Transcript-derived Fragments Research Articles

Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection.

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu).Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work.Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-613) contains supplementary material, which is available to authorized users.

Development of molecular markers linked to barley heterosis

The use of a three-line breeding system based on cytoplasmic male sterility has a great potential for hybrid production in barley. Hybrid vigour, or heterosis, which is an advantage of the offspring as compared to two parental lines, is one of the major factors for the success of the use of hybrids. In this study, gene differential expression profiles between barley hybrids and their parents were analysed by cDNA-AFLP analysis using forty eight crosses. Five transcript-derived fragments (TDFs) showed significant effects on heterosis of 1,000-kernel-weight, one of the major yield components. These five TDFs were successfully validated in different crosses by eliminating crosses with lower heterosis and thus can be used in breeding programs.

Identification of desiccation tolerance transcripts potentially involved in rape (Brassica napus L.) seeds development and germination

To investigate regulatory processes and protective mechanisms leading to desiccation tolerance (DT) in seeds, cDNA amplified fragment length polymorphism (cDNA-AFLP) in conjunction with 128 primer combinations was used to detect differential gene expression in rape seeds in response to DT during seed development and germination. We obtained approximately 8000 transcript-derived fragments (TDFs), of which 394 TDFs with differential expression patterns (“sustained expression”, “up-regulated”, “couple with seed DT”, and “down-regulated”) were excised from gels and re-amplified by polymerase chain reaction (PCR). After sequencing and comparison with the National Center for Biotechnology Information database, 176 TDFs presented significant similarity with known genes that could be classified into the following categories: metabolism and energy, stress resistance and defense, storage, signal transduction, and other functional categories. Using semiquantitative reverse-transcription PCR and real-time PCR approaches, the significance of the differences was further confirmed in fresh seeds and dehydrated seeds. The genes that encode superoxide dismutase, peroxiredoxin, caleosin, oleosin S3, steroleosin, late embryogenesis abundant protein, glutathione reductase, β-glucosidase, S23 transcriptional repressor, and some heat-shock proteins could be associated with DT. The results of this study will aid in the identification of candidate genes for future experiments that seek to understand seed DT.

Mapping QTL for cotton fiber quality traits using simple sequence repeat markers, conserved intron-scanning primers, and transcript-derived fragments

Cotton is an important fiber crop worldwide. Improved fiber quality is a driving force for cotton genetic studies and breeding. A BC1 population containing 115 individuals from a cross between Gossypium hirsutum cv. CCRI8 and G. barbadense cv. Pima 90-53 was established, and 519 simple sequence repeat (SSR) markers, two conserved intron-scanning primers (CISPs), and transcript-derived fragments (TDFs) amplified from 156 ApoI/TaqI selective primer combinations were used to construct a genetic linkage map. The map included 579 markers distributed on 56 linkage groups. Accounting for 83.4 % of the cotton genome, it covered 4,168.72 cM, with an average distance of 7.19 cM between markers. Lengths of the linkage groups ranged from 1.25 to 255.79 cM, with 2 to 44 markers per group. Of these 56 groups, 43 were assigned to 26 chromosomes, with the remaining 13 unknown. Based on this newly constructed map of tetraploid cotton, we performed quantitative trait loci (QTL) mapping and analysis of fiber quality traits from the BC1 and its derived BC1F2 lines. A total of 44 fiber quality QTL were detected on 17 chromosomes, explaining 7.72–23.73 % of the phenotypic variation. Pima 90-53 offered 13 QTL alleles with positive additive effects and four with negative additive effects for fiber quality traits. The results from this study may provide useful information for breeders to transfer desirable fiber traits from cotton types, such as the Sea Island strain, to Upland cotton that is primarily cultivated currently.

Oligo-dT anchored cDNA-SCoT: a novel differential display method for analyzing differential gene expression in response to several stress treatments in mango (Mangifera indica L.).

