• BMB Reports
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• 제40권6호
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• pp.986-1001
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• 2007

Cold acclimation improves freezing tolerance in plants. In higher plants, many advances have been made toward identifying the signaling and regulatory pathways that direct the low-temperature stress response; however, similar insights have not yet been gained for simple nonvascular plants, such as bryophytes. To elucidate the pathways that regulate cold acclimation in bryophytes, we used two PCR-based differential screening techniques, cDNA amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridization (SSH), to isolate 510 ESTs that are differentially expressed during cold acclimation in Physcomitrella patens. We used realtime RT-PCR to further analyze expression of 29 of these transcripts during cold acclimation. Our results show that cold acclimation in the bryophyte Physcomitrella patens is not only largely similar to higher plants but also displays distinct differences, suggests significant alteration during the evolution of land plants.

• Journal of Photoscience
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• 제7권2호
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• pp.73-78
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• 2000

To analyze molecular traits determining pigmentation between Pharbitis nill violet and white, Amplified Fragment Length Polymorphism(AFLP) and Differential Display Reverse Transcription(DDRT) experiments were carried out with either genomic DNAs or total RNAs isolated from both plants. Results of AFLP experiment in combination of 8 EcoRⅠ primers with 6 MseⅠ primers showed 41 violet-and 60 white-specific DNA bands. In the subsequent experiment, 22 violet-and 22 white-specific DNA fragments were amplified by PCR with DNAs eluted. The sizes of the fragments range from 200 to 600bp. DDRT using total RNA produced 19 violet-and 17 white-specific cDNA fragments, ranging from 200 to 600bp. The fragments obtained by both AFLP and DDRT had been cloned into pGEM T-easy vector, amplified and subjected to the nucleotide sequence analyses. As a result of Blast sequence analysis, most of them sequenced up to date showed no similarity to any Known gene, while few has similarity to known animal or plant genes. An AFLP clone V6, for example, has a strong sequence similarity to the human transcription factor LZIP-alpha mRNA and a DDRT clone W19 to Solanum tuberosum 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA.

• The Plant Pathology Journal
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• 제25권2호
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• pp.184-188
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• 2009

Identification of differently expressed genes under specific tissues and/or environments provides insights into the nature and underlying mechanisms of cellular processes. Although cDNA-AFLP (Amplified Fragment Length Polymorphism) is a powerful method for analyzing differentially expressed genes, its use has been limited to the requirement of radioactive isotope use and the difficulty of isolating the bands of interest from a gel. Here, we describe a modified method for cDNA-AFLP that uses a fluorescence dye for detection and isolation of bands directly from a small size polyacrylamide gel. This method involves three steps: (i) preparation of cDNA templates, (ii) PCR amplification and differential display, and (iii) identification of differentially expressed genes. To demonstrate its utility and efficiency, differentially expressed genes during vegetative growth and appressorial development of Magnaporthe oryzae were analyzed. This method could be applied to compare gene expression profiles in a diverse array of organisms.

• Journal of Microbiology
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• 제40권4호
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• pp.254-259
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• 2002

The Phytophthora infestans requires two mating types for sexual reproduction. Amplified fragment length polymorphism (AFLP) was used to specifically detect different mating types of P. infestans. The AFLP primers E+AA (5'-GACTGCGTACCAATTCAA-3') and M+CAA (5'-GATGAGTCCTGAG-TAAC AA-3') detected a fragment that is specific in the A2 mating type of P. infestans. This fragment was cloned and sequenced. Based on the sequence data, PHYB-1 and PHYB-2 primer were designed to detect the A2 mating type of P. infestans. A single 347 bp segment was observed in the A2 mating type of P. infestans, but not in the A1 mating type of P. infestans or other Phytophthora spp. Identification of mating type was performed with phenotype (sexual reproduction) and genotype (CAPs marker) methods. Two factors, the annealing temperature and template DNA quantity, were investigated to determine the optimal conditions. Using mating type-specific primers, a unique band was obtained within annealing temperatures of 57$^{\circ}C$-62$^{\circ}C$ and DNA levels of 10pg-100 ng (data not shown).

• 한국약용작물학회지
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• 제21권6호
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• pp.455-460
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• 2013

Collected germplasms of five representative species belonging to Curcuma genus (C. longa, C. aromatica, C. zedoaria, C. phaeocaulis and C. kwangsiensis) were 52 samples from different farmhouse in Korea and China. To elucidate the genetic diversity among the species, 52 samples were analyzed by genomic fingerprinting method using amplified fragment length polymorphism (AFLP). AFLP results of 6 primer combinations were revealed 643 total DNA fragments and 349 polymorphic bands with the 54.3% ratio of polymorphism. In the analysis of coefficient similarity using unweight pair group method with arithmetic averages (UPGMA), 52 Curcuma germplasm lines were ranged from 0.60 to 0.99 and clustered distinct five groups according to the species and collected geographical levels. However, the result of principal coordinate analysis (PCA) by multi-variate analysis was shown significantly greater differences among species than geographical origins based on AFLP profiling data of these samples.

