf-19, and the moneymaker (MM) tomato line (without Cf gene) were used as plant materials. The changes in reactive oxygen species (ROS) levels and antioxidant activities of plant leaves after inoculation of C. fulvum were measured. Ethylene (ETH), abscisic acid (ABA) and salicylic acid (SA) contents were also determined. The results revealed that the ROS levels in Cf-19 tomato were the most sensitive to C. fulvum inoculation, while in MM they were the least. The antioxidant enzymes of Cf-carrying plants exhibited different activities as compared to those of MM. In the cDNA-AFLP analysis data, the expression of ERF109, sedoheptulose-1,7-bisphosphatase (SBPase), L-ascorbic acid oxidase (AAO) and Thioredoxin H2 (TRX2) were different between Cf-gene carriers and MM plants. In summary, in the process of leaf–mold interaction, the outbreak of ROS and development of systemic acquired resistance (SAR) are tightly combined with antioxidant enzymes, including thioredoxin. Tomato plants carrying the Cf-19 gene appear to have stronger C. fulvum resistance.

Citrus fruits produced in China are often affected by granulation. Granulation is an altered physiological state of citrus fruits occurring usually before harvest but whose underlying mechanisms remain elusive. In this study, cDNA-AFLP technology enabled the identification of 116 granulation-associated genes in pummelo (C. grandis) juice sacs. Differentially expressed transcript-derived fragments (TDFs) were shown to be mainly involved in biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid, protein metabolism, stress responses, and cell metabolism. Therefore, granulation in pummelo juice sacs seems to involve the following alterations: (1) changes in hormone levels; (2) activation of metabolic pathways related to ATP and sugar synthesis to produce more energy; (3) nucleic acid accumulation and increased protein degradation; (4) activation of stress-responsive metabolic pathways; (5) accelerated juice sac senescence. Our findings provide an overview of differential responses occurring at the transcriptional level in granulated juice sacs, thus revealing new insights into the adaptive mechanisms underlying this altered physiological state in 'Guanximiyou' pummelo (C. grandis) juice sacs.

Natural rubber, an important industrial raw material with wide applications, is harvested in the form of latex (cytoplasm of rubber-producing laticifers) from Hevea brasiliensis (para rubber tree) by the way of tapping. Conspicuous stimulation on latex production is observed for the first few tappings conducted on virgin (untapped before) or resting (tapped before but no tapping for a period) rubber trees. To understand the underlying mechanisms, an integrative analysis of the latex transcriptome and proteome was conducted on virgin or resting Hevea trees for the first five tappings. A total of 505 non-redundant differentially expressed (DE) transcript-derived fragments (TDFs) were identified by silver-staining cDNA-AFLP, with 217 exhibiting patterns of upregulated, 180 downregulated and 108 irregularly-regulated. Meanwhile, 117 two dimensional gel electrophoresis DE-protein spots were isolated and subjected to mass spectrometry analysis, with 89 and 57 being successfully identified by MALDI-TOF and MALDI-TOF/TOF, respectively. About 72.5% DE-TDFs and 76.1% DE-proteins were functionally annotated and categorized. Noteworthily, most of the DE-TDFs implicated in sugar transport and metabolism as well as rubber biosynthesis were upregulated by the tapping treatment. The importance of sugar metabolism in harvesting-induced latex production was reinforced by the identification of abundant relevant DE-protein spots. About 83.8% of the randomly selected DE-TDFs were validated for expression patterns by semi-quantitative RT-PCR, and an 89.7% consistency for the 29 latex regeneration-related DE-TDFs examined by quantitative RT-PCR analysis. In brief, our results reveal extensive physiological and molecular changes in Hevea laticifers incurred by the tapping treatment, and the vast number of DE genes and proteins identified here contribute to unraveling the gene regulatory network of tapping-stimulated latex production.

Natural rubber, an important industrial raw material with wide applications, is harvested in the form of latex (cytoplasm of rubber-producing laticifers) from Hevea brasiliensis (para rubber tree) by the way of tapping. Conspicuous stimulation on latex production is observed for the first few tappings conducted on virgin (untapped before) or resting (tapped before but no tapping for a period) rubber trees. To understand the underlying mechanisms, an integrative analysis of the latex transcriptome and proteome was conducted on virgin or resting Hevea trees for the first five tappings. A total of 505 non-redundant differentially expressed (DE) transcript-derived fragments (TDFs) were identified by silver-staining cDNA-AFLP, with 217 exhibiting patterns of upregulated, 180 downregulated and 108 irregularly-regulated. Meanwhile, 117 two dimensional gel electrophoresis DE-protein spots were isolated and subjected to mass spectrometry analysis, with 89 and 57 being successfully identified by MALDI-TOF and MALDI-TOF/TOF, respectively. About 72.5% DE-TDFs and 76.1% DE-proteins were functionally annotated and categorized. Noteworthily, most of the DE-TDFs implicated in sugar transport and metabolism as well as rubber biosynthesis were upregulated by the tapping treatment. The importance of sugar metabolism in harvesting-induced latex production was reinforced by the identification of abundant relevant DE-protein spots. About 83.8% of the randomly selected DE-TDFs were validated for expression patterns by semi-quantitative RT-PCR, and an 89.7% consistency for the 29 latex regeneration-related DE-TDFs examined by quantitative RT-PCR analysis. In brief, our results reveal extensive physiological and molecular changes in Hevea laticifers incurred by the tapping treatment, and the vast number of DE genes and proteins identified here contribute to unraveling the gene regulatory network of tapping-stimulated latex production. [ABSTRACT FROM AUTHOR]

Tomato (Solanum lycopersicum) is one of the highest-value vegetable crops worldwide, but tomato planting can be easily affected by leaf mold. Tomato leaf mold disease is caused by Cladosporium fulvum, and plants carrying the Cf genes have effective resistance to C. fulvum. To identify leaf mold resistant varieties and screen for specific cDNA amplified fragment length polymorphism (cDNA-AFLP) molecular markers, tomato plants harbouring genes Cf-5, Cf-11, Cf-19, and the moneymaker (MM) tomato line (without Cf gene) were used as plant materials. The changes in reactive oxygen species (ROS) levels and antioxidant activities of plant leaves after inoculation of C. fulvum were measured. Ethylene (ETH), abscisic acid (ABA) and salicylic acid (SA) contents were also determined. The results revealed that the ROS levels in Cf-19 tomato were the most sensitive to C. fulvum inoculation, while in MM they were the least. The antioxidant enzymes of Cf-carrying plants exhibited different activities as compared to those of MM. In the cDNA-AFLP analysis data, the expression of ERF109, sedoheptulose-1,7-bisphosphatase (SBPase), L-ascorbic acid oxidase (AAO) and Thioredoxin H2 (TRX2) were different between Cf-gene carriers and MM plants. In summary, in the process of leaf–mold interaction, the outbreak of ROS and development of systemic acquired resistance (SAR) are tightly combined with antioxidant enzymes, including thioredoxin. Tomato plants carrying the Cf-19 gene appear to have stronger C. fulvum resistance. [ABSTRACT FROM AUTHOR]