Differential display is a powerful technique for analyzing differences in gene expression. Oligo-dT cDNAstart codon targeted marker (cDNA-SCoT) technique is a novel, simple, cheap, rapid, and efficient method for differential gene expression research. In the present study, the oligo-dT anchored cDNA-SCoT technique was exploited to identify differentially expressed genes during several stress treatments in mango. A total of 37 primers combined with oligo-dT anchor primers 3side amplified approximately 150 fragments of 150 bp to 1500 bp in length. Up to 100 fragments were differentially expressed among the stress treatments and control samples, among which 92 were obtained and sequenced. Out of the 92 transcript derived fragments (TDFs), 70% were highly homologous to known genes, and 30% encoded unclassified proteins with unknown functions. The expression pattern of nine genes with known functions involved in several abiotic stresses in other species was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) under cold (4 °C), salinity (NaCl), polyethylene glycol (PEG, MW 6000), and heavy metal treatments in leaves and stems at different time points (0, 24, 48, and 72 h). The expression patterns of the genes (TDF4, TDF7, TDF23, TDF45, TDF49, TDF50, TDF57, TDF91 and TDF92) that had direct or indirect relationships with cold, salinity, drought and heavy metal stress response were analyzed through qRT-PCR. The possible roles of these genes are discussed. This study suggests that the oligo-dT anchored cDNA-SCoT differential display method is a useful tool to serve as an initial step for characterizing transcriptional changes induced by abiotic stresses and provide gene information for further study and application in genetic improvement and breeding in mango.

Identification of Genes Expressed Differentially in Grapefruit Infected with Candidatus Liberibacter asiaticus in the Late Stage of Disease

Citrus greening or Huanglongbing (HLB), a destructive disease of citrus worldwide, was reported from south of Iran in 2007. The molecular basis of compatibility and disease development in this system is poorly understood. We have carried out a cDNA-AFLP analysis to analyse gene expression of grapefruit infected by Candidatus Leiberibacter asiaticus in the late infection stage. We have applied a cDNA-AFLP approach on grafted infected grapefruit trees at the representing symptoms stage in susceptible host. Selective amplifications with 10 primer combinations allowed the visualization of approximately 24 transcript-derived fragments (TDFs) in the leaves of graft-inoculated trees, which were differentially expressed. We sequenced 14 fragments, which were identified as grapefruit transcripts after homology searching, whereas 12 were not homologous to sequences in NCBI databases. Many grapefruit genes spanning almost all functional categories were upregulated during infection, especially genes involved in ATP synthesize, cytochrome P450 synthesize, isoflavone 2'-hydroxylase, zeaxanthin epoxidase, cellulose synthase, DNA repair protein, aconitate hydratase 2 and citrus tristeza virus resistance gene. This study provides the first global catalogue of grapefruit genes expressed during inoculation, together with their functional annotations. This will help to elucidate the molecular basis of the resistance process and identify genes and chemicals that could help to inhibit the pathogen.

Identification of photoperiod-regulated gene in soybean and functional analysis in Nicotiana benthamiana.

Soybean (Glycine max) is a short-day crop and the photoperiod is a crucial factor regulating its flowering time. To investigate the molecular mechanism controlling the flowering time by photoperiod in soybean, cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify photoperiod-regulated genes in leaves of soybean growing under short-day length, neutral photoperiod and long-day length. A total of 36 transcript-derived fragments (TDFs) were identified to be regulated by photoperiod. Among them, 26 TDFs were homologues of genes with known function. These genes are involved in secondary metabolism, cellular metabolism, cell wall components metabolism, ion transport and hormone signalling. Silencing of the homologue genes in Nicotiana benthamiana for 14 TDFs was conducted by virus-induced gene silencing. The flowering time was delayed by silencing of the genes encoding rhodanese and 40S ribosomal protein S4 (RPS4). The results indicated that rhodanese and RPS4 probably play important roles in regulating flowering time.

Identification of Genes Differentially Expressed between Resistant and Susceptible Tomato Lines during Time-Course Interactions with Xanthomonas perforans Race T3

Bacterial spot caused by several Xanthomonas sp. is one of the most devastating diseases in tomato (Solanum lycopersicum L.). The genetics of hypersensitive resistance to X. perforans race T3 has been intensively investigated and regulatory genes during the infection of race T3 have been identified through transcriptional profiling. However, no work on isolating regulatory genes for field resistance has been reported. In this study, cDNA-amplified fragment length polymorphism technique was used to identify differentially expressed transcripts between resistant tomato accession PI 114490 and susceptible variety OH 88119 at 3, 4 and 5 days post-inoculation of the pathogen. Using 256 selective primer combinations, a total of 79 differentially expressed transcript-derived fragments (TDFs) representing 71 genes were obtained. Of which, 60 were up-regulated and 4 were down-regulated in both tomato lines, 4 were uniquely up-regulated and 2 were uniquely down-regulated in PI 114490, and 1 was specifically up-regulated in OH 88119. The expression patterns of 19 representative TDFs were further confirmed by semi-quantitative and/or quantitative real time RT-PCR. These results suggested that the two tomato lines activated partly similar defensive mechanism in response to race T3 infection. The data obtained here will provide some fundamental information for elucidating the molecular mechanism of response to race T3 infection in tomato plants with field resistance.

