Amplified Fragment Length Polymorphism DNA Research Articles

Caspofungin resistance in clinical Aspergillus flavus isolates

Introduction and Aims: The present study was conducted to determine the candidate genes involved in caspofungin (CAS) resistance in clinical isolates of Aspergillus flavus (A. flavus).Materials and Methods: The antifungal susceptibility assay of the CAS was performed on 14 clinical isolates of A. flavus using the CLSI-M-38-A2 broth micro-dilution protocol. Since CAS had various potencies, the minimum effective concentration (MEC) of anidulafungin (AND) was also evaluated in the present study. The FKS1 gene sequencing was conducted to assess whether mutations occurred in the whole FKS1 gene as well as hot spot regions of the FKS1 gene of the two resistant isolates. A complementary DNA-amplified fragment length polymorphism (CDNA-AFLP) method was performed to investigate differential gene expression between the two resistant and two sensitive clinical isolates in the presence of CAS. Furthermore, quantitative real-time PCR (QRT-PCR) was utilized to determine the relative expression levels of the identified genes.Results: No mutations were observed in the whole FKS1 gene hot spot regions of the FKS1 genes in the resistant isolates. A subset of two genes with known biological functions and four genes with unknown biological functions were identified in the CAS-resistant isolates using the CDNA-AFLP. The QRT-PCR revealed the down-regulation of the P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 in the CAS-resistant isolates, compared to the susceptible isolates.Conclusion: The findings showed that P-type ATPase and ubiquinone biosynthesis methyltransferase COQ5 might be involved in the CAS-resistance A. flavus clinical isolates. Moreover, a subset of genes was differentially expressed to enhance fungi survival in CAS exposure. Further studies are recommended to highlight the gene overexpression and knock-out experiments in A. flavus or surrogate organisms to confirm that these mentioned genes confer the CAS resistant A. flavus.

CDNA-AFLP analysis of differentially expressed genes during microspore embryogenesis in non-heading Chinese cabbage

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.

Association between seed yield-related traits and cDNA-AFLP markers in cumin (Cuminum cyminum) under drought and irrigation regimes

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 EcoR 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.

Isolation and expression pattern analysis of CmRNF5 and CmNPH3L potentially involved in graft compatibility in cucumber/pumpkin graft combinations

Abstract 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.

The effect of antimicrobial photodynamic therapy on the expression of novel methicillin resistance markers determined using cDNA-AFLP approach in Staphylococcus aureus

BackgroundWidespread 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 methodsWe 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. ResultsAccording 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. ConclusionsPDT 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.

CDNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica

Non-heading Chinese cabbage (Brassica rapa ssp. chinensis) is one of the main green leafy vegetables in the world, especially in China, with significant economic value. Hyaloperonospora parasitica is a fungal pathogen responsible for causing downy mildew disease in Chinese cabbage, which greatly affects its production. The objective of this study was to identify transcriptionally regulated genes during incompatible interactions between non-heading Chinese cabbage and H. parasitica using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP). We obtained 129 reliable differential transcript-derived fragments (TDFs) in a resistant line ‘Suzhou Qing’. Among them, 121 upregulated TDFs displayed an expression peak at 24–48 h post inoculation (h.p.i.). Fifteen genes were further selected for validation of cDNA-AFLP expression patterns using quantitative reverse transcription PCR. Results confirmed the altered expression patterns of 13 genes (86.7%) revealed by the cDNA-AFLP. We identified four TDFs related to fungal resistance among the 15 TDFs. Furthermore, comparative analysis of four TDFs between resistant line ‘Suzhou Qing’ and susceptible line ‘Aijiao Huang’ showed that transcript levels of TDF14 (BcLIK1_A01) peaked at 48 h.p.i. and 25.1-fold increased in the resistant line compared with the susceptible line. Similarly, transcript levels of the other three genes, TDF42 (BcCAT3_A07), TDF75 (BcAAE3_A06) and TDF88 (BcAMT2_A05) peaked at 24, 48 and 24 h.p.i. with 25.1-, 100- and 15.8-fold increases, respectively. The results suggested that the resistance genes tended to transcribe at higher levels in the resistance line than in the susceptible line, which may provide resistance against pathogen infections. The present study might facilitate elucidating the molecular basis of the infection process and identifying candidate genes for resistance improvement of susceptible cultivars.

