cDNA-amplified Fragment Length Polymorphism Research Articles

Identification of differentially expressed genes related to coloration in red/green mutant pear (Pyrus communis L.)

Fruit skin color is an important parameter of outer quality and plays an important role in attracting customers. In many plants, it is the result of coordinative regulation of anthocyanin pathway genes. In our study, the differential expression of cDNA library in a pair of pear mutant with red and green color was investigated to find candidate genes which might regulate the anthocyanin biosynthesis and control the coloration of pear. We constructed a cDNA library using the cDNA-amplified fragment length polymorphism approach to analyze the transcriptional differences between the original cultivar “Early red Doyenne du Comice” with high anthocyanin content in the peel and its green color mutant with comparatively low anthocyanin content. Altogether, 47 transcript-derived fragments, putatively involved in anthocyanin biosynthesis, primary metabolism, stress, and defense responses, were identified. The relationships of differentially expressed genes and coloration were investigated by quantitative real-time PCR with fruit skin samples at different developmental stages. A gene putatively involved in anthocyanin biosynthesis was found and named as PyMADS18. Its sequence is similar to genes reported in the literature as regulators of anthocyanin biosynthesis. The expression results indicate that PyMADS18 is likely to be involved in anthocyanin accumulation and regulation of anthocyanin synthesis in early fruit development of pear.

Genome-wide identification of differentially expressed genes under water deficit stress in upland cotton (Gossypium hirsutum L.).

BackgroundCotton is the world’s primary fiber crop and is a major agricultural commodity in over 30 countries. Like many other global commodities, sustainable cotton production is challenged by restricted natural resources. In response to the anticipated increase of agricultural water demand, a major research direction involves developing crops that use less water or that use water more efficiently. In this study, our objective was to identify differentially expressed genes in response to water deficit stress in cotton. A global expression analysis using cDNA-Amplified Fragment Length Polymorphism was conducted to compare root and leaf gene expression profiles from a putative drought resistant cotton cultivar grown under water deficit stressed and well watered field conditions.ResultsWe identified a total of 519 differentially expressed transcript derived fragments. Of these, 147 transcript derived fragment sequences were functionally annotated according to their gene ontology. Nearly 70 percent of transcript derived fragments belonged to four major categories: 1) unclassified, 2) stress/defense, 3) metabolism, and 4) gene regulation. We found heat shock protein-related and reactive oxygen species-related transcript derived fragments to be among the major parts of functional pathways induced by water deficit stress. Also, twelve novel transcripts were identified as both water deficit responsive and cotton specific. A subset of differentially expressed transcript derived fragments was verified using reverse transcription-polymerase chain reaction. Differential expression analysis also identified five pairs of duplicated transcript derived fragments in which four pairs responded differentially between each of their two homologues under water deficit stress.ConclusionsIn this study, we detected differentially expressed transcript derived fragments from water deficit stressed root and leaf tissues in tetraploid cotton and provided their gene ontology, functional/biological distribution, and possible roles of gene duplication. This discovery demonstrates complex mechanisms involved with polyploid cotton’s transcriptome response to naturally occurring field water deficit stress. The genes identified in this study will provide candidate targets to manipulate the water use characteristics of cotton at the molecular level.

Sequence-independent VIDISCA-454 technique to discover new viruses in canine livers

In many mammals, viruses cause hepatitis. Despite many efforts a specific virus responsible for canine idiopathic hepatitis has not been identified. The discovery of a viral etiology in canine hepatitis will promote the development of specific drugs and vaccines for the treatment of idiopathic hepatitis in dogs. The objective of this study was the application of the sequence-independent Virus Discovery cDNA-amplified fragment length polymorphism (VIDISCA) technique combined with high through-put sequencing on a Roche-454 sequencer to identify unknown viruses. Liver tissue of a dog with idiopathic acute hepatitis was cultured on a canine liver cell line and the cell culture medium was submitted to the VIDISCA-454 technique. Without prior knowledge of the viral species involved, this technique identified Canine adenovirus type 1 (CAV-1) as the infecting agent. This demonstrates the power of VIDISCA-454 to identify viruses, independent of preliminary information about the genomic sequence. Consequently, the strategy of propagation in this cell line followed by the VIDISCA-454 technique is valuable to identify the viral etiology of idiopathic hepatitis in dogs.