Genetic and gene expression analysis of dm1, a dwarf mutant from Cucurbita maxima Duch. ex Lam, based on the AFLP method

Wang, R., Huang, H., Lin, Y., Chen, Q., Liang, Z. and Wu, T. 2014. Genetic and gene expression analysis of dm1, a dwarf mutant from Cucurbita maxima Duch. ex Lam, based on the AFLP method. Can. J. Plant Sci. 94: 293–302. dm1, a dwarf mutant from Cucurbita maxima (Duch. ex Lam) by natural mutation, showed distinct dwarf phenotypes such as shorter vines and fewer and shorter internodes. Genetic analysis indicated that the dm1 mutation was recessive, and the dwarfing character was controlled by a single locus. DNA-AFLP analysis showed that a fragment (MCAG/ETT) was linked with the dwarfing character of dm1 and that the fragment contained 152 base pairs (bp). It was investigated in F2 populations of dm1 and vine plants, and the genetic distance between the MCAG/ETT fragment and dwarf gene in dm1 was 11.2 cM, calculated by JoinMap 3.0 software. In addition, the result of cDNA-AFLP analysis showed that there were 52 differential transcript derived fragments (TDFs) found between dm1 and vine plants. Only four TDFs, A16T12, A16T9, A6T14 and A6T16, were expressed stably and specifically in dm1 plants in subsequent investigation. The four fragments share 71, 79, 87 and 79% nucleic acid sequence similarity with the complete coding sequence of Arabidopsis thaliana histidine kinase 3 (AHK3) mRNA, nucleic acid sequence of Vitis vinifera dihydroflavonol-4-reductase-like (DFRL), nucleic acid sequence of Glycine max histone-lysine N-methyltransferase ATX4-like and nucleic acid sequence of Arabidopsis thaliana histidinol dehydrogenase (HDH), respectively. Bioinformatics analysis indicated that AHK3, DFRL and HDH were respectively related to Cytokinin signaling, indole acetic acid signaling and Ni accumulation, which played important roles in plant growth, so the expression of the four TDFs may contribute to form dwarfism in dm1.

Isolation, molecular cloning and characterization of a cold-responsive gene, AmDUF1517, from Ammopiptanthus mongolicus

Temperature is one of important factors that influence plant growth and development. Using cDNA-amplified fragment length polymorphism approach, we previously screened 344 temperature-related transcript-derived fragments (TDFs) from Ammopiptanthus mongolicus. In this study, we confirmed that 15 of these TDFs were upregulated in response to low- or high-temperature by using semi-quantitative RT-PCR. Based on the rapid amplification of cDNA ends, PCR and genome walking approaches, full-length cDNA and promoter sequence of AmDUF1517 was cloned and identified. The 906 bp open reading frame of the AmDUF1517 gene encoded for a protein of 301 amino acids residues. The corresponding genomic DNA sequence contains two exons and one intron. Bioinformatic analysis showed that a predicted cleavage site for chloroplast transit peptide, a DUF1517 domain, two transmembrane domains and two putative sumoylation sites were conserved between AmDUF1517 and its homolog from Arabidopsis thaliana (AtDUF1517). We further showed that GFP-tagged AmDUF1517 was indeed targeted to the chloroplast in Arabidopsis protoplast. The transcript levels of AmDUF1517 were increased specifically in leaves in response to cold stress. In addition, treatment of ethylene, salicylic acid, gibberellic acid or NaCl induced the transcription of AmDUF1517. Mutation of DUF1517 in Arabidopsis exhibited enhanced sensitivity to cold stress, which was coupled with increased electrolyte leakage, malondialdehyde content and decreased contents of soluble sugar, proline. Interestingly, heterologous expression of AmDUF1517 in Arabidopsisatduf1517 mutants significantly rescued their cold-sensitive phenotypes. Altogether, our data suggest the potential roles of both AmDUF1517 and AtDUF1517 in the regulation of cold stress tolerance.