The interpopulation differences between nucleotide sequence polymorphisms in alleles of the STR-loci of human chromosomal DNA

The objective of the present work was to study the phenomenon of nucleotide sequence polymorphism in alleles of the STR-loci of human chromosomal DNA and to estimate its interpopulation differences with a view to the search for the molecular-genetic markers to be used as an efficient tool for the determination of belonging of the subjects of interest to a given population. We undertook the comprehensive analysis of amplified DNA fragment sequence polymorphism (AFSP) and amplified DNA fragment length polymorphism (AFLP) with the use of the PLEX-ID-TM analytical mass-spectrometry platform (Abbott Molecular, USA). The interpopulation differences were estimated in terms of the presence or the absence of single nucleotide replacements (SNP) in the STR markers based on a few population samples. Some of the loci of interest were found to have allelic variants the occurrence of which was significantly different in individual samples. Such alleles are of importance for the further investigation since they can be regarded as potential ethno-geographical markers. Their application opens up the new promising prospects for the expert detection of the ethnic affiliation of individual subjects.

New taxane biosynthetic gene encoding a β-phenylalanine-CoA ligase identified in jasmonate-elicited Taxus cells

The data-generating power of “omics” technologies is increasingly being harnessed in studies of plant biology to shed light on aspects such as metabolite production patterns, gene sequences and protein expression. Taxol and related taxanes, produced by several Taxus species, are high-value secondary metabolites widely used in cancer therapy due to their antineoplastic activity. To date, several biosynthetic steps of these compounds remain undefined. After carrying out a genome-wide expression analysis of jasmonate-elicited Taxus baccata cell cultures using complementary DNA-amplified fragment length polymorphism (cDNA-AFLP), we found that the increased taxane production in the target cultures was correlated with an extensive elicitor-induced genetic reprogramming. Subsequent in silico analysis allowed us to identify 15 genes with jasmonate-induced differential expression putatively involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene and its predicted 3D structure showed a strong homology with other genes previously reported to encode acyl-CoA ligases, suggesting it was involved in the formation of the taxol lateral chain. In vitro analysis confirmed that the TB768 gene encodes an acyl-CoA ligase, localized in the cytoplasm and able to convert b-phenylalanine into its CoA ester derivative. The latter is then attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. In determining that the TB768 gene encodes a cytosolic CoA ligase with an important role in the taxol biosynthetic pathway, we have corroborated that the combination of a cDNA-AFLP transcriptome profiling approach with bioinformatics analysis constitutes an efficient set of tools for the identification of new genes involved in the metabolism of valuable bioactive compounds. The newly described gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

Applying genotoxicology tools to identify environmental stressors in support of river management

Although bioassay approaches are useful for identifying chemicals of potential concern, they provide little understanding of the mechanisms of chemical toxicity. Without this understanding, it is difficult to address some of the key challenges that currently face aquatic ecotoxicology. To overcome this, the toxicity potential of the water samples was assessed and surviving organisms (Physa acuta) were used for protein activity measurements and gene expression profiling by making use of complementary DNA amplified fragment length polymorphism (cDNA-AFLP) analysis. From the data it was evident that the impacts of specific pollutants (e.g. sewage) on organisms at the cellular level could be identified, and that the expressed stressor genes can be used as bioindicators/markers/genetic signatures or fingerprints during identification of point source pollution. From an ecosystem management point of view these insights could assist with the forecasting and reduction of environmental risks on catchment level by implementing suitable management interventions.

Antibiosis functions during interactions of Trichoderma afroharzianum and Trichoderma gamsii with plant pathogenic Rhizoctonia and Pythium.