Key message: The expression of terpenoid and defense response genes elevated during agarwood formation. TDFs obtained from these genes can serve as biomarkers to distinguish resin-loaded Aquilaria plant from healthy plants. Agarwood develops in a fraction of the wild Aquilaria population as a result of natural infections and wounding in the live vascular tissues of the Aquilaria malaccensis tree. Only an expert agarwood farmer or trader can identify resin-loaded trees by detecting the hole in the trunk, but this approach is less reliable and non-scientific. This must be replaced with a scientific approach to remove ambiguity and establish a standard protocol to assess the quality and hence the economic value of the specific Aquilaria tree. Each year, a significant number of trees are harvested due to high market demand and indiscriminate felling of Aquilaria tree. This study involves a cDNA-AFLP approach for transcript profiling using 64 selective primer combinations. This produced 2760 transcript-derived fragments (TDFs), of which 50 differentially expressed TDFs (DE-TDFs) were sequenced and identified. Amongst these seven were linked to terpenoid biosynthesis, one found in each LOX, abscisic acid, jasmonic acid, and defense response pathway. TDF-1–3, 5, and 6 were identified as sesquiterpene synthase, TDF-4 as a branch point enzyme, TDF-48 as a MAP kinase, TDF-47, and TDF-49 as transcription factors (TFs). To validate the cDNA-AFLP results qRT-PCR and semi-quantitative PCR was performed for 11 DE-TDFs. These DE-TDFs were expressed only in infected plants and thus can be used as a biomarker to identify and delineate economically valuable resin-containing plants. [ABSTRACT FROM AUTHOR]

Drought seriously threatens tomato production worldwide. Despite much research on exogenous salicylic acid (SA) and Ca2+ improving plant resistance to biotic and abiotic stress, the molecular mechanisms of exogenous SA- and Ca2+-mediated drought resistance response in tomato remain unclear. In this study, we analyzed SA- and Ca2+ -induced differentially expressed transcripts under drought in tomato plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP). In total, 34 transcript derived fragments (TDFs) were differentially expressed. The functions identified through NCBI BLAST alignment mainly involved signal transduction, amino acid metabolism, transcription factors, transfer transport and stress response. The quantitative real-time polymerase chain reaction results of 12 TDFs associated with drought response were consistent with the patterns of changes observed with cDNA-AFLP analysis. These differentially expressed transcripts may be used for functional verification, transgenic research and breeding of drought-resistant tomato varieties.

Analysis of differentially expressed genes has assisted discovery of gene sets involved in particular biological processes. The purpose of this study was to identify genes involved in appressorium formation in the rice blast fungus Magnaporthe oryzae via analysis of cDNA–amplified fragment length polymorphisms. Amplification of appressorial and vegetative mycelial cDNAs using 28 primer combinations generated over 200 differentially expressed transcript-derived fragments (TDFs). TDFs were excised from gels, re-amplified by PCR, cloned, and sequenced. Forty-four of 52 clones analyzed corresponded to 42 genes. Quantitative real-time PCR showed that expression of 23 genes was up-regulated during appressorium formation, one of which was the MCK1 gene that had been shown to be involved in appressorium formation. This study will be providing valuable resources for identifying the genes such as pathogenicity-related genes in M. oryzae.

Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and vegetative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect 'genetic' information for both species, indicating influence of the genetic background on the epigenetic variation. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of floral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear-cut distinctive patterns, nonetheless profiles of young leaves and floral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa´s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application. [ABSTRACT FROM AUTHOR]

Gummosis (Phytophthora root and crown rot) is an important and destructive disease that affects fruit trees like pistachio (Pistacia vera L.). Although many studies have been performed on the possibility of biological control of gummosis, up to now few studies have been done on transcriptionally induced resistance genes in pistachio against Phytophthora disease. In the present research, the cDNA-AFLP method was used to analyze the transcriptionally regulated genes during the interaction between susceptible (Sarakhs) and resistant (Badami-Rize Zarandi) pistachio cultivars with Pseudomonas fluorescens and/or Phytophthora parsiana. Using 12 AFLP primer combinations, approximately 740 different cDNA-AFLP fragments with sizes ranged from 50 to 700 bp were generated for susceptible and resistant pistachio cultivars. Forty six TDFs (transcript derived fragments) were then selected for susceptible cultivar and 24 TDFs for resistant cultivar. Accordingly, each one of these TDFs was recognized by similarity search using the BlastX program against the NCBI GenBank nucleotide database. Notably, 18.5% of TDFs were hypothetical or uncharacterized proteins and 1.4% of them indicated no significant similarity to the known genes. Among the TDFs with known function, disease resistance proteins were identified, which were newly regulated in response to pathogen or rhizobacterium. Furthermore, we found that the expression level of different growth-regulatory or defense-related genes was higher after the exposure to Pseudomonas fluorescens and/or Phytophthora parsiana. To better understand the defense mechanism of pistachio plants and recognition of P. fluorescens-activated genes, which induce resistance against P. parsiana, would significantly assist in the development of new resistant pistachio cultivars. [ABSTRACT FROM AUTHOR]

Currently, a global demand exists forlavender as a significant medicinal plant and source of essential oils. Freshwater and arable lands are two major factors that inhibit extensive farming of medicinal plants in Iran. Saline water from seas and salty soil may be new resources for agricultural use, especially for medicinal plants. We sought to extend our knowledge of the Lavandula angustifolia genome and molecular basis of its salinity tolerance by using cDNA amplified fragment length polymorphism (cDNA-AFLP) to investigate the changes in plant transcriptomes in response to NaCl. All identified transcript derived fragments (TDF) were assigned as novel L. angustifolia genes related to signal transduction, regulation of gene expression, alternative splicing, autophagy, and secondary metabolite biosynthesis. qRT-PCR analysis of the TDFs in response to different concentrations of NaCl revealed various levels of mRNA of the identified genes in this plant. Our findings provided primary insights into the molecular response of L. angustifolia to salinity.

Ricinoleic acid, one of the biggest components in castor oil, is an unusual fatty acid with numerous industrial applications. Despite much research on the oil content of Ricinus communis L., the genes encoding the oil biosynthesis function in the seed developmental stages are still poorly known. Here, we analyzed differentially expressed transcripts during seed development using cDNA-amplified fragment length polymorphism. Almost all ingredients significantly decreased along with seed maturation, except for ricinoleic acid, which increased greatly from 6.7% to 86.8%. A total of 534 distinguished fragments were differentially expressed in six development stages. Among these, 67 fragments showed high homology with known-function genes, and 26 sequences were successfully annotated with fatty acid synthesis or storage proteins in castor and other species. The results showed that fatty acid synthesis and accumulation in castor seeds displayed various temporal patterns. Different cDNA-fragment patterns were mainly found in later development stages, coinciding with the onset of oil synthesis.