Differentially expressed TDFs by cDNA-AFLP in tomato infected withFusarium oxysporumf.sp. lycopersici

To elucidate the tomato defense responses during the interaction of tomato and Fusarium oxysporum f. sp. lycopersici (Fol), a cDNA-AFLP experiment of the fungal-induced changes at the transcriptional level was performed to identify up- or down-regulated genes. Transcript derived fragments (TDF) from leaves, stems and roots were used for accurate gene expression profiles by visual observation and analysis of bands and their intensities in Fol infected and non-infected tomato lines (AVTO 9802 and AVTO 1002). Using 10 AFLP primer combinations, an average of 163 scored TDFs was identified as differentially expressed genes, corresponding to about 55.7% of all visualized transcripts. These TDFs were consistent with the extensive physiological changes that affect infected tissues from leaves, stems and roots. Although most of the visualized transcripts were affected by infection, about 46.6% of the identified differentially expressed TDFs were down-regulated during infection, possibly reflecting the management of cellular resources and/or the repression of defense responses. Understanding the defense mechanism of plant against pathogens would significantly assist in the development of new resistant tomato cultivars.

Gene expression profiling by cDNA-AFLP reveals potential candidate genes for partial resistance of 'Président Roulin' against Venturia inaequalis.

BackgroundScab, caused by the fungus Venturia inaequalis, is one of the most important diseases of cultivated apple. While a few scab resistance genes (R genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance, supposed to be more durable, are still unknown. This study aims to investigate the molecular mechanisms involved in the partial resistance of the old Belgian apple cultivar ‘Président Roulin’ against V. inaequalis.ResultsA global gene expression analysis was conducted in ‘Président Roulin’ (partially resistant) and in ‘Gala’ (susceptible) challenged by V. inaequalis by using the cDNA-AFLP method (cDNA-Amplified Fragment Length Polymorphism). Transcriptome analysis revealed significant modulation (up- or down-regulation) of 281 out of approximately 20,500 transcript derived fragments (TDFs) in ‘Président Roulin’ 48 hours after inoculation. Sequence annotation revealed similarities to several genes encoding for proteins belonging to the NBS-LRR and LRR-RLK classes of plant R genes and to other defense-related proteins. Differentially expressed genes were sorted into functional categories according to their gene ontology annotation and this expression signature was compared to published apple cDNA libraries by Gene Enrichment Analysis. The first comparison was made with two cDNA libraries from Malus x domestica uninfected leaves, and revealed in both libraries a signature of enhanced expression in ‘Président Roulin’ of genes involved in response to stress and photosynthesis. In the second comparison, the pathogen-responsive TDFs from the partially resistant cultivar were compared to the cDNA library from inoculated leaves of Rvi6 (HcrVf2)-transformed ‘Gala’ lines (complete disease resistance) and revealed both common physiological events, and notably differences in the regulation of defense response, the regulation of hydrolase activity, and response to DNA damage. TDFs were in silico mapped on the ‘Golden Delicious’ apple reference genome and significant co-localizations with major scab R genes, but not with quantitative trait loci (QTLs) for scab resistance nor resistance gene analogues (RGAs) were found.ConclusionsThis study highlights possible candidate genes that may play a role in the partial scab resistance mechanisms of ‘Président Roulin’ and increase our understanding of the molecular mechanisms involved in the partial resistance against apple scab.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1043) contains supplementary material, which is available to authorized users.

Sugarcane Transcript Profiling Assessed by cDNA-AFLP Analysis during the Interaction with <i>Sugarcane Mosaic Virus</i>

Sugarcane mosaic caused by Sugarcane Mosaic Virus (SCMV) is one of the most important virus diseases of sugarcane. In the present study, changes in the transcription profile obtained by cDNA-AFLP analysis were investigated in two sugarcane varieties contrasting to SCMV resistance, when challenged with a severe virus strain. Healthy plants derived from meristem tip tissue culture were mechanically inoculated under greenhouse controlled conditions and sampled at 24, 48 and 72 hours after inoculation. A total of 392 transcript-derived fragments (TDFs) were verified in the resistant variety against 380 in the susceptible one. The two sugarcane genotypes showed differential behavior in the number of induced and repressed TDFs along the time-course samplings. Ten out of 23 sequenced TDFs (unique from the resistance variety), showed identity with known plant sequences, mostly related to plant defense mechanisms against pathogens. The cDNA-AFLP technique was effective in revealing changes in the transcription profile within and between contrasting varieties when challenged by SCMV.