CDNA-AFLP analysis of gene expression changes in apple trees induced by phytoplasma infection during compatible interaction

In order to gain insight into molecular and physiological changes in apple trees during compatible interaction with two ‘Candidatus Phytoplasma mali’ strains (AP and AT), cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was used. A rootstock of apple (MM106) susceptible to ‘Ca. P. mali’ was used to extend the range of the potential host responses by the maximum number of identified genes that will be deregulated by phytoplasma in apple. Gene expression comparisons were studied in three directions: healthy versus infected samples, symptomatic versus non-symptomatic sample, and AP-infected versus AT-infected sample. Forty-five genes whose steady-state levels of expression significantly changed in response to phytoplasma infection were identified. Among their partial cDNA sequences, only 27 showed similarity to DNA or protein data bases; of these, 18 were related to known genes in plants, and the rest were related to unknown or hypothetical proteins. Eighteen out of 45 did not show any similarity with sequences in data bases (potential novel genes). Quantitative real-time RT-PCR (qRT-PCR) was used to confirm differential expression of AFLP identified genes, and showed the similar profile expression for 11 known genes among 18, and for 13 unknown, hypothetical or novel genes among 27. Changes in gene expression involved a wide spectrum of biological functions, including processes of metabolism, cell defence, senescence, photosynthesis, transport, transcription, signal transduction and protein synthesis. This is the first study of global gene profiling in plants in response to phytoplasma infections using cDNA-AFLP, and a model is proposed to explain the mode of action of the ‘Ca. P. mali’ in apple.

Early gene expression in the walnut tree occurring during stimulation of leaf hydraulic conductance by irradiance

Leaf hydraulic conductance (Kleaf) plays a significant part in plant-water regulation. In walnut leaves, Kleaf is stimulated by irradiance and closely relates to the accumulation of JrPIP2s aquaporin transcripts, but it is independent of stomatal aperture. To provide an insight into the early molecular events occurred during light-induced Kleaf, a large-scale transcriptomic analysis consisting of the cDNA-amplified fragment length polymorphism (AFLP) was carried out on walnut leaves maintained under irradiance or in darkness. Of the total 12 000 transcript-derived fragments (TDFs) obtained using cDNA-AFLP with 128 primer pairs, 187 TDFs were selected after sequencing, and only 93 (49 %) that had been ascribed known functions through BLAST searching of the GenBank databases. Most of these TDFs correspond to genes whose protein products are involved in cellular regulation (57.9 %) and global metabolism (39.8 %). To validate cDNA-AFLP expression patterns, 30 TDFs were further analyzed using real-time quantitative polymerase chain reaction. Moreover, exposure of leaves to irradiance was accompanied by the modification of the Ca2+-signaling pathway, ubiquitin-proteasome pathway, vesicle trafficking process and expression of multiple transcription factors.

Screening of differentially expressed genes during the end of endogenous dormancy of flower buds in Prunus armeniaca L.

An insufficient endogenous dormancy period of apricot flower buds can result in reduced frost resistance. This is one of the causes of lower fruit yields in certain years. The aim of this work was the analysis of gene expression profiles in flower buds of four apricot cultivars (‘Sundrop’, ‘Stark Early Orange’, ‘Vestar’ and ‘Betinka’) that differ in the duration of their dormancy periods. The investigation was carried out before and during exit of buds from endogenous dormancy. Transcriptomes from bud tissues were analyzed by cDNA amplified fragment length polymorphism. Amplified fragments showing a significant change in their intensity within obtained expression profiles were considered to be derived from genes associated with the exit of flower buds from endogenous dormancy. All selected fragments were sequenced and compared with sequences recorded in public databases, yielding 147 sequences with similarity to previously described genes. The majority of identified genes match with theoretical expectations for events happening in dormant tissues. Moreover, some of the identified genes are included in the category of cellular or physiological processes previously reported to directly influence the exit from endogenous dormancy (e.g. aquaporin, GTP-binding proteins, elongation factor 1-alpha, ATP-dependent ubiquitin, xyloglucan endotransglycosylase hydrolase, and EXGT-A1).