Olive is one of the important oil-produce fruit species with long history of cultivation. Despite high economic importance of olive, the molecular mechanisms that are involved in the oil production were not investigated completely in this fruit crop. In this study, the trend of oil accumulation, its composition and the transcriptional profiles of two important olive cultivars of Iran ('Mari' and 'Shenge') was screened during different fruit developmental stages (60, 90, 120, 150 and 180 days after flowering [DAF]). These cultivars showed the same trend for oil accumulation but a quite different fatty acid composition during the growing season. According to results, 60–120 DAF was identified as the most critical stages for oil biosynthesis in these olive cultivars. A cDNA-AFLP procedure was employed to detect the transcripts with differential expression during the critical stages of fatty acid biosynthesis in the fruit mesocarp. Altogether 195 distinguished fragments were identified as the transcripts with differential expression in these cultivars during the stages that were mostly important for oil biosynthesis. Out of which, twenty high quality clear transcript-derived fragments (TDFs) were cloned, sequenced and subjected to homology search analysis in GenBank database. Based on the homology search results, the entire differentially expressed TDFs in current investigation were classified into four main categories including oil production, carbohydrate metabolism, defense responsive and signaling related transcripts. Altogether, the majority of TDFs that were involved in the fatty acid biosynthesis and carbohydrate metabolism had higher expression in 'Mari', the cultivar which is superior in oil percentage and its composition. The expression of these TDFs coincided with the critical stages of oil accumulation (60–120 DAF). On the contrary, most of the defense related TDFs had higher expression in 'Shenge', an olive cultivar which is highly tolerant to the environmental stresses but inferior in the oil percentage and its composition. In conclusion, our finding provided primary information about the molecular basis of oil production in olive mesocarp and revealed new insight into the mechanisms that may be involved in the stress resistance in this fruit crop and shed some light on the probable candidate transcripts that may contribute to these two processes. [ABSTRACT FROM AUTHOR]

Siberian wildrye (Elymus sibiricus L.) is widely cultivated in the central and western regions of China. It is an important agricultural produce and utilized in animal feed and is also important for ecological environment protection. It is of great significance for the preservation and production of germplasm resource to elucidate the seed aging mechanism, such as delaying seed aging, prolonging seed life as well as repairing seed vigor. Information on seed aging including aging conditions, physiological and biochemical characteristics, damage to genomic DNA and genetic integrity of germplasm resource is limited but necessary for germplasm collection, conservation and effective commercial use. However, it is rarely reported on the transcriptome analysis of seed aging in Siberian wildrye. In this study, we analyzed the differentially accumulated TDF (Transcript-Derived Fragments) by cDNA-AFLP technique using 136 pairs primers, a total of 653 TDFs were identified. These up-regulated TDFs were picked for sequence alignment and functional analysis. The results showed that the sequences of TDFs are highly homologous in plants, which are pyruvate kinase, metal transport protein, DNA damage repair protein, lectin receptor kinase, and leucine zip transcription factor. Additionally, chloroplast structure was destroyed and energy consumption was increased in seed aging, at the same time, it could resist aging by increasing transcripts of relevant defense genes transporter genes and corresponding DNA damage repair genes when Siberian wildrye is aged. The purpose of this study was to provide a theoretical basis for revealing the potential aging mechanism of Siberian wildrye from the transcriptional level. [ABSTRACT FROM AUTHOR]

Drought seriously threatens tomato production worldwide. Despite much research on exogenous salicylic acid (SA) and Ca2+ improving plant resistance to biotic and abiotic stress, the molecular mechanisms of exogenous SA- and Ca2+-mediated drought resistance response in tomato remain unclear. In this study, we analyzed SA- and Ca2+ -induced differentially expressed transcripts under drought in tomato plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP). In total, 34 transcript derived fragments (TDFs) were differentially expressed. The functions identified through NCBI BLAST alignment mainly involved signal transduction, amino acid metabolism, transcription factors, transfer transport and stress response. The quantitative real-time polymerase chain reaction results of 12 TDFs associated with drought response were consistent with the patterns of changes observed with cDNA-AFLP analysis. These differentially expressed transcripts may be used for functional verification, transgenic research and breeding of drought-resistant tomato varieties. [ABSTRACT FROM AUTHOR]

This work presents a new method and tool to solve a common problem of molecular biologists and geneticists who use molecular markers in their scientific research and developments: curation of sequences. Omic studies conducted by molecular biologists and geneticists usually involve the use of molecular markers. AFLP, cDNA-AFLP, and MSAP are examples of markers that render information at the genomics, transcriptomics, and epigenomics levels, respectively. These three types of molecular markers use adaptors that are the template for PCR amplification. The sequences of the adaptors have to be eliminated for the analysis of the results. Since a large number of sequences are usually obtained in these studies, this clean-up of the data could demand long time and work. To automate this work, an R package, named CleanBSequences, was created that allows the sequences to be curated massively, quickly, without errors and can be used offline. The curating is performed by aligning the forward and/or reverse primers or ends of cloning vectors with the sequences to be removed. After the alignment, new subsequences are generated without biological fragments not desired by the user, i.e., sequences needed by the techniques. In conclusion, the CleanBSequences tool facilitates the work of researchers, reducing time, effort, and working errors. Therefore, the present tool would respond to the problems related to the curation of sequences obtained from the use of some types of molecular markers. In addition to the above, being an open source, CleanBSequences is a flexible tool that has the potential to be used in future improvements to respond to new problems. [ABSTRACT FROM AUTHOR]

In this study, the molecular mechanism underlying ethephon-induced shortened internodes in maize was investigated using Zhengdan958 maize variety. The ethephon treatment was performed by spraying 225 mL/ha Ethephon 40% SL onto the foliage at the eight-expanded-leaves stage. The differentially expressed genes in the young internodes under ethephon treatment were identified through methylation-sensitive amplified fragment length polymorphism (MSAP), start codon targeted polymorphism (SCoT) and cDNA-amplified fragment length polymorphism (AFLP) analyses. MSAP results showed the methylation sites were widely distributed in both the ethephon-treated (at 27.8%) and control plants (30.1%). This suggested that ethephon treatment modified the methylation patterns; with 3.0% of the sites being hyper-methylated and 7.3% demethylated compared with the control. Based on SCoT analysis, 148 transcript derived fragments (TDFs) were obtained in the ethephon-treated plants. Among them, 38 were up-regulated (25.7%) and 47 down-regulated (31.8%). cDNA-AFLP analysis using 70 primer pairs identified 1635 TDFs in the ethephon-treated and the control plants. Of these, 600 and 564 TDFs were up- and down-regulated by the ethephon treatment, respectively. BLASTX analysis on 50 (randomly selected) differentially expressed TDFs divided them into several categories based on their putative biological functions: signal transduction (6%), resistance-related (14%), energy and metabolism (22%), transcription (4%), cell apoptosis (2%), unknown functional proteins (42%) and unknown genes (10%). Our results revealed that ethephon treatment could induce DNA methylation variation principally by increasing the demethylation tendency. This is suggested to play roles in stress-defence genes expression regulation and the differentially expressed genes could be associated with shortened internodes in maize.