CDNA-AFLP transcriptional profiling reveals genes expressed during flower development in Oncidium Milliongolds

The flower developmental process, which is crucial to the whole lifecycle of higher plants, is influenced by both environmental and endogenous factors. The genus Oncidium is commercially important for cut flower and houseplant industry and is ideal for flower development studies. Using cDNA-amplified restriction fragment length polymorphism analysis, we profiled transcripts that are differentially expressed during flower development of Oncidium Milliongolds. A total of 15,960 transcript-derived fragments were generated, with 114 primer sets. Of these, 1248 were sequenced, producing 993 readable sequences. BLASTX/N analysis showed that 833 of the 993 transcripts showed homology to genes in the NCBI databases, exhibiting functions involved in various processes, such as signal transduction, energy conversion, metabolism, and gene expression regulation. The full-length mRNAs of SUCROSE SYNTHASE 1 (SUS1) and LEAFY (LFY) were cloned, and their expression patterns were characterized. The results showed that the expression levels of SUS1 and LFY were similar during flower development. To confirm the function of SUS1 in flower buds, carbohydrate content and sucrose synthase activity were determined. The results showed that changes in sucrose content and sucrose synthase activity reflected SUS1 expression levels. Collectively, these results indicate that SUS1 influences flower development by regulating LFY expression levels through changing the sucrose content of flower buds.

Transcriptomic clues to understand the growth of Lactobacillus rhamnosus in cheese

BackgroundLactobacillus rhamnosus is a non-starter lactic acid bacterium that plays a significant role during cheese ripening, leading to the formation of flavor. In long-ripened cheeses it persists throughout the whole time of ripening due to its capacity to adapt to changing environmental conditions. The versatile adaptability of L. rhamnosus to different ecosystems has been associated with the capacity to use non-conventional energy sources, regulating different metabolic pathways. However, the molecular mechanisms allowing the growth of L. rhamnosus in the cheese dairy environment are still poorly understood. The aim of the present study was to identify genes potentially contributing to the growth ability of L. rhamnosus PR1019 in cheese-like medium (CB) using a transcriptomic approach, based on cDNA-amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time reverse transcription-PCR (qPCR).ResultsUsing three primer combinations, a total of 89 and 98 transcript-derived fragments were obtained for L. rhamnosus PR1019 grown in commercial MRS medium and CB, respectively. The cDNA-AFLP results were validated on selected regulated genes by qPCR. In order to investigate the main adaptations to growth in a cheese-mimicking system, we focused on 20 transcripts over-expressed in CB with respect to MRS. It is worth noting the presence of transcripts involved in the degradation of pyruvate and ribose. Pyruvate is a intracellular metabolite that can be produced through different metabolic routes starting from the carbon sources present in cheese, and can be released in the cheese matrix with the starter lysis. Similarly the ribonucleosides released with starter lysis could deliver ribose that represents a fermentable carbohydrate in environments, such as cheese, where free carbohydrates are lacking.Both pyruvate degradation and ribose catabolism induce a metabolite flux toward acetate, coupled with ATP production via acetate kinase. Taking into account these considerations, we suggest that the energy produced through these pathways may concur to explain the great ability of L. rhamnosus PR1019 to grow on CB.ConclusionsBy a transcriptomic approach we identified a set of genes involved in alternative metabolic pathways in L. rhamnosus that could be responsible for L. rhamnosus growth in cheese during ripening.