• Molecules and Cells
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• 제25권2호
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• pp.205-210
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• 2008

To develop molecular markers linked to the $L^4$ locus conferring resistance to tobamovirus pathotypes in pepper plants, we performed AFLP with 512 primer combinations for susceptible (S pool) and resistant (R pool) DNA bulks against pathotype 1.2 of pepper mild mottle virus. Each bulk was made by pooling the DNA of five homozygous individuals from a T10 population, which was a near-isogenic $BC_4F_2$ generation for the $L^4$ locus. A total of 19 primer pairs produced scorable bands in the R pool. Further screening with these primer pairs was done on DNA bulks from T102, a $BC_{10}F_2$ derived from T10 by back crossing. Three AFLP markers were finally selected and designated L4-a, L4-b and L4-c. L4-a and L4-c each underwent one recombination event, whereas no recombination for L4-b was seen in 20 individuals of each DNA bulk. Linkage analysis of these markers in 112 $F_2$ T102 individuals showed that they were each within 2.5 cM of the $L^4$ locus. L4-b was successfully converted into a simple 340-bp SCAR marker, designated L4SC340, which mapped 1.8 cM from the $L^4$ locus in T102 and 0.9 cM in another $BC_{10}F_2$ population, T101. We believe that this newly characterized marker will improve selection of tobamovirus resistance in pepper plants by reducing breeding cost and time.

• The Plant Pathology Journal
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• 제30권4호
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• pp.425-431
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• 2014

Leaf scald caused by the infection of Rhynchosporium secalis, is a worldwide crop disease resulting in significant loss of barley yield. In this study, a systematic sequencing of expressed sequence tags (ESTs) was chosen to obtain a global picture of the assembly of genes involved in pathogenesis. To identify a large number of plant ESTs, which are induced at different time points, an amplified fragment length polymorphism (AFLP) display of complementary DNA (cDNA) was utilized. Transcriptional changes of 140 ESTs were observed, of which 19 have no previously described function. Functional annotation of the transcripts revealed a variety of infection-induced host genes encoding classical pathogenesis-related (PR) or genes that play a role in the signal transduction pathway. The expression analyses by a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that Rar1 and Rpg4 are defense inducible genes, and were consistent with the cDNA-AFLP data in their expression patterns. Hence, the here presented transcriptomic approach provides novel global catalogue of genes not currently represented in the EST databases.

• Molecules and Cells
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• 제25권2호
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• pp.196-204
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• 2008

Despite increasing awareness of the importance of WRKY genes in plant defense signaling, the locations of these genes in the Capsicum genome have not been established. To develop WRKY-based markers, primer sequences were deduced from the conserved sequences of the DNA binding motif within the WRKY domains of tomato and pepper genes. These primers were derived from upstream and downstream parts of the conserved sequences of the three WRKY groups. Six primer combinations of each WRKY group were tested for polymorphisms between the mapping parents, C. annuum 'CM334' and C. annuum 'Chilsung-cho'. DNA fragments amplified by primer pairs deduced from WRKY Group II genes revealed high levels of polymorphism. Using 32 primer pairs to amplify upstream and downstream parts of the WRKY domain of WRKY group II genes, 60 polymorphic bands were detected. Polymorphisms were not detected with primer pairs from downstream parts of WRKY group II genes. Half of these primers were subjected to $F_2$ genotyping to construct a linkage map. Thirty of 41 markers were located evenly spaced on 20 of the 28 linkage groups, without clustering. This linkage map also consisted of 199 AFLP and 26 SSR markers. This WRKY-based marker system is a rapid and simple method for generating sequence-specific markers for plant gene families.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.502-509
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• 2005

Sexual reproduction plays an important role in the biology and epidemiology of oomycete plant pathogens such as the heterothallic species Phytophthora infestans. Recent worldwide dispersal of A2 mating type strains of P. infestans resulted in increased virulence, gene transfer, and genetic variation, creating new challenges for disease management. To develop a genetic assay for mating type identification in P. infestans, we used the Amplified Fragment Length Polymorphism (AFLP) technique. The primer combination E+AT/M+CTA detected a fragment specific to A1 mating type (Mat-A1) of P. infestans. This fragment was cloned and sequenced, and a pair of primers (INF-1, INF-2) were designed and used to differentiate P. infestans Mat-A1 from Mat-A2 strains. The Mat A1-specific fragment was detected using Southern blot analysis of PCR products amplified with primers INF-1 and INF-2 from genomic DNA of 14 P. infestans Mat-A1 strains, but not 13 P. infestans Mat-A2 strains or 8 other isolates representing several Phytophthora spp. Southern blot analysis of genomic DNAs of P. infestans isolates revealed a 1.6 kb restriction enzyme (EcoRI, BamHI, AvaI)-fragment only in Mat-A1 strains. The A1 mating type-specific primers amplified a unique band under stringent annealing temperatures of $63^{\circ}C-64^{\circ}C$, suggesting that this PCR assay could be developed into a useful method for mating type determination of P. infestans in field material.

• Parasites, Hosts and Diseases
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• 제51권4호
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• pp.413-419
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• 2013

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$^{(R)}$) 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 resistant isolates compared to sensitive ones. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.