Herb dodder ( Cuscuta spp.) is one of the most important parasitic plants that can severely affect crop yields in the world. So far, interactions of this parasitic plant with hosts were not investigated adequately. Here, we conducted a differential expression analyzes and identified a number of genes that were differentially expressed in haustorium tissue compared with the stem of Cuscuta campestris growing on Alfalfa. We obtained 439 cDNA fragments from haustoria (parasite-host connection zone) and stems (25 cm away from connections zones) using the cDNA-AFLP (Amplified Fragment Length Polymorphism) method with eight different primer combinations. Of 439 transcript-derived fragments (TDFs) that were detected, 145 fragments were identified as differentially expressed genes. Five TDF sequences were similar to known functional genes involved in signal transduction, metabolism, respiration, and stress responses. Genes encoding DEAD-box ATP-dependent RNA helicase, potential heme-binding protein, lysine-specific demethylase 5A were selected for qRT-PCR. The qRT-PCR analyzes confirmed the results obtained using cDNA-AFLP. Our findings shed light on the elicitation of dodder defense responses in the connection zone to overcome plant defense reactions.

Stress can induce microspores to change their developmental pathway from the gametophytic to the embryogenic pathway. To explore the molecular mechanisms of microspore embryogenesis, complement DNA-amplified fragment length polymorphism was used to isolate the transcript-derived fragments during microspore embryogenesis of the non-heading Brassica campestris L. ssp. chinensis 'Wuyueman'. With 256 primer combinations screened, a total of 94 transcript-derived fragments were identified, and 15 were successfully sequenced. Based on a BLAST search in the Brassica database, 12 of the 15 sequenced transcript-derived fragments were homologous to genes with annotations; the remaining three transcript-derived fragments did not match any sequences. Transcript-derived fragments with annotations were involved in cell wall formation, hormones, and resistance. Analysis of cis-elements indicated that there were heat shock-related and stress-related cis-elements in the promoter sequences of 12 transcript-derived fragments. TDF1(Bra040720), TDF6(Bra013664), and TDF15(Bra022587) were selected for validation of complement DNA-amplified fragment length polymorphism expression patterns by real-time quantitative PCR. Results confirmed the altered expression patterns of three genes revealed by the complement DNA-amplified fragment length polymorphism. This study provides novel information on the molecular mechanism of microspore embryogenesis in non-heading Chinese cabbage. [ABSTRACT FROM AUTHOR]

L-Ascorbic acid (AsA) is an important nutrient in non-heading Chinese cabbage (Brassica rapa ssp. chinensis). To understand the regulatory mechanism of AsA accumulation, we crossed "Wutacai" and "Erqing" to obtain F2 plants and then constructed two pools (AsA-High and AsA-Low) of F2-generation plants to further analyze the differentially expressed genes (DEGs) by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technology. Among the DEGs, BcERF070 showed a significantly higher expression level in "Wutacai" and F2 plants with high AsA than in "Erqing" and F2 plants with low AsA, suggesting that BcERF070 is involved in the accumulation of AsA. To further understand the function of BcERF070 in AsA accumulation, we obtained BcERF070-silenced plants and BcERF070-overexpressed Arabidopsis thaliana plants. It was found that the downregulation of BcERF070 resulted in a decrease in AsA content, and the upregulation of BcERF070 resulted in an increase in AsA content. To evaluate the mechanism of BcERF070 involvement in the accumulation of AsA, the leaves of BcERF070-silenced and control plants were subjected to transcriptome sequencing. Twelve DEGs including BrAPXs were found to be associated with ascorbic acid. The qRT-PCR analysis on BcERF070-silenced plants and BcERF070-overexpressed plants further demonstrated the reliability of the transcriptome sequencing. Finally, based on analysis of the cis-acting elements of the promoter of 12 AsA-related genes, we speculated that BcERF070 may act on the DRE (dehydration-responsive element) of seven target gene promoters to regulate AsA content. In conclusion, a novel transcription factor, BcERF070, was revealed in this study, which affected the accumulation of AsA by regulating genes involved in the ascorbate biosynthesis and metabolic pathway. This information will help in understanding the regulatory mechanism of BcERF070 involvement in the accumulation of AsA in plants. [ABSTRACT FROM AUTHOR]

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• Piriformospora indica mediate host resistance against early blight by priming of a versatile host-signaling component. • P. indica modulate downstream defense related genes. • P. indica primed transcriptome reprogramming fine tunes jasmonate/ethylene mediated basal defense against pathogen infection. Piriformospora indica is an adaptable mycorrhiza-like fungus belonging to the Sebacinales order that can colonize roots of a wide range of plant species. Studies have shown that P. indica improves growth and enhances systemic defense against pathogens in host plants. However, the mechanism(s) through which these effects occur remain unclear. Therefore to gain more insight into the molecular basis of P. indica induced resistance, cDNA-AFLP (Amplified fragment length polymorphism) based transcript profiling was done to identify differentially expressed genes in P. indica -colonized tomato plants infected with Alternaria solani. Our results demonstrated that pre-colonization of tomato roots with P. indica systemically induced resistance against early blight. Transcript profiling of P. indica pre-colonized tomato plants revealed systemic modulation of several key components of signaling network transcriptional regulators including CBL-interacting protein kinase (CIPK), Mitogen activated protein kinases (MPKs), Lipid transfer proteins (LTP s), WRKY1 , ethylene responsive transcription factors (ERF), and Jasmonate Zim Domain 1 (JAZ1), a negative regulator of jasmonic acid (JA) signaling. Expression of downstream defense related genes like Thaumatin-like protein, β-1, 3-glucanase and chitinases was also affected in leaves upon challenge inoculation with pathogen. Interestingly, P. indica pre-colonization alone was unable to induce transcript levels for most of the genes studied. However, pathogen attack on P. indica pre-colonized plants induced strong defense responses. In conclusion, P. indica induces transcriptome reprogramming in a manner that allows rapid and efficient activation of JA/ET (jasmonic acid/ethylene)-mediated basal defenses against pathogen infection by altering the expression of JA/ET related genes. P. indica colonization appears to potentiate the complete signal transduction cascade leading to the systemic expression of defense genes against foliar pathogens. It thus presents itself as a potential and sustainable method of activating multiple components of defense signaling thereby conveying durable horizontal defense against a range of pathogens. [ABSTRACT FROM AUTHOR]

Osmanthus fragrans is an important traditional fragrant flower in China. Floral color and scent as the important ornamental characters are influenced by flowering process. Reports on the components of floral color and scent and related synthetic genes have been reported. However, studies on the synthesis and molecular basis of floral color and scent during flowering process are rarely reported. The study established a cDNA-AFLP system suitable for multiple samples and analyzed genes differential expression at different flowering stages. Finally, 283 TDFs with specific expression in petals and differential expression during flowering were successfully cloned and sequenced. Public databases blast showed that 120 TDFs had no homologous sequences, 12 TDFs had homologous sequences but their functions were unknown, and 150 TDFs had known biological functions, including the secondary metabolism, primary metabolism and development process, ect. qRT-PCR results of 6 TDFs with known functions comparing with cDNA-AFLP displayed that 4 TDFs with higher transcriptional level had consistent expression level. These results would be helpful to understand the gene expression of petals during flowering processes, and provide useful information for exploiting the mechanism of the floral color and scent formation of O. fragrans from molecular level. [ABSTRACT FROM AUTHOR]