Trichoderma afroharzianum is one of the best characterized Trichoderma species, and strains have been utilized as plant disease suppressive inoculants. In contrast, Trichoderma gamsii has only recently been described, and there is limited knowledge of its disease suppressive efficacies. Comparative studies of changes in gene expression during interactions of these species with their target plant pathogens will provide fundamental information on pathogen antibiosis functions. In the present study, we used complementary DNA amplified fragment length polymorphism (cDNA-AFLP) analysis to investigate changes in transcript profiling of T. afroharzianum strain LTR-2 and T. gamsii strain Tk7a during in vitro interactions with plant pathogenic Rhizoctonia solani and Pythium irregulare. Considerable differences were resolved in the overall expression profiles of strains LTR-2 and Tk7a when challenged with either plant pathogen. In strain LTR-2, previously reported mycoparasitism-related genes such as chitinase, polyketide synthase, and non-ribosomal peptide synthetase were found to be differentially expressed. This was not so for strain Tk7a, with the only previously reported antibiosis-associated genes being small secreted cysteine-rich proteins. Although only one differentially expressed gene was common to both strains LTR-2 and Tk7a, numerous genes reportedly associated with pathogen antibiosis processes were differentially expressed in both strains, including degradative enzymes and membrane transport proteins. A number of novel potential antibiosis-related transcripts were found from strains LTR-2 and Tk7a and remain to be identified. The expression kinetics of 20 Trichoderma (10 from strain LTR-2, 10 from strain Tk7a) transcript-derived fragments (TDFs) were quantified by quantitative reverse transcription PCR (RT-qPCR) at pre- and post-mycelia contact stages of Trichoderma-prey interactions, thereby confirming differential gene expression. Collectively, this research is providing information to elucidate the antibiosis mechanisms and disease suppressive activities of T. afroharzianum and T. gamsii against soilborne fungal and oomycete plant pathogens.

Epigenetic Patterns in Genetically Imbalanced Polyploid Dog Rose Hybrids (Rosa L. sect. Caninae (DC.) Ser.) Revealed by cDNA–Amplified Fragment Length Polymorphisms and Methylation-Sensitive Amplified Polymorphisms

Premise of research. Spontaneous hybrids between species of Rosa sect. Caninae represent a unique example to study the genetic and epigenetic consequences of an imbalanced parental contribution to polyploid hybrids. Due to the unique canina meiosis, pentaploid or hexaploid dog rose hybrids contain a single subgenome transmitted by the haploid pollen grain of the paternal species and four to five subgenomes transmitted by the tetraploid or pentaploid egg cell of the maternal species. During this study, we investigated epigenetic changes in spontaneous reciprocal hybrids between the subsections Caninae and Rubigineae compared to their parental species in a natural population in eastern Germany.Methodology. For investigating epigenetic patterns, we applied cDNA–amplified fragment length polymorphisms (AFLPs) and methylation-sensitive amplified polymorphisms (MSAPs). Additionally, we analyzed microsatellite data and measured morphological characters.Pivotal results. In line with expectations deduced from the genetic composition of the pentaploid and hexaploid hybrids, we observed a maternal bias of DNA methylation and expression patterns. We did not detect silencing of the respective paternal subgenomes. However, the percentage of paternally expressed fragments differed between plant organs. We found a higher number of paternally expressed fragments in sepals compared to leaves, suggesting that the parental genetic information underwent a rapid subfunctionalization in recently formed hybrids. Moreover, epigenetic data obtained from cDNA-AFLPs and MSAPs correlated stronger with morphology than with genetic data, based on microsatellites.Conclusions. The genetic contribution of subgenomes is strongly biased toward the maternal parent in dog rose hybrids, leading to a nearly apomictic mode of inheritance. However, we found that the underrepresented paternal genome is not silenced but transcribed and differentially expressed between plant organs. The correlation between epigenetic data and morphological data implies that the probably fast-evolving epigenetic changes have a major impact on dog rose phenotypes and may affect the establishment of hybrids in natural populations.

Transcript profiling of jasmonate-elicited Taxus cells reveals a β-phenylalanine-CoA ligase.

Plant cell cultures constitute eco-friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate-elicited Taxus baccata cell cultures by complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) indicated a correlation between an extensive elicitor-induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate-induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl-CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl-CoA ligase that localizes to the cytoplasm and is able to convert β-phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β-phenylalanyl-CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.