Identification of genes involved in a water stress response in timothy and mapping of orthologous loci in perennial ryegrass

In order to characterize the response of selected grasses to water stress, relative water content (RWC) in leaves and quantum efficiency of photosystem 2 (Fv/Fm) were measured in Phleum pratense L., P. bertolonii DC. and P. phleoides H. Karst. during 6 d of water stress. The results indicated differential responses to water stress among the three Phleum species with higher water deficit sensitivity of P. pratense and P. bertolonii than that of P. phleoides. The cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was applied to identify differentially expressed genes responding to water stress in P. pratense. Cloned and sequenced differentially expressed fragments (DEFs) were used for primer design in order to identify orthologous genes in Lolium perenne L. Twelve genes orthologous to P. pratense DEFs were mapped in the L. perenne mapping population VrnA based on a high resolution melting curve analysis (HRM). This study provides genomic information about 29 differentially expressed genes after water stress in P. pratense and reports on the identification and mapping of twelve orthologs in L. perenne.

CDNA- AFLP analysis of the response of tetraploid black locust (Robinia pseudoacacia L.) to salt stress

Soil salinity is a major abiotic stress that limits plant growth. In this study, to understand the tolerance mechanism associated with salinity stress in tetraploid black locust ( Robinia pseudoacacia L.), 2-yearold plants were treated by salinity. cDNA amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to study the gene expression of tetraploid black locust in response to 5, 10 and 15 days after salinity stress (300 mM NaCl) treatment. One hundred and ten (110) transcript derived fragments (TDFs) with up-regulation expression in leaves of tetraploid black locust in response to salinity stress were characterized and classified into seven groups. The putative functions of these TDFs were related to energy metabolism, material metabolism, signal transduction, transcription factor, stress and defense-related proteins and transport facilitation. The expression patterns of six genes having direct or indirect relation with salt stress response were analyzed through qRT-PCR. The possible roles of these genes are discussed. These data may enhance the understanding of the salinity tolerance mechanisms in polyploid plants. Keywords : cDNA-AFLP, salt stress, gene expression, qRT-PCR; tetraploid black locust.

Sequence characterization and expression pattern of BcMF21, a novel gene related to pollen development in Brassica campestris ssp. chinensis

In this report a full length cDNA, Brassica campestris Male Fertile 21 (BcMF21) was successfully isolated from one of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript-derived fragments (TDFs), BBP10, that was found down-regulated in the flower buds of sterile plants in Brassica campestris L. ssp. chinensis Makino genic male sterile (GMS) AB line system (Bcajh 97-01A/B). BcMF21 protein structure analysis showed a signal peptide at the N-terminus; two protein kinase C phosphorylation sites, five N-myristoylation sites and one casein kinase II phosphorylation site. The promoter region of BcMF21, a 779 bp upstream of ATG was isolated by thermal asymmetric interlaced-PCR (TAIL-PCR). Bioinformatics analysis revealed that the promoter of BcMF21 contained several classical cis-acting elements and three pollen specific elements. Transient expression analysis showed that the promoter could drive green fluorescence protein (GFP) expression. Quantitative reverse transcript-PCR analysis revealed that BcMF21 was specifically expressed in flower buds. The transcript level of BcMF21 was much lower in the sterile flower buds than in the fertile flower buds in 'Bcajh 97-01A/B' system. In situ hybridization further showed that BcMF21 was only expressed in the tetrads and the microspores at the tetrad stage and the uninucleate stage. In addition, phylogenetic analysis demonstrated that the BcMF21 was relative conserved within family Crucifereae and might be originated from the ancestor diploid B. campestris within genus Brassica according to the Triangle of U theory.