Highlights • Variation in seed yield traits was assessed in cumin under drought and irrigation. • Molecular markers were used to analyze association of traits and genomic regions. • Differentially expressed markers associated with traits were identified in cumin. • No common marker-trait association was identified in two water status conditions. • Several marker combinations were associated with multiple agronomic traits. Abstract Cumin (Cuminum cyminum L.) is an aromatic medicinal herb with beneficial essential oil being used in antibacterial activities and industry of creams, lotions, and perfumes. Use of functional markers expedites breeding cumin for drought tolerance. The objective of this study was to identify association of plant height and several seed yield- related traits with complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) markers in 15 cumin (Cuminum cyminum) accessions under normal irrigation (NI) and drought stress (DS) conditions. Cumin accessions were grown in the field and subjected to NI practice and post-heading DS in two years in Kerman, Iran. The mean seed yield ranged between 448.72 and 994.18 kg/ha in NI and was from 211.05 to 452.05.06 kg/ha in DS. Reduction in plant height and seed yield-related traits ranged 27–28% in the first and 29–50% in the second year in DS compared with NI. Twelve Eco R I- Mse I primer combinations were used to amplify AFLP markers in cDNA fragments isolated from RNA samples. The identified markers were subjected to analysis of marker-trait associations (MTAs) using the generalized linear model (GLM) and mixed linear model (MLM). A number of 239 and 208 reliable transcript-derived fragments (TDFs) were amplified in cumin genotypes tested in NI and DS, respectively. Of the identified TDFs, highly significant markers were used for association analysis, only. Higher number of MTAs was identified for traits in NI compared with DS. Of the identified linked markers, 5 in NI and 3 in DS were significantly associated with more than one trait under two water status conditions. None of markers were identified as common MTAs between the two moisture conditions. Analysis of population structure demonstrated cumin accessions could be divided into 3 and 4 sub-populations. Overall, high variation was identified in cumin with respect to biological yield, seed yield and seed related traits and the identified linked markers can be incorporated into marker-assisted selection programs for the improvement of seed yield in drought condition. [ABSTRACT FROM AUTHOR]

Highlights • Gene expression under drought and recovery in Sphagnum denticulatum ecotypes. • cDNA-AFLP and RT-qPCR analysis of gene expression. • Expression enhanced by water deficiency in aquatic plants, not in terrestrial ones. • Identified genes can be used as indicators of stress tolerance in peat mosses. Abstract Adverse changes in vegetation occur as the effect of the progressive drying of peatlands, leading to the disappearance of peat mosses (Sphagnum), a key component of these ecosystems. We report gene expression divergence for two putative ecotypes of S. denticulatum collected from contrasted habitats under the threat of drought. We used cDNA-amplified fragment length polymorphism (cDNA-AFLP) as the first screening method. We then used real-time, quantitative PCR to identify genes whose transcription changes in response to drought and rehydration treatments. Using 133 of 900 pretested cDNA-AFLP primer combinations, we have identified 949 drought-induced transcript-derived fragments, 6 (0.6%) of which are significantly differentially expressed genes (DEGs) in the putative ecotypes. We cloned and sequenced 378 fragments using designed fragment-specific primers. Transcription patterns were quantified for 21 target genes using qRT-PCR. When upregulated under drought, nine genes showed fold changes in a range of 4.64–150.03. The DEGs represented functional categories including protein/nucleotide binding, catalytic activity, transporter activity, and enzyme regulation activity. These genes are known to be involved in various metabolic pathways and physiological processes which emphasize the complexity of plant response to stress. Most of the significant differences in transcription patterns were evident in the aquatic plants, suggesting induced responses to drought in these plants. The expression was significantly less pronounced in the terrestrial plants, indicating that they are preconditioned to withstand drought stress. We detected positive selection on individual codons for specific branches using the ratio of substitution rates at nonsynonymous and synonymous sites (d N /d S). For the first time, this study provides insight into the genetic basis of the putative adaptive evolution in Sphagnum and allows for the discovery of genes involved in drought stress response. [ABSTRACT FROM AUTHOR]

The transition of a plant from vegetative to reproductive stage is controlled by a large group of genes, which respond to environmental and endogenous stimuli. Application of methyl jasmonate (MeJA) and gibberellins (GA3) to oilseed plants (Brassica napus L.) interrupts the delicate endogenous balance and results in various floral organ abnormalities. Exogenous MeJA or GA3 influences the transcriptome at the initial flowering stage in Arabidopsis, but the corresponding changes of transcriptome in floral tissues of oilseed rape remain unknown. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) was analyzed to identify genes whose expression was modulated by application of MeJA and GA3 to flower buds. A total of 2 787 cDNA fragments were counted using 64 primer pair combinations, and bands larger than 50 bp were compared among four treatments, namely, water control, MeJA (50 μmol L−1), MeJA (100 μmol L−1), and GA3 (50 μmol L−1). Overall, 168 transcript-derived fragments (TDFs) were differentially expressed among the treatments. The expression pattern of some TDFs was confirmed by semi-quantitative RT-PCR analysis, and a group of 106 differentially displayed TDFs was cloned and sequenced. Homologs of Arabidopsis genes were identified and classified into 12 functional categories. A total of 34, 39, and 24 TDFs were responsive to GA3, MeJA, and both GA3 and MeJA, respectively. This finding indicated that cross-talk between these two hormones may be involved in regulating flower development. This study provides potential target genes for manipulation in terms of flowering time and floral organ initiation, important agronomic traits of oilseed rape.

As aquatic animals, fishes often encounter various situations of low oxygen, and they have evolved the ability to respond to hypoxia stress. Studies of physiological and molecular responses to hypoxia stress are essential to clarify genetic mechanisms underlying hypoxia tolerance in fish. In this study, we performed acute hypoxia treatment in juvenile bighead carp (Hypophthalmicthys nobilis) by decreasing water O2 from 6.5 mg/L to 0.5 mg/L in three hours. This hypoxia stress resulted in a significant increase in blood lactate and serum glucose. Comparisons of heart transcriptome among hypoxia tolerant (HT), hypoxia sensitive (HS), and normoxia control (NC) groups showed that 820, 273, and 301 differentially expressed genes (DEGs) were identified in HS vs. HT, NC vs. HS, and NC vs. HT (false discovery rate (FDR) < 0.01, Fold Change> 2), respectively. KEGG pathway enrichment showed that DEGs between HS and HT groups were mainly involved in mitogen-activated protein kinase (MAPK) signaling, insulin signaling, apoptosis, tight junction and adrenergic signaling in cardiomyocytes pathways, and DEGs in MAPK signaling pathway played a key role in cardiac tolerance to hypoxia. Combined with the results of our previous cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of hypoxia stress in this species, such genes as stbp2, ttn, mapk, kcnh, and tnfrsf were identified in both studies, representing the significance of these DEGs in hypoxia tolerance in bighead carp. These results provide insights into the understanding of genetic modulations for fish heart coping with hypoxia stress and generate basic resources for future breeding studies of hypoxia resistance in bighead carp.