Identification of Azole Resistance Markers in Clinical Isolates of Candida tropicalis Using cDNA‐AFLP Method

Global reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism(') s drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs. Two susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates. Five genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)-belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes. Our findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development.

Two distinct classes of protein related to GTB and RRM are critical in the sclerotial metamorphosis process of Rhizoctonia solani AG-1 IA.

Sheath blight of rice, caused by Rhizoctonia solani Kühn AG-1 IA [teleomorph: Thanatephorus cucumeris (Frank) Donk], is one of the major diseases of rice (Oryza sativa L.) worldwide. Sclerotia produced by R. solani AG-1 IA are crucial for their survival in adverse environments and further dissemination when environmental conditions become conducive. Differentially expressed genes during three stages of sclerotial metamorphosis of R. solani AG-1 IA were investigated by utilizing complementary DNA amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 258 transcript derived fragments (TDFs) were obtained and sequenced, among which 253 TDFs were annotated with known functions through BLASTX by searching the GenBank database and 19 annotated TDFs were assigned into 19 secondary metabolic pathways through searching the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database. Moreover, the results of quantitative real-time PCR (qRT-PCR) analysis showed that the expression patterns of eight representative annotated TDFs were positively correlated with sclerotial metamorphosis. Sequence annotation of TDFs showed homology similarities to several genes encoding for proteins belonging to the glycosyltransferases B (GTB) and RNA recognition motif (RRM) superfamily and to other development-related proteins. Taken together, it is concluded that the members of the GTB and RRM superfamilies and several new genes involved in proteolytic process identified in this study might serve as the scavengers of free radicals and reactive oxygen species (ROS) and thus play an important role in the sclerotial metamorphosis process of R. solani AG-1 IA.

Characterization of maize B-chromosome-related transcripts isolated via cDNA-AFLP.

The maize B-chromosome consists mainly of heterochromatin and is considered to be genetically inert. However, the B-chromosome contains euchromatin that carries control elements that direct its behaviors during cell division, such as nondisjunction during the second pollen mitosis. To determine the transcriptional activity of the B-chromosome, complementary DNA-amplified fragment length polymorphism analysis was applied to five inbred maize lines with and without B-chromosomes. Six putative B-chromosome-related transcripts were identified, four of which were cloned and characterized via Southern hybridization, fluorescence in situ hybridization, and sequence comparison to further confirm their B-chromosome origin. All the analyzed B-chromosome-related transcript sequences were repetitive and showed homology to A-chromosomes. Quantitative real-time reverse transcriptase-polymerase chain reaction revealed that the B-chromosome-specific transcribed sequences B3547-179 and B3849-212 were transcribed in a B-chromosome-dosage-dependent manner. Expression of B3849-189 and B3849-147 was not specific to the B-chromosome; however, the former showed a transcriptional pattern with B-chromosome dosage compensation, and the latter displayed down-regulation of transcription due to higher B-chromosome numbers. Using four B-10L translocations, B3849-212 was mapped to the B-chromosome region that contains the nondisjunction control elements of the B-chromosome. Taken together, our results suggested that the maize B-chromosome harbors few transcriptionally active sequences and might influence the transcription of A-chromosomes.

Identification of Genes Involved in Wild Crucifer Rorippa indica Resistance Response on Mustard Aphid Lipaphis erysimi Challenge

Mustard aphid, Lipaphis erysimi (L.) Kaltenbach is a perpetual annual threat in the cultivation of rapeseed- mustard (Brassica spp.) crop in tropical and sub-tropical climate. Cultivated Brassica germplasm has failed so far to provide any source of resistance. Wild germplasm is a potential source of resistance against many threatening herbivores. On wild germplasm screening, we noted that the wild crucifer Rorippa indica (L.) Hiern confers resistance against L. erysimi. In the present study L. erysimi challenged transcriptome of R. indica was compared to un-infested R. indica sample to get a molecular insight about the aphid resistance mechanism and identify the candidate defense response genes. Cloning, sequencing and in silico sequence analysis of complimentary DNA amplified fragment length polymorphism identified 116 differentially expressed transcript derived fragments revealed thirty candidates which are from different functional categories including redox regulation, signalling, photosynthesis, structure, metabolism, defense response as well as a few of unknown function. Twenty four identifications were then studied by quantitative real time RT PCR analysis at 6, 12, 24 and 48 hour time point post infestation to understand the early-to-late defense response through their relative gene expression profiles. Seventeen fragments showed significant up or down regulation at p<0.05 level. The response was influenced by different phytohormonal signalling pathways simultaneously. The candidate defense response expressed sequence tags specifically for the resistance genes identified in this study have implication in building desired mustard aphid resistance in susceptible rapeseed-mustard plants in future. This is the first molecular report on crucifer defense response against mustard aphid L. erysimi.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Characterization and mapping of QTL used in breeding of Scots pine (Pinus sylvestris L.)

This paper reports the construction a map b ased on Amplified Fragment Length Polymorphic DNA (AFLP) in Scots pine (Pinus sylvestris L.). The main purpose of map construction was its application to quantitative traits loci (QTL) mapping for breeding traits economically important in Scots pine breeding program such as tree height and diameter at breast height, number of needles and their length, width, and area. Genomic DNA of needles and haploid megagamethophytes from seeds originating from a sing le tree were amplified with 25 AFLP primer-enzyme combinations with three or four selective nucleotides. Sixteen of them generated easily readable patterns and revealed a poly morphism. Each analyzed marker was tested for the expected 1 : 1 seg regation ratio using χ 2 – test and only 6 were significant with (α ≤ 0.05). The total map size equaled 291,7 cM an d all markers were distributed within one linkage group. For all traits only one QTL associated with tree height (H) was detected.

Differential gene expression in East African highland bananas (Musa spp.): Interactions between non-pathogenic Fusarium oxysporum V5w2 and Radopholus similis

Endophytic non-pathogenic Fusarium oxysporum isolates are known to control Radopholus similis through induced resistance. Using complementary (c)DNA-amplified fragment length polymorphism (cDNA-AFLP), we identified genes induced in nematode-susceptible and -tolerant bananas following colonization by non-pathogenic endophytic F. oxysporum isolate V5w2. Quantitative real-time polymerase chain reactions (qRT-PCR) were then used to analyze expression profiles of selected genes. Accumulated gene transcripts included those involved in signal transduction, cell wall strengthening, Jasmonic acid (JA) pathway and transport of defense molecules. Endophyte colonization primed the expression of β-1,3-glucan synthase and Coronatine insensitive 1 (COI1) in the susceptible cultivar, and that of COI1 and Lipoxygenase (LOX) in the tolerant. We isolate and identify for the first time genes involved in the interaction between endophytic non-pathogenic F. oxysporum and banana.

Gene expression profiling of defense-related genes resistant to Septoria tritici blotch in wheat

To recognize defense-related genes in wheat plants resistant to Septoria tritici leaf blotch (STB) caused by Mycosphaerella graminicola, we utilized complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) analysis. An analysis of transcript-derived fragments’ (TDFs) patterns of the studied resistant wheat cultivar, Frontana, was compared with that of the susceptible cultivar, Seri 82, and related controls. Sampling was done at nine timepoints from 0 h to 24 days post inoculation. Transcripts from Frontana cultivar that had enhanced expression post inoculation (170) were isolated, recovered, cloned, sequenced and analyzed via BLAST. A total of 47 TDFs had significant protein homology. The expression profile of nine genes was obtained by real-time PCR. These genes contained PCI domain, protein tyrosine kinase, zinc-finger-homeodomain (ZF- HD) protein dimerization region, leucine-rich repeat receptor-like protein kinase, WD40 domain, diacylglycerol kinase catalytic domain and geranylgeranyl transferase type II, ATPases associated with a wide variety of cellular activities and FKBP-type peptidyl prolyl cis-trans isomerase. The function of these genes relative to defense response against the pathogen was described. Key words: Triticum aestivum, Mycosphaerella graminicola, cDNA-AFLP, resistant wheat, transcript-derived fragments, Septoria tritici.