Diversity and Genetic Differentiation of the Whitefly Bemisia tabaci Species Complex in China Based on mtCOI and cDNA-AFLP Analysis

The whitefly Bemisia tabaci are considered as a taxonomically complex that contained some destructive pests. Two of the most prevalent cryptic species are B. tabaci Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED). In an extensive field survey of the B. tabaci complex present throughout part of China from 2004 to 2007, we obtained 93 samples of B. tabaci from 22 provinces. We determined that these Chinese haplotypes included 2 invasive species (MEAM1 and MED), and 4 indigenous cryptic species (Asia II 1, Asia II 3, China 3 and Asia II 7) by sequencing mitochondrial cytochrome oxidose one gene (mtCOI). The diversity and genetic differentiation of a subset of 19 populations of B. tabaci were studied using cDNA amplified fragment length polymorphism (AFLP). Prior to 2007, MEAM1 was a dominant species in many provinces in China. By 2007, MED was dominant in 11 provinces. Both invasive and indigenous species were simultaneously found in some regions. Indigenous species of B. tabaci were found in six provinces in southern China. MED and MEAM1 have broad ranges of host plants, and indigenous species appeared to have much narrower host ranges. All Asia II 3 samples were found on cotton except one on aubergine. China 3 has more host plants than Asia II 3. Twelve samples of China 3 were collected from sweet potato, Japanese hop, squash and cotton. A total of 677 reproducible bands amplified with 5 AFLP primer combinations were obtained. The highest proportion of polymorphic bands was 98.7% and the lowest was 91.9%. Unweighted pair-group method analysis indicated that the clustering was independent of the different species. MED showed the lowest degree of similarity than the other species. The data indicate that both MEAM1and MED were rapidly established in China.

CDNA-AFLP analysis reveals a set of new genes differentially expressed in cucumber root apexes in response to iron deficiency

Cucumber (Cucumis sativus L.) is considered a model plant for the investigation of Fe deficiency responses, since it strongly exhibits typical strategy I, i.e. increased activities of Fe(III)-chelate reductase, H+-ATPase and iron regulated transporters. In this study, cDNA amplified fragment length polymorphism analysis was employed to identify genes differentially expressed in the root apex following Fe deficiency. The expression patterns of the most interesting transcript derived fragments were validated by semiquantitative reverse trascriptase - polymerase chain reaction. A set of new genes overexpressed under Fe deficiency, such as those coding for calmodulin, SNAP, TIM23 and V-PPase were identified. Furthermore, we also observed that calmodulin protein accumulated in Fe-deficient root apexes.

Differential gene expression in response to cold stress in Lepidium apetalum during seedling emergence

Germination of Lepidium apetalum Wild. seeds is invariably arrested by cold stress. cDNA-amplified fragment length polymorphism (AFLP) technique was used to isolate genes relevant to chilling stress (4 °C) during seedling emergence. 43 transcript-derived fragments (TDFs) were found to be up-regulated and 17 down-regulated during chilling stress. Eighteen TDF of up-regulated genes were cloned and sequenced. Some of these genes are involved in the stress response, some play important roles in energy and substrate metabolism, and some encode unknown proteins such as TDF119. Two sequences, designated TDF217 and TDF223, may correspond to novel genes. The expression profiles of 6 from 18 TDFs were analyzed by quantitative real-time PCR under chilling and abscisic acid (ABA) stress. It was demonstrated that all 6 genes were significantly induced by chilling and their expression was decreased when the temperature was shifted from 4 to 25 °C. The transcriptional levels of 5 TDFs were strongly enhanced also in response to exogenous ABA. Based on the characteristics of genes isolated from seedlings exposed to cold stress, we conclude that Lepidium adapts to cold stress by regulating many signal transduction pathways, including both ABA-dependent and ABA-independent signaling pathways.

CDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C.×morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3h, 12h, 24h and 48h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.