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Ricinoleic acid, one of the biggest components in castor oil, is an unusual fatty acid with numerous industrial applications. Despite much research on the oil content of Ricinus communis L., the genes encoding the oil biosynthesis function in the seed developmental stages are still poorly known. Here, we analyzed differentially expressed transcripts during seed development using cDNA-amplified fragment length polymorphism. Almost all ingredients significantly decreased along with seed maturation, except for ricinoleic acid, which increased greatly from 6.7% to 86.8%. A total of 534 distinguished fragments were differentially expressed in six development stages. Among these, 67 fragments showed high homology with known-function genes, and 26 sequences were successfully annotated with fatty acid synthesis or storage proteins in castor and other species. The results showed that fatty acid synthesis and accumulation in castor seeds displayed various temporal patterns. Different cDNA-fragment patterns were mainly found in later development stages, coinciding with the onset of oil synthesis. [ABSTRACT FROM AUTHOR]

Background and study aims Homeobox-containing genes are composed of a group of regulatory genes encoding transcription factors involved in the control of developmental processes. The homeodomain proteins could activate or repress the expression of downstream target genes. This study was conducted to in vivo identify the potential target gene(s) of TGIF2LX in colorectal adenocarcinoma. Methods A human colorectal adenocarcinoma cell line, SW48, was transfected with the recombinant pEGFPN1-TGIF2LX. The cells were injected subcutaneously into the flank of the three groups of 6-week-old female athymic C56BL/6 nude mice (n = 6 per group). The transcript profiles in the developed tumours were investigated using the cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. Results The real-time RT-PCR and DNA sequencing data for the identified genes indicated that the N-terminal domain-interacting receptor 1 (Nir1) gene was suppressed whereas Nir2 and fragile histidine triad (FHIT) genes were upregulated followed by the overexpression of TGIF2LX gene. Conclusion Downregulation of Nir1 and upregulation of Nir2 and FHIT genes due to the overexpression of TGIF2LX suggests that the gene plays an important role as a suppressor in colorectal adenocarcinoma. [ABSTRACT FROM AUTHOR]

The ethanol-inducible system derived from Aspergillus nidulans has been used for gene expression analysis in plants. However, the response of non-transformed plants to ethanol suggests that this system may be present in plants. Therefore, it is possible to use an ethanol-inducible promoter derived from plants for gene regulation. In this study, effects of ethanol on rice growth were examined and an ethanol-inducible gene from rice was identified. The ethanol-inducible transcript-derived fragments (TDFs) were identified using cDNA-AFLP. These TDFs corresponded to genes involving different pathways including stress response. Using semi-quantitative RT-PCR, five TDFs, which were homologous to Os07g0240300, Os02g0175700, Os05g0392100, Os03g0569000, Os07g0627300, and adh2, were up-regulated. Among them, Os07g0627300 is highly conserved and expressed in several tissues; therefore, the promoter of Os07g0627300 was isolated and analyzed for motifs and promoter activity. The results indicated that it was a TATA-less promoter containing a number of motifs involved in stress responses. Transient expression results revealed that this promoter had ethanol-inducible promoter activity. These findings suggested that the Os07g0627300 promoter could be used as a useful tool for regulating gene expression in plants and could be used to generate transgenic crops with desirable traits. [ABSTRACT FROM AUTHOR]

Russian wheat aphid (Diuraphis noxia, Kurdjumov) feeding on susceptible Triticum aestivum L. leads to leaf rolling, chlorosis and plant death – symptoms not present in resistant lines. Although the effects of several D. noxia (Dn) resistance genes are known, none have been isolated or characterized. Wheat varieties expressing different Dn genes exhibit distinct modes of D. noxia resistance, such as antibiosis (Dn1), tolerance (Dn2), and antixenosis (Dn5). However, the mechanism whereby feeding aphids are perceived, and how subsequent transcriptional responses are partitioned into resistance categories, remains unclear. Here we report on downstream events in near-isogenic wheat lines containing different Dn genes after D. noxia biotype SA1 feeding. Transcripts involved in stress, signal transduction, photosynthesis, metabolism and gene regulation were differentially regulated during D. noxia feeding. Expression analyses using RT-qPCR and RNA hybridization, as well as enzyme activity profiling, provide evidence that the timing and intensity of pathways induced are critical in the development of particular modes of resistance. Pathways involved include the generation of kinase signalling cascades that lead to a sustained oxidative burst, and a hypersensitive response that is active during antibiosis. Tolerance is a passive resistance mechanism that acts through repair or de novo synthesis of photosystem proteins. Results further suggest that ethylene-mediated pathways are possibly involved in generating volatile compounds and cell wall fortification during the antixenosic response.

Transcriptome analysis reveals novel insights in the plant responses against biotic and abiotic stresses. cDNA-AFLP is appropriate and repeatable method for genome wide expression analysis when genomic information is limited. Although several factors may affect sensitivity, accuracy and repeatability of cDNA-AFLP technique so attention to these factors increases quality of results. In this study cDNA-AFLP technique was optimized for analysis of potato against Ralstonia solanacearum under in vitro conditions through examining various factors and their interactions, that affecting the results of such analysis. Results showed that RNA extraction method and the use of mRNA has large effects on the reliability of results. RNAs extracted based on using a purification column were monotonous. Ribosomal RNA and inhibitors alleviated by mRNA extraction, Also optimization of factors such as concentrations of template RNA, Mgcl2 and Taq DNA polymerase, in pre and selective amplification has significant effects on number, clarity and concentration of amplificated cDNA fragments.

Abstract: Abiotic stressors are main limiting factors for agricultural production around the world. Plant growth‐promoting bacteria have been successfully used to improve abiotic stress tolerance in several crops including wheat. However, the molecular changes involved in the improvement of stress management are poorly understood. The present investigation addressed some molecular factors involved in bacterially induced plant abiotic stress responses by identifying differentially expressed genes in wheat (Triticum aestivum) seedlings treated with the beneficial bacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5113 prior to challenge with abiotic stress conditions such as heat, cold or drought. cDNA‐AFLP analysis revealed differential expression of more than 200 transcript‐derived fragments (TDFs) in wheat leaves. Expression of selected TDFs was confirmed using RT‐PCR. DNA sequencing of 31 differentially expressed TDFs revealed significant homology with both known and unknown genes in database searches. Virus‐induced gene silencing of two abscisic acid‐related TDFs showed different effects upon heat and drought stress. We conclude that treatment with B. amyloliquefaciens 5113 caused molecular modifications in wheat in order to induce tolerance against heat, cold and drought stress. Bacillus treatment provides systemic effects that involve metabolic and regulatory functions supporting both growth and stress management. [ABSTRACT FROM AUTHOR]

Metals released into the environment continue to be of concern for human health. Using white-rot fungi as biosorbents for heavy metals removal is an attractive alternative owing to its good performance and low cost. However, the molecular mechanism underlying heavy metal tolerance in white-rot fungi has not yet been fully elucidated. This study identified and analyzed the lead (Pb)-induced transcriptional changes in Phanerochaete chrysosporium , a well-known heavy metal hyperaccumulating white-rot fungus. The results confirmed its outstanding ability in Pb tolerance and effective defense system. By comparative analysis of gene expression profiles obtained from cDNA-amplified fragment length polymorphism (cDNA-AFLP), we isolated 43 transcript-derived fragments (TDFs) differentially regulated by Pb exposure in P. chrysosporium , and 23 TDFs presented significant similarities to genes encoding known or putative proteins which belong to different functional categories involving ion binding, energy and carbohydrate metabolism, and signal transduction. The detailed characterization of these Pb-responsive genes was presented and the expression patterns of some interesting genes were validated by quantitative RT-PCR. This work provides the first evidence of Pb-responsive genes along with their putatively functional annotations in P. chrysosporium , which may help to understand the mechanism underlying heavy metal accumulation and tolerance in P. chrysosporium . [ABSTRACT FROM AUTHOR]

The ripening stage at harvest time determines the tomato fruit quality. After the fruit achieves its maximum size several metabolic changes of typically climacteric fruits are produced. Two cultivated genotypes of Solanum lycopersicum (Caimanta and 804627), with normal and altered fruit ripening, respectively and two accession, LA1385 of S. lycopersicum var. cerasiforme and LA722 of S. pimpinellifolium , with genes that prolong fruit shelf life, were tested to: 1) characterize and make a comparatively analysis for the transcriptome at different fruit ripening stages in genotypes that differ in fruit shelf life by cDNA-AFLP; and 2) provide further insight into the relationship between the extreme phenotypic differences for ripening among the genotypes through changes at transcriptomic level. Fruits at the breaker stage (B) were evaluated for fruit weight, firmness and fruit shelf life. The elapsed days between mature green (MG) and breaker stages Days (MG-B) as well as the elapsed days between B and red ripe (RR) stages Days (B-RR) were recorded. Comparison among ripening stages showed a great polymorphism related to the changes in gene expression. For all genotypes the transition from B to RR stages had higher polymorphism than the transition from MG to B. It was observed a great genetic variability for the phenotypic traits in agreement with the changes of gene expression. Moreover, it was observed that the transcriptome expression profiles in the initial and intermediate stages during ripening (MG and B) are more important to characterize genotypes. The wild species which have long shelf life do not show as drastic changes in gene expression as the cultivar with altered ripening that carrythe nor gene. These results suggest that the expressed or silenced genes could be involved, in some way, in the determination of the phenotypic traits evaluated in this study. [ABSTRACT FROM AUTHOR]

In this study, the molecular mechanism underlying ethephon-induced shortened internodes in maize was investigated using Zhengdan958 maize variety. The ethephon treatment was performed by spraying 225 mL/ha Ethephon 40% SL onto the foliage at the eight-expanded-leaves stage. The differentially expressed genes in the young internodes under ethephon treatment were identified through methylation-sensitive amplified fragment length polymorphism (MSAP), start codon targeted polymorphism (SCoT) and cDNA-amplified fragment length polymorphism (AFLP) analyses. MSAP results showed the methylation sites were widely distributed in both the ethephon-treated (at 27.8%) and control plants (30.1%). This suggested that ethephon treatment modified the methylation patterns; with 3.0% of the sites being hyper-methylated and 7.3% demethylated compared with the control. Based on SCoT analysis, 148 transcript derived fragments (TDFs) were obtained in the ethephon-treated plants. Among them, 38 were up-regulated (25.7%) and 47 down-regulated (31.8%). cDNA-AFLP analysis using 70 primer pairs identified 1635 TDFs in the ethephon-treated and the control plants. Of these, 600 and 564 TDFs were up- and down-regulated by the ethephon treatment, respectively. BLASTX analysis on 50 (randomly selected) differentially expressed TDFs divided them into several categories based on their putative biological functions: signal transduction (6%), resistance-related (14%), energy and metabolism (22%), transcription (4%), cell apoptosis (2%), unknown functional proteins (42%) and unknown genes (10%). Our results revealed that ethephon treatment could induce DNA methylation variation principally by increasing the demethylation tendency. This is suggested to play roles in stress-defence genes expression regulation and the differentially expressed genes could be associated with shortened internodes in maize. [ABSTRACT FROM PUBLISHER]

Graft compatibility between the rootstock and scion is a prerequisite for grafting and is presumably regulated by multiple genes involved in a complicated regulatory network. To better understand the mechanism of graft compatibility, it is necessary to analyze the function of differentially expressed genes in graft combinations with different degrees of graft compatibility. The objective of this study was to identify transcriptionally regulated genes and their possible functions in graft compatibility. Using cucumber ( Cucumis sativus L.) as scions, pumpkin ( Cucurbita L.) with different degrees of graft compatibility as rootstocks, and both self-grafting and non-grafting as controls, 18 different transcript-derived fragments (TDFs) in the leaves of grafted cucumber plants were cloned and sequenced using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technique. qRT-PCR showed that 6 TDFs exhibited significant differences between compatible and incompatible grafted cucumber leaves, and two of these TDFs also displayed differences between the two compatible and incompatible rootstocks at 25 days after grafting (DAG). Subsequently, a RING finger protein gene named CmRNF5 and a BTB domain-containing protein gene named CmNPH3L were cloned from pumpkin. Amino acid sequence alignments and phylogenetic analysis showed that these two genes were highly orthologous with those in other plants. The expression patterns of the two genes were investigated in rootstocks with different degrees of compatibility over time (5, 10, 15, 20, and 25 DAG). The results indicated that CmRNF5 and CmNPH3L transcripts were differentially expressed between compatible and incompatible unions throughout graft development. The expression differences of the two genes observed in the scion and different compatibility rootstocks suggested that these two genes could be related to graft compatibility/incompatibility responses. To gain more insight into the regulation of graft development, analysis of the expression patterns of the two genes under different environmental stimuli was performed via qRT-PCR, and the results showed that both genes are stress-induced genes. Based on the bioinformatics analysis of CmRNF5 and CmNPH3L , we hypothesized that CmRNF5 and CmNPH3L may trigger a compatibility/incompatibility response between the two graft partners that constitutes a compatible/incompatible combination via the ABA and auxin signal transduction pathways, respectively. [ABSTRACT FROM AUTHOR]

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, is one of the most devastating disease of wheat worldwide. Due to time and rate of defense genes expression, cultivar reaction to disease is different. So time and rate of defense genes expression can lead to develop an in vitro test for screening relative resistance of cultivars against pathogen. Forty wheat genotypes were screened by artificial infection and Marvdasht and Chamran as a resistant and Darab2 and Tajan as a susceptible cultivar were selected. Two treatments including scratched and not scratched leaves were considered for each cultivar. The soluble components were extracted 24 and 48 h after scratching the leaves and effect of extracts on sporulation and germination of M.graminicola spores was investigated. Increment of spore population in resistant cultivars was lower than susceptible ones. Also germination percent on media with resistant cultivars extract was lower as compared with media with extracts of very susceptible cultivars extract. The cDNA-AFLP experiment showed that expression of peroxidase 3 and 8 genes was increased in Chamran and Marvdasht cultivars respectively 48 hour after scratching the leaves. So it seems that we can use in vitro plant extract for screening relative resistance of wheat cultivars against M.graminicola.

Drought is the single largest abiotic stress factor leading to reduced crop yields. The identification of diff erentially expressed genes and the understanding of their functions in environmentally stressful conditions are essential to improve drought tolerance. Transcriptomics is a powerful approach for the global analysis of molecular mechanisms under abiotic stress. To identify genes that are important for drought tolerance, we analyzed mRNA populations from untreated and drought-stressed leaves of Triticum durum by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Overall, 76 transcript-derived fragments corresponding to differentially induced transcripts were successfully sequenced. Most of the transcripts identified here, using basic local alignment search tool (BLAST) database, were genes belonging to different functional categories related to metabolism, energy, cellular biosynthesis, cell defense, signal transduction, transcription regulation, protein degradation and transport. The expression patterns of these genes were confirmed by quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) based on ten selected genes representing different patterns. These results could facilitate the understanding of cellular mechanisms involving groups of genes that act in coordination in response to stimuli of water deficit. The identification of novel stress-responsive genes will provide useful data that could help develop breeding strategies aimed at improving durum wheat tolerance to field stress.

The purpose of the present study was to understand the molecular mechanisms during the graft union healing process. In the present study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was used to examine gene expression in Pyrus ussuriensis Maxim micro-grafting plantlets at four time points (at 0, 4, 7, and 11 days) during grafting tissue healing process. Twenty-six transcript-derived fragments (TDFs) involved in the graft healing mechanism were successfully obtained, which comprised 14 genes of known function, 9 genes of unknown function, and 3 novel genes. These 14 genes of known functions were involved in the functions of kinase, carbohydrate metabolism, protein metabolism, nuclear metabolism, cell activity and development, energy, and others. The expression analysis of 3 TDFs by qPCR showed that the results analyzed by cDNA-AFLP were basically reliable. These results suggest that P. ussuriensis undergoes complex metabolic processes during the graft union healing process. Detailed characterization of the genes involved in the specific process can help us uncover the molecular mechanisms for the healing process in plants. [ABSTRACT FROM AUTHOR]

Background Widespread methicillin resistant Staphylococcus aureus (MRSA) and absence of effective antimicrobial agents has led to limited therapeutic options for treating MRSA infection. We aimed to evaluate the effect of antimicrobial photodynamic therapy (aPDT) on the expression of novel identified methicillin resistance markers (NIMRMs) in S. aureus using complementary DNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) approaches to address the therapeutic alternatives for MRSA infections. Materials and methods We used cDNA-AFLP to compare MRSA and methicillin susceptible S. aureus (MSSA) for identification of target genes implicated in methicillin resistance. To determine the sub-lethal aPDT (sPDT), MRSA and MSSA clinical isolates photosensitized with toluidine blue O (TBO), and then were irradiated with diode laser. After sPDT, the colony forming units/mL was quantified. Antimicrobial susceptibility against methicillin was assessed for cell-surviving aPDT. Effects of sPDT on the expression of NIMRMs were evaluated by real-time quantitative reverse transcription PCR. Results According to our results, serine hydrolase family protein (Shfp) encoding gene and a gene encoding a conserved hypothetical protein (Chp) were implicated in methicillin resistance in MRSA. sPDT reduced the minimum inhibitory concentrations of methicillin by 3-fold in MRSA. sPDT could lead to about 10- and 6.2- fold suppression of expression of the Chp and Shfp encoding genes, respectively. Conclusion sPDT would lead to reduction in resistance to methicillin of MRSA in surviving cells by suppressing the expression of the Shfp and Chp encoding genes associated with methicillin resistance. This may have potential implications of aPDT for the treatment of MRSA infections. [ABSTRACT FROM AUTHOR]

Herb dodder (Cuscuta spp.) is one of the most important parasitic plants that can severely affect crop yields in the world. So far, interactions of this parasitic plant with hosts were not investigated adequately. Here, we conducted a differential expression analyzes and identified a number of genes that were differentially expressed in haustorium tissue compared with the stem of Cuscuta campestris growing on Alfalfa. We obtained 439 cDNA fragments from haustoria (parasite-host connection zone) and stems (25 cm away from connections zones) using the cDNA-AFLP (Amplified Fragment Length Polymorphism) method with eight different primer combinations. Of 439 transcript-derived fragments (TDFs) that were detected, 145 fragments were identified as differentially expressed genes. Five TDF sequences were similar to known functional genes involved in signal transduction, metabolism, respiration, and stress responses. Genes encoding DEAD-box ATP-dependent RNA helicase, potential heme-binding protein, lysine-specific demethylase 5A were selected for qRT-PCR. The qRT-PCR analyzes confirmed the results obtained using cDNA-AFLP. Our findings shed light on the elicitation of dodder defense responses in the connection zone to overcome plant defense reactions. [ABSTRACT FROM AUTHOR]

Drought is a major stressor that severely hampers the production of many important crops. The short arm of rye (Secale cereale) chromosome 1 contains favourable stress-resistance alleles and is thus widely used in wheat (Triticum aestivum) breeding to generate 1BL.1RS translocation lines. In this study, 1BL.1RS-specific drought-responsive genes were identified using cDNA-amplified fragment length polymorphism (AFLP) analysis. Among the 144 transcript-derived fragments (TDF) differentially expressed in the 1BL.1RS line, we identified the function and chromosomal position of 84 TDF using public databases. Real-time PCR was performed to validate the results of cDNA-AFLP, and four TDF were significantly up-regulated. Furthermore, we developed two wheat-rye translocation-specific markers by comparing the TDF from the short arms of wheat and rye chromosome 1. One is specific to 1BL.1RS and the other is specific to 1AL.1RS and 1BL.1RS. Combinational use of both markers will be helpful to breed wheat possessing 1RS. [ABSTRACT FROM AUTHOR]

Biomarkers of exposure can be used to identify specific contaminants that are adversely affecting aquatic organisms. However, it remains prohibitively costly to investigate multiple novel biomarkers of exposure in a non-model species, despite the development of next-generation sequencing technology. In this study, we focused on the use of cDNA-amplified fragment length polymorphism (AFLP) as a cost-effective biomarker discovery tool to test whether it could identify biomarkers of exposure in the non-model amphipod species Grandidierella japonica . Loci were identified that were differentially expressed in amphipods exposed to reference chemicals (Cu, Zn, and nicotine) and to an environmental sample (road dust) at sublethal concentrations. Eight loci were shown to respond consistently to nicotine at different concentrations, but not to Cu or Zn. Some of the loci also responded to an environmental road dust sample containing nicotine. These findings suggest that loci identified using cDNA-AFLP could be used as biomarkers of nicotine exposure in environmental samples with complex matrices. Further studies with other organisms and toxicants are needed, but we have demonstrated that the use of cDNA-AFLP to identify biomarkers for ecotoxicological studies of non-model species is at least feasible. [ABSTRACT FROM AUTHOR]