CDNA-AFLP analysis reveals heat shock proteins play important roles in mediating cold, heat, and drought tolerance in Ammopiptanthus mongolicus

Ammopiptanthus mongolicus (Maxim.ex kom.) Cheng F. is the only evergreen broadleaf shrub endemic to the desert of central Asian and it can survive at drought, salt, and alkali stress. It is believed that A. mongolicus is an important germplasm containing abiotic-tolerance genes. In order to identify drought-, cold-, and heat-responsive genes and to gain a better understanding of stress responses in A. mongolicus, genome-wide investigation of drought-, cold-, and heat-responsive genes was performed in A. mongolicus using cDNA-amplified fragment length polymorphism. Selective amplification with 240 primer combinations generated 5,000 differentially expressed transcript derived fragments (TDFs). Of these, 201 TDFs with differential expression patterns were excised from gels, reamplified by PCR, and sequenced. The gene expression patterns of 11 regulated genes were further investigated by semiquantitative reverse transcriptase polymerase chain reaction analysis. Sequencing and similarity analysis revealed that TDFs present homologies chiefly with proteins involved in various abiotic and biotic stress and developmental responses. The information presented in this study reveals that heat shock proteins play an active role in mediating drought, cold, and heat tolerance in A. mongolicus.

Histological and transcriptional characterization of a novel recessive genic male sterility mutant in sesame (Sesamum indicum L.)

Recessive genic male sterility (RGMS) is an important approach to utilize heterosis, but the molecular mechanism of RGMS is not well understood in Sesamum indicum L. 95ms-5AB is a novel male sterility two-type line, which is controlled by a single RGMS gene. Histological observations revealed that abnormal inhibited male gametogenesis appeared during the mononuclear microspore stage. It was characterized by collapsed microspores, expanded vacuoles and the delayed degeneration of tapetum at the pollen maturation stage. To elucidate the mechanism of this RGMS, the transcriptional profiles of the sterile flower buds in 95ms-5A and the fertile flower buds in 95ms-5B were investigated using the cDNA-AFLP (amplified fragment length polymorphism) technique. About 30 reproducible differentially expressed transcript-derived fragments (TDFs) were detected, including 27 up-regulated from fertile buds and three from sterile buds. The functional analysis of 27 successfully cloned TDFs indicated that the corresponding genes of 11 TDFs may participate in the processes of energy metabolism, signal transduction and plant cell development. The other 16 TDFs were homologous to the proteins with unknown functions or with no homology in GenBank. Real-time PCR analysis of 21 genes in fertile and sterile buds at four different stages was used to validate and further investigate their expression patterns identified by cDNA-AFLP. Our results suggested that several genes related to callose wall or tapetum degeneration were suppressed in 95ms-5A even before the microspore mother cell stage, which led to delayed tapetum degeneration and blocked male gametogenesis. Several RGMS-related novel genes, including one stamen specially expressed gene, had also been identified that showed significantly different expression level between fertile and sterile buds along sesame male gametogenesis.

Differential Gene Expression for Curvularia eragrostidis Pathogenic Incidence in Crabgrass (Digitaria sanguinalis) Revealed by cDNA-AFLP Analysis

Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

Comparative analysis of latex transcriptome reveals putative molecular mechanisms underlying super productivity of Hevea brasiliensis.

Increasing demand for natural rubber prompts studies into the mechanisms governing the productivity of rubber tree ( Hevea brasiliensis ). It is very interesting to notice that a rubber tree of clone PR107 in Yunnan, China is reported to yield more than 20 times higher than the average rubber tree. This super-high-yielding (SHY) rubber tree (designated as SY107), produced 4.12 kg of latex (cytoplasm of rubber producing laticifers, containing about 30% of rubber) per tapping, more than 7-fold higher than that of the control. This rubber tree is therefore a good material to study how the rubber production is regulated at a molecular aspect. A comprehensive cDNA-AFLP transcript profiling was performed on the latex of SY107 and its average counterparts by using the 384 selective primer pairs for two restriction enzyme combinations (ApoI/MseI and TaqI/MseI). A total of 746 differentially expressed (DE) transcript-derived fragments (TDFs) were identified, of which the expression patterns of 453 TDFs were further confirmed by RT-PCR. These RT-PCR confirmed TDFs represented 352 non-redundant genes, of which 215 had known or partially known functions and were grouped into 10 functional categories. The top three largest categories were transcription and protein synthesis (representing 24.7% of the total genes), defense and stress (15.3%), and primary and secondary metabolism (14.0%). Detailed analysis of the DE-genes suggests notable characteristics of SHY phenotype in improved sucrose loading capability, rubber biosynthesis-preferred sugar utilization, enhanced general metabolism and timely stress alleviation. However, the SHY phenotype has little correlation with rubber-biosynthesis pathway genes.

Differential gene expression in response to Papaya ringspot virus infection in Cucumis metuliferus using cDNA-amplified fragment length polymorphism analysis.

A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future.