Molecular Analysis of Differentially Expressed Genes in Birch (Betula Platyphylla) Inflorescence

ABSTRACTBirch (Betula platyphylla) has been widely applied in furniture, paper-making, and architecture for its special properties. One of the major problems is it has a long juvenile phase which limits the possibilities for production and application in these industries. To date, the molecular mechanism and biochemical pathways of the long juvenile phase are poorly understood. In this study, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique was used to analyze genes specifically expressed in the male or female inflorescence of birch. More than 7,000 TDFs (Transcript Derived Fragments) were screened by using 100 primer combinations. Of them, 168 male and 103 female inflorescence specific TDFs were isolated, subcloned and sequenced. About 54% of these TDFs were found to be similar with known genes in NCBI databases. Through GO annotation and KEGG pathway analysis, the results indicated that the male inflorescence of B. platyphylla possesses nearly two-fold more transcripts than the female inflorescence in energy production and conversion.

Differential gene expression during somatic embryogenesis in the maize (Zea mays L.) inbred line H99

Somatic embryogenesis is a complex developmental process that offers great potential for plant propagation. Although many studies have shown that the generation of embryonic cells from somatic cells is accompanied by the synthesis of RNA and DNA and by elevated enzymatic activity, the mechanism of the onset of somatic embryogenesis is not well understood. cDNA-amplified fragment length polymorphism analysis was used to evaluate the gene expression pattern in embryogenic and non-embryogenic of the inbred maize line H99 during the process of embryogenesis. We identified a total of 101 candidate genes associated with the formation of maize embryonic calli. Based on the sequence analysis, these genes included 53 functionally-annotated TDFs involved in such processes as energy production and conversion, cell division and signal transduction, suggesting that somatic embryogenesis undergoes a complex process. Two full-length cDNA sequences, encoding KHCP (kinesin heavy chain like protein) and TypA (tyrosine phosphorylation protein A), and partial sequences, encoding ARF-GEP (guanine nucleotide-exchange protein of ADP ribosylation factor) homologs, were isolated from embryonic calli of maize and named ZmKHCP, ZmTypA and ZmARF-GEP, respectively. Finally, the real-time qRT-PCR results showed that the expression levels of the three genes were significantly higher in the embryonic calli than the non-embryonic calli. Thus, this study provides important clues to understanding the induction of somatic embryogenesis in maize. The candidate genes associated with the formation of embryonic calli may offer additional insights into the mechanism of somatic embryogenesis, and further research on the three candidate genes may determine their role in increasing the rate of induction of embryonic calli, which may aid in the development of cultivars through transgenic breeding.

Effects of heat stress on gene expression in eggplant (Solanum melongema L.) seedlings

In order to identify differentially expressed genes involved in heat shock response, cDNA amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time polymerase chain reaction (QPCR) were used to study gene expression of eggplant seedlings subjected to 0, 6 and 12 h at 43°C. A total of 53 of over 2400 bands amplified were differentially expressed between the control and heat treatment; 42 up-regulated and 11 corresponding to genes repressed by heat stress. 24 transcriptderived fragments (TDFs) were successfully isolated, cloned and sequenced. BLAST searching revealed that 15 heat stress response transcripts presented similarities with those that encoded small molecular shock protein, disease resistance protein, stress-related protein, enzymes related to photosynthesis as well as biosynthesis of protein, and so on. The expression patterns of five randomly selected TDFs were confirmed by using Q-PCR experiments. The direct and indirect relationships of genes corresponding to the 15 TDFs with stress tolerance are discussed. Key words : Eggplant, heat stress, cDNA amplified fragment length polymorphism (cDNA-AFLP), transcriptderived fragment (TDF), quantitative real-time polymerase chain reaction (Q-PCR).

Differential gene expression in incompatible interaction between turnip mosaic virus and non-heading Chinese cabbage

Viral disease, caused by turnip mosaic virus (TuMV), is considered the most destructive disease of non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino). Although several TuMV resistance loci/genes have been mapped or characterized in Brassica vegetables, the mechanism of molecular interaction between TuMV and non-heading Chinese cabbage is poorly understood. Additionally, TuMV response genes need to be identified. The objectives of this study were to identify differentially expressed genes during the incompatible interaction between TuMV and non-heading Chinese cabbage, and validate their expressions. A total of 200 transcript-derived fragments (TDFs) obtained by complementary DNA-amplified fragment length polymorphism were recovered and sequenced. The results revealed that 176 (88.0%) TDFs produced specific sequences, among which 48 (27.3%) sequences were predicted with putative functions using NCBI BLAST. Among the 48 available TDFs, 22 (45.8%) sequences belonging to different functional groups were selected to monitor the changes in their expression in incompatible and compatible interactions by quantitative real-time polymerase chain reaction. To the best of our knowledge, this study provides the first global transcriptomic analysis of non-heading Chinese cabbage genes during an incompatible interaction. The results are expected to aid in characterizing TuMV response genes and clarifying the molecular mechanism of TuMV–host interaction.

Differential gene expression in response to cold stress in Lepidium apetalum during seedling emergence

Germination of Lepidium apetalum Wild. seeds is invariably arrested by cold stress. cDNA-amplified fragment length polymorphism (AFLP) technique was used to isolate genes relevant to chilling stress (4 °C) during seedling emergence. 43 transcript-derived fragments (TDFs) were found to be up-regulated and 17 down-regulated during chilling stress. Eighteen TDF of up-regulated genes were cloned and sequenced. Some of these genes are involved in the stress response, some play important roles in energy and substrate metabolism, and some encode unknown proteins such as TDF119. Two sequences, designated TDF217 and TDF223, may correspond to novel genes. The expression profiles of 6 from 18 TDFs were analyzed by quantitative real-time PCR under chilling and abscisic acid (ABA) stress. It was demonstrated that all 6 genes were significantly induced by chilling and their expression was decreased when the temperature was shifted from 4 to 25 °C. The transcriptional levels of 5 TDFs were strongly enhanced also in response to exogenous ABA. Based on the characteristics of genes isolated from seedlings exposed to cold stress, we conclude that Lepidium adapts to cold stress by regulating many signal transduction pathways, including both ABA-dependent and ABA-independent signaling pathways.

A mini-scale hot borate method for the isolation of total RNA from a large number of cotton tissue samples

Genetic genomics in cotton requires isolating RNA from a large number of plant tissue samples, in a mapping population. However, traditional methods for the extraction of RNA from cotton tissues is time consuming and not suitable for processing many samples at the same time. Here, we present a miniscale protocol for quick isolation of total RNA from cotton ovaries with high yield and quality suitable for gene expression studies. By modifying and scaling down a hot borate extraction method, a quick and easy method was developed to extract total RNA from ovaries of three genotypes ranging in ages from -1 to 10 days post anthesis (dpa) with an average yield of 600 µg/gfw (gram fresh weight) and ten dpa ovaries of 64 genotypes. The method was also successfully tested using cotton leaves and anthers. The quality and quantity of total RNA were suitable for reverse transcription-polymerase chain reaction (RT-PCR) and cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis.

A mini-scale hot borate method for the isolation of total RNA from a large number of cotton tissue samples

Genetic genomics in cotton requires isolating RNA from a large number of plant tissue samples, in a mapping population. However, traditional methods for the extraction of RNA from cotton tissues is time consuming and not suitable for processing many samples at the same time. Here, we present a miniscale protocol for quick isolation of total RNA from cotton ovaries with high yield and quality suitable for gene expression studies. By modifying and scaling down a hot borate extraction method, a quick and easy method was developed to extract total RNA from ovaries of three genotypes ranging in ages from -1 to 10 days post anthesis (dpa) with an average yield of 600 µg/gfw (gram fresh weight) and ten dpa ovaries of 64 genotypes. The method was also successfully tested using cotton leaves and anthers. The quality and quantity of total RNA were suitable for reverse transcription-polymerase chain reaction (RT-PCR) and cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis.