Cetyltrimethylammonium Bromide Protocol Research Articles

Effects of energy-gathered and energy-divergent ultrasound on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch.

The identification of adulterated sweet potato vermicelli faces significant challenges, seriously hindering the development of the vermicelli industry. Herein, we investigate effects of energy-gathered ultrasound (EGU) and energy-divergent ultrasound (EDU) (30, 40 and 50 W L-1) on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch, thereby exploring their potential in adulteration of sweet potato vermicelli. EGU-assisted modified cetyltrimethylammonium bromide (CTAB) protocol with β-mercaptoethanol significantly improved DNA extraction from sweet potato vermicelli (223.7-249.2 ng μL-1) and its starch (133.4-186.4 ng μL-1), followed by EDU-assisted DNA extraction from sweet potato vermicelli (115.1-209.3 ng μL-1) and its starch (33.4-61.0 ng μL-1). Both EGU and EDU treatments resulted in the destruction of microstructure and crystalline structure, as well as changes in pasting and thermal properties of sweet potato vermicelli and its starch. Real-time polymerase chain reaction (PCR) amplification results revealed that EGU and EDU enhanced the efficiency of DNA amplification, and EDU showed smaller cycle threshold (Ct) values than EGU. In addition, EDU-assisted CTAB protocol combined with real-time PCR could detect levels of less than 1% of cassava and maize starches in sweet potato vermicelli. EDU-assisted CTAB protocol combined with real-time PCR shows promise as a sensitive and reliable analytical tool for vermicelli adulteration. © 2024 Society of Chemical Industry.

Perbandingan Teknik Isolasi DNA pada Daun dan Kayu Sonokeling (Dalbergia latifolia)

Sonokeling (Dalbergia latifolia) is a hardwood plant, resistant to termite attack, its wood has beautiful patterned fibers and textures, so it is widely used for furniture in luxury categories with high economic value. According to CITES (Convention on International Trade in Endagered Species of Wild Fauna and Flora), rosewood trade status is included in appendix II. In the trade of rosewood in both domestic and international markets, there are relatively many obstacles and difficulties in identifying the origin of rosewood. It is very important to establish a reference on the identity of wood based on the location of origin as an effort to prevent illegal trade, namely by using a molecular-based identification approach (DNA fingerprinting). In the isolation of rosewood DNA, special techniques are required due to its hard wood characteristics. This study aims to compare DNA isolation techniques from rosewood and rosewood leaves using the CTAB (Cetyl Trimethyl Ammonium Bromide) protocol and the DNA Mini Kit Plant protocol. The research used molecular biology-based identification methods. PCR results showed that the DNA isolation protocol using the commercial kit outperformed the CTAB protocol for both leaf and wood samples. DNA isolation from wood samples was more difficult than leaf samples. Overall, the best protocol for DNA isolation from rosewood leaves and wood was to use a commercial kit.

What is the "modified" CTAB protocol? Characterizing modifications to the CTAB DNA extraction protocol.

Cetyltrimethylammonium bromide (CTAB)-based methods are widely used to isolate DNA from plant tissues, but the unique chemical composition of secondary metabolites among plant species has necessitated optimization. Research articles often cite a "modified" CTAB protocol without explicitly stating how the protocol had been altered, creating non-reproducible studies. Furthermore, the various modifications that have been applied to the CTAB protocol have not been rigorously reviewed and doing so could reveal optimization strategies across study systems. We surveyed the literature for modified CTAB protocols used for the isolation of plant DNA. We found that every stage of the CTAB protocol has been modified, and we summarized those modifications to provide recommendations for extraction optimization. Future genomic studies will rely on optimized CTAB protocols. Our review of the modifications that have been used, as well as the protocols we provide here, could better standardize DNA extractions, allowing for repeatable and transparent studies.

Optimizing the lysis step in CTAB DNA extractions of silica-dried and herbarium leaf tissues.

The use of cetyltrimethylammonium bromide (CTAB) is an effective and inexpensive method of extracting DNA from plants. The CTAB protocol is frequently modified to optimize DNA extractions, but experimental approaches rarely perturb a single variable at a time to systematically infer their effect on DNA quantity and quality. We investigated how chemical additives, incubation temperature, and lysis duration affected DNA quantity and quality. Altering those parameters influenced DNA concentrations and fragment lengths, but only extractant purity was significantly affected. CTAB and CTAB plus polyvinylpyrrolidone buffers produced the highest DNA quality and quantity. Extractions from silica gel-preserved tissues had significantly higher DNA yield, longer DNA fragments, and purer extractants compared to herbarium-preserved tissues. We recommend DNA extractions of silica gel-preserved tissues that include a shorter and cooler lysis step, which results in purer extractions compared to a longer and hotter lysis step, while preventing fragmentation and reducing time.

A comparison of total RNA extraction methods for RT-PCR based differential expression of genes from Trichoderma atrobrunneum

The genus Trichoderma is ubiquitous in various niches and is currently used for biocontrol, biofertilizer, enzyme production and bioremediation. However, molecular mechanisms underlying its diverse biological functions are yet not fully elucidated. Extraction of high-quality RNA for downstream applications such as quantitative polymerase chain reaction is a prerequisite. The current study aims to optimize a total RNA extraction protocol for high-quality and quantity RNA from Trichoderma atrobrunneum. Seven RNA extraction protocols including Trizol, RiboEx PureLink RNA mini kit, high salt CTAB (cetyltrimethylammonium bromide), modified high salt CTAB, SDS (sodium dodecyl sulfate) and CTAB-PVP (polyvinylpyrrolidone) were performed separately, to extract RNA. Quality and quantity of extracted RNA samples were further analyzed by Nanodrop spectrophotometry, agarose gel electrophoresis and RT-PCR analysis. The results of quantitative and qualitative analysis of RNA samples showed that more intact, high-quality RNA was extracted using the modified high salt CTAB as compared to other methods. The RT-PCR results for the amplification of the genes encoding β-tubulin and Ubiquitin carrier protein also showed lowest threshold cycle (Ct) and coefficient of variation (CV) for RNA samples extracted with the modified high salt CTAB method as compared to RNA samples extracted with other protocols. Therefore, it is proposed that the modified high salt CTAB protocol is an excellent method to obtain high-quality RNA with good yield from T. atrobrunneum for its downstream applications. Moreover, the optimized protocol is very economical and can be used to extract total RNA from a large number of samples.

First report of Cotton leaf curl Multan virus infecting Millettia pinnata in Pakistan

Millettia pinnata (Pongame oiltree, Sukh chain; family Fabaceae) is an important leguminous tree widely grown and utilised as a herb in Southeast Asia and the Indian subcontinent. In October 2020, symptoms of stunted growth, leaf curling (upward and downward) and vein thickening (Figures 1 and 2), which are associated with infection by Begomovirus species in other hosts, were observed on M. pinnata, growing near cotton fields (30.3°N, 73.0°E), in Faisalabad, Punjab Province, Pakistan. DNA was isolated from ten leaves (S1 to S10), one each from ten diseased plants, and one leaf from two asymptomatic plants (AS11 and AS12) using a cetyl trimethyl ammonium bromide protocol (Doyle & Doyle, 1990). Rolling circle amplification in combination with PCR using begomovirus universal primers (BegomoF/BegomoR) (Briddon & Markham, 1994) amplified a c. 2.8 kb product from all the diseased samples. PCR assays for the Begomovirus alphasatellite (primer pair DNA101F/DNA101R) (Bull et al., 2003) and betasatellite (primer pair Beta01F/Beta02F) (Briddon et al., 2002) each amplified a c. 1.4 kb product from all the diseased samples. Neither begomovirus genome nor satellite DNA was detected in the asymptomatic samples. Amplicons from the begomovirus genome (AV-2 and AV-3 from S1 and S2; MZ268122-MZ268123), alphasatellite (AV-36 from S1; MZ057253) and betasatellite (AV-8 and AV-10 from S1, AV-11 and AV-12 from S2, and AV-13 from S3; MZ057231- MZ057235), were cloned in a general purpose PTZ57R/T cloning vector (Thermo Fisher Scientific, USA), Sanger sequenced, and the sequences deposited in GenBank. The genome sequences shared >99% nucleotide identity with Cotton leaf curl Multan virus (CLCuMuV) and were most closely related to CLCuMuV strain NAS-14 (MK357257), which was found in cotton in the same region of Faisalabad (Ahmed et al., 2021). They also had 94% and 87% nt similarity with the CLCuMuV-Raj (AF363011) and CLCuMuV-Dar strains (EU365613) respectively. Moreover, the alphasatellite and betasatellite sequences shared 98% similarity with Cotton leaf curl Multan alphasatellite (CLCuMuA; MK357286.1) and betasatellite (CLCuMuB; MT920327.1), respectively. The five CLCuMuB betasatellite sequences (MZ057231-MZ057235) shared 99% identity and the two Begomovirus genomic sequences (MZ268122-MZ268123) from the M. pinnata samples had 99% identity with each other. Southern hybridization (Zubair et al., 2017) confirmed the detection of Begomovirus genomic DNA and quantitative real-time PCR (Shafiq et al., 2017) assays confirmed the detection of CLCuMuV in three samples, S1, S2 and S3, selected for further analysis. To the best of our knowledge, this is the first report of CLCuMuV in Millettia pinnata which may serve as an alternative host of the virus in the off season when cotton is not grown. Routine monitoring and detection of CLCuMuV is essential to prevent the devastating leaf curl disease it causes from spreading worldwide. The authors acknowledge support in Sanger sequencing from Dr. Brian Scheffler and his group from USDA-ARS.

Optimization of the Cetyltrimethylammonium bromide (CTAB) DNA extraction protocol using forest elephant dung samples

Among non-invasive biological samples, feces offer an important source of DNA and can easily be collected. However, working with fecal DNA from highly vegetarians species such as elephant is more challenging because plant secondary compounds have an inhibitory effect on PCR reactions.Working with forest elephant dung samples, we tested and adapted a protocol of DNA extraction developed on plants based on the Cetyltrimethylammonium bromide (CTAB) protocol.The protocol is relatively simple and yields a high DNA concentration. It is five-time less expensive compared to the methods of Benbouza et al. The extracted DNA is of good quality and easily amplified by PCR. The high-amplification percentage of mitochondrial genes in fecal DNA and subsequent sequencing of PCR products indicate that the proposed optimized method is reliable for molecular analysis of forest elephant dung samples.•Our optimized CTAB protocol has been adjusted by the addition of Sodium Dodecyl Sulfate (SDS) and proteinase K during the lysis phase. The combined effect of these reagents was capable of lysing cell walls and removing proteins efficiently.•Moreover, the prolonged time of incubation (overnight incubation at room temperature followed by 3 hours of incubation in a water bath) enhanced the increase of DNA yield but make the optimized protocol more time-consuming.

First Report of Leaf Spot on Industrial Hemp (Cannabis sativa L.) Caused by Alternaria alternata in China.

Industrial hemp is an economically important plant with traditional uses for textiles, paper, building materials, food and medicine (Li 1974; Russo et al. 2008; Zlas et al. 1993). In August 2020, an estimated 80% of the industrial hemp plants with leaf spots were observed in greenhouse in Minzhu town, Harbin City, Heilongjiang Province, China (45.8554°N, 126.8167°E), resulting in yield losses of 20%. Leaf symptoms began as small spots on the upper surface of leaves and gradually developed into brown spots with light yellow halos. These irregular spots expanded gradually and eventually covered the entire leaf; the center of the spots was easily perforated. To identify the pathogen, 20 diseased leaves were collected, and small sections of (3 to 5 mm) were taken from the margins of lesions of infected leaves. The pieces were sterilized with 75% alcohol for 30 s, a 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water. Samples were then cultured on potato dextrose agar at 28℃ in darkness for 4 days. A single-spore culture was obtained by monosporic isolation. Conidiophores were simple or branched, straight or flexuous, brown, and measured 22 to 61 μm long × 4 to 5 μm wide (n = 50). Conidia were solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid. Conidia ranged from 23 to 55 μm long × 10 to 15 μm wide (n = 50) with one to eight transverse and several longitudinal septa. For molecular identification (Jayawardena et al. 2019), genomic DNA of pathogenic isolate (MZ1287) was extracted by a cetyltrimethylammonium bromide protocol. Four gene regions including the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosplate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1) and RNA polymerase II beta subunit (RPB2) were amplified with primers ITS1/ITS4, GDF1/GDR1, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR, respectively (White et al. 1990). Resulting sequences were deposited in GenBank with accession numbers of MW272539.1, MW303956.1, MW415414.1 and MW415413.1, respectively. A BLASTn analysis showed 100% homology with A. alternata (GenBank accession nos. MN615420.1, MH926018.1, MN615423.1 and KP124770.1), respectively. A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate MZ1287 clustered in the A. alternata clade with 100% bootstrap support. Thus, based on morphological (Simmons 2007) and molecular characteristics, the pathogen was identified as A. alternata. To test pathogenicity, leaves of ten healthy, 2-month-old potted industrial hemp plants were sprayed using a conidial suspension (1×106 spores/ml). Control plants were sprayed with sterile water. All plants were incubated in a greenhouse at 25℃ for a 16 h light and 8 h dark period at 90% relative humidity. The experiment was repeated three times. After two weeks, leaf spots of industrial hemp developed on the inoculated leaves while the control plants remained asymptomatic. The A. alternata pathogen was re-isolated from the diseased leaves on inoculated plants, fulfilling Koch's postulates. Based on morphology, sequencing, and pathogenicity test, the pathogen was identified as A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spot disease of industrial hemp (Cannabis sativa L.) in China and is worthy of our attention for the harm it may cause to industrial hemp production.

Development of an SSR-based DNA fingerprinting method for black wattle (Acacia mearnsii De Wild)

Background: The most commonly used method for extracting DNA from plant leaf tissue involves cetyl trimethylammonium bromide but some species, such as Acacia mearnsii, contain high levels of secondary metabolites and polysaccharides that interfere with this process. Various modifications have been proposed for effective removal of these biomolecules but these methods can be time consuming. Therefore, this study was initiated to optimise the cetyl-trimethylammonium bromide protocol for the extraction of high-quality genomic DNA and to develop a fingerprinting tool using cross species transferable simple sequence repeat markers for genetic diversity studies in A. mearnsii.
 Methods: Five CTAB-based modification were examined and 49 cross-species microsatellite markers, developed for several Acacia species, were tested in four multiplex panels of A. mearnsii populations.
 Results: The modified protocol yields high quantity and quality DNA from A. mearnsii leaves using high concentration of NaCl to remove polysaccharides and polyvinylpolypyrrolidone (PVPP) to eliminate polyphenols during DNA purification. In addition, omitting the selective precipitation and NaCl gradient steps in the extraction protocol, enabled us to extract DNA 10–20 min faster than the normal protocol. Of the tested microsatellite loci, 11 were successful in amplifying sharp and high-intensity bands in all the four multiplex panels and were polymorphic. The level of polymorphism ranged from 0.115 to 0.794, with a mean 0.50 and mean number of alleles varied from 2 to 10, with overall mean of 6 alleles per locus. The mean observed and expected heterozygosity ranged from 0.058 to 0.970 and 0.102 to 0.796, respectively. The 11 microsatellite loci that were effectively amplified from A. mearnsii DNA were adequate in detecting genetic variation among the tested populations.
 Conclusions: These PCR-based, multi-allelic, co-dominant microsatellite markers provide a powerful tool for genetic, breeding and conservation studies in A. mearnsii.

Isolation of genomic DNA from groundnut plant by modified, rapid and efficient protocol

DNA isolation is prerequisite to study the crop at molecular level. However, DNA (Deoxyribonucleic Acid) isolation is difficult in groundnut due to presence of large amount of polyphenols and polysaccharides. The age and growth stage of the plant also affect the DNA purity during isolation. In this study, newly expanded leaves and stems of five days old seedlings and matured leaves of 20 days old seedlings were used for optimization of extraction protocol. High quality DNA was obtained from all plant materials by this modified CTAB (Cetyltrimethylammonium bromide) protocol without separate purification. Optimized protocol was used to isolate DNA from newly expanded leaves of 5 days old seedlings of 100 groundnut genotypes. DNA obtained ranged from 6.3-22.1 µg/ g of sample when quantified by Nano Drop spectrophotometer and was able to amplify with Simple sequence repeats (SSR) primers during Polymerase Chain Reaction (PCR).

DNA de Solenidium lunatum (Lindl.) Kraenzl (Orchidaceae)

Estudo de caracterização molecular e de diversidade genética com base em marcadores moleculares requer DNA de qualidade, livre de contaminantes como fenóis e polissacarídeos, e também em quantidade suficiente para realização de reações em cadeia da polimerase (PCR). Os protocolos de extração de DNA vegetal são, em sua maioria, baseados no método CTAB (Brometo de Cetiltrimetilamônio), contudo devido às particularidades de cada planta, são necessários ajustes quanto aos reagentes utilizados e à quantidade dos mesmos. Diante do exposto, este estudo teve por objetivo avaliar diferentes concentrações de CTAB e de β-mercaptoetanol no protocolo de extração de DNA de Solenidium lunatum (Lindl.) Kraenzl (Orchidaceae), visando futuros estudos de diversidade genética com utilização de marcadores moleculares. Para o CTAB foram testadas as concentrações 2% e 5%, enquanto que para o β-mercaptoetanol, foram testados 0% e 2%. Para verificar se o material extraído era passível de amplificação, realizam-se testes com três primers ISSR (Inter Simple Sequence Repeats). Os resultados indicam que todos os métodos foram eficientes na extração do DNA em quantidade e qualidade necessárias para realização de PCRs. Todavia, recomenda-se a utilização do protocolo CTAB 2% sem adição de β-mercaptoetanol, uma vez que não foram verificadas diferenças significativas nos resultados de amplificações. A utilização desse protocolo produz material de qualidade, passível de amplificação, com redução de custos e de riscos de intoxicação.

First Report of Brown Leaf Blight of Shenguyou (Staphylea bumalda) Caused by Alternaria alternata in China

Staphylea bumalda DC. (Staphyleaceae) is a woody bush distributed widely in China. The tender leaves and blooms of S. bumalda have high nutritional value and have been used as a cough remedy, antidiarrheal, and blood refresher after delivery. Furthermore, the seed oil content and quality are excellent (Yu et al. 2005). During June 2017, brown leaf blight was observed on this plant in Xiaogan, Hubei Province, and the disease incidence was about 30%. The disease could be prevalent in large-scale planting conditions, and it is potentially a large risk for the development of Shenguyou industry. Thus, the identification and detection of this disease are important. The symptoms emerged from the tip of leaves at the early stage and extended to the middle or even the whole leaves subsequently. The blight was brown and caused defoliation of the infected leaves at the end. Five 3- × 3-mm pieces of leaf tissue were excised from the margins of individual lesions from five 2-year-old S. bumalda trees with typical symptoms. Excised tissues were surface disinfested by using 0.1% mercury bichloride for 3 min, rinsed three times with sterilized water, plated on potato dextrose agar, and incubated at 25°C (Robak et al. 2014). Five fungal isolates with consistent morphology were obtained. The colonies were gray white to black in obverse and dark blue in reverse. Hyphae were achromatic, branched, and septated, with a diameter of 2.1 to 4.6 μm. The conidia were 3.5 to 15.8 μm in width and 7.6 to 46.5 μm in length with 2 to 10 transverses and 0 to 3 longitudinal septa (n = 100) (Simmons 2007). DNA was extracted from 100 mg of freeze-dried mycelium following the cetyltrimethylammonium bromide protocol (O’Donnell et al. 2010). The internal transcribed spacer (ITS) region of rDNA and a histone gene were amplified using the primers ITS1/ITS4 and H3-1a/H3-1b (Glass et al. 1995), respectively, and sequenced (GenBank accessions MF614038 and MF991905). These sequences had ≥99% nucleotide identity with the corresponding sequences (KX926578.1 and KY548071.1) of the reference strains of Alternaria alternata in GenBank. Supported by the molecular identification and morphological characteristics (Simmons 2007), the pathogen was identified as A. alternata. Koch’s postulates were fulfilled on 10 healthy leaves of 2-year-old S. bumalda tree in an incubator. Spore suspension (2 ml, 1.0 × 10⁶ conidia/ml) was inoculated onto leaves until runoff by a spray method, and sterilized water was applied on 10 additional leaves as a control. The incubator was set at 25°C and 12-h light/dark alternation with 192 lux. It was observed that the symptoms on the inoculated leaves 14 days postinoculation were similar to the diseased trees in the field, whereas the control plants remained healthy. The fungus was consistently reisolated from the inoculated symptomatic leaves and identified as the same. To our knowledge, this is the first report of S. bumalda brown leaf blight disease caused by A. alternata in China and across the world. Furthermore, our findings extended the host range of the pathogen A. alternata on characteristic plants.

First Report of Charcoal Rot on Zebra Plant (Aphelandra squarrosa) Caused by Macrophomina phaseolina.

The Zebra plant (Aphelandra squarrosa) is native to tropical forests of Brazil and is a popular decorative pot plant in Serbia. Charcoal rot-like symptoms were first observed on a zebra plant in August 2010 in Novi Sad (Vojvodina Province, Serbia). The diseased plant showed symptoms of stem and root rot, loss of stem turgor, and premature death. In the cross section of the stem, mycelia with black microsclerotia were observed from the base of the plant up to 50% of the plant stem. To isolate the causal agent, 10 cuttings of the infected stem tissue were surface disinfected with 2% sodium hypochlorite solution for 5 min, rinsed three times in sterile distilled water, air dried on sterilized filter paper, and plated on potato dextrose agar (PDA) and water agar (WA) amended with streptomycin sulfate. After 7 days of incubation at 30°C in the dark, colonies with Macrophomina phaseolina morphology (gray aerial mycelia with black pigmentation in agar caused by homogenous black microsclerotia formation) were observed from all cuttings. The colonies had average growth rates of 33 mm/day on PDA and 8 mm/day on WA and produced microsclerotia with average diameters of 119.6 µm (PDA) and 49.0 µm (WA), which is in accordance with Watanabe (2010) and earlier findings in Serbia (Acimovic 1998). A representative isolate was purified by a hyphal tip transfer technique for further analyses (Leslie and Summerell 2006). The pathogenicity was determined according to Koch’s postulate. Plants were longitudinally cut at the stem base, artificially inoculated with a 5 mm² plug of PDA with developed microsclerotia or without fungus for the control, sealed with Parafilm, and placed in a growth chamber at 30°C with a 12 h/12 h light/dark regime. Plants were regularly watered every third day with 200 ml of water per pot, and the humidity level was artificially raised by humidity trays around the plants. The first charcoal rot symptoms occurred 6 days after inoculation as brown spots on the leaves and stems around the incision site, along with loss of turgor. On day 9, symptoms advanced with gray mycelia present, and browning stems and complete loss of turgor were observed. The fungus was reisolated as previously described. No symptoms were observed on the control plants. To confirm the identification, DNA from the 7-day-old M. phaseolina isolate used for inoculation was extracted using the cetyltrimethylammonium bromide protocol (Permingeat et al. 1998). Polymerase chain reaction amplification was performed in 25-µl reaction volume according to the protocol of Nagl et al. (2011). DNA of the M. phaseolina isolate originating from the zebra plant was compared with 15 M. phaseolina isolates originating from sunflower, soybean, common bean, and flax from different sites in Serbia by random amplification of polymorphic DNA (RAPD) analyses based on 154 polymorphic bands obtained with 14 (10-mer) OPA primers (Operon Technologies, Alameda, CA): OPA-02 to OPA-05, OPA-07 to OPA-13, and OPA-18 to OPA-20. So far, OPA primers and the RAPD technique have proved useful for distinguishing M. phaseolina isolates from species common in mixed infections with M. phaseolina (Jana et al. 2003). Genetic similarity between the isolate from the zebra plant with other M. phaseolina isolates was confirmed through cluster analyses using the unweighted pair group method with arithmetic mean and the Jaccard similarity coefficient. The tested isolate showed 80% genetic similarity with some sunflower-originating isolates. To the best of our knowledge, this is the first report of M. phaseolina causing charcoal rot on zebra plant in the world.

Analysis of genetic diversity of Laeliinae (Orchidaceae) in the State of Sergipe using ISSR markers

The Orchidaceae represent one of the largest and most diverse families on the planet. However, this family is constantly threatened by predators and by the advancement of urban centers over its natural habitats. The objective of this study was to use inter-simple sequence repeat markers to evaluate the genetic diversity between orchid accessions of the Laeliinae subtribe, which comprise part of the Orchidaceae study collection at the Department of Agronomic Engineering of the Federal University of Sergipe. DNA was extracted from each specimen by using an adapted 2% cetyltrimethyl ammonium bromide protocol. Similarity between individuals was calculated using the Jaccard method. Clustering was carried out by the unweighted pair group method with arithmetic mean method, with resampling and 10,000 bootstraps. Eighty-seven fragments were obtained, all of which were polymorphic, revealing high variability between accessions. The mean similarity was 35.77% between Encyclia sp individuals, and 35.90% between specimens of Cattleya tigrina. For Epidendrum secundum, a relationship between geographic and genetic distances was observed, and the accession collected in the southern part of the State of Sergipe (Serra de Itabaiana National Park) was more divergent than that of the other parts of the state. The data generated in this study will guide further research aimed at the ex situ conservation of these materials.

English

  Pomegranate (Punica granatum L.) is a plant rich in polysaccharides, polyphenols and secondary  metabolites, which makes it difficult to obtain high quality DNA. The present study reports a quick,  simple and inexpensive method to isolate genomic DNA suitable for amplified fragment length  polymorphism (AFLP) analysis and other PCR-based applications. This method is a modification of a  protocol described by Doyle and Doyle (1990). It is a cetyl trimethyl ammonium bromide (CTAB)-based  protocol modified by the use of potassium acetate (KoAc) and polyvinylpyrrolidone (PVP) to remove  polyphenols and polysaccharides and a high concentration of β-mercaptoethanol to reduce oxidation.  Moreover, the final optimized protocol was then compared with three different methods, which are  routinely used for many plant species. The results show that our modified CTAB protocol produced a  high yield (>500 ng/μl) of good-quality DNA (A260/A280 >1.8) compared to the other three methods. The  DNA purity was further confirmed by complete digestion with EcoRI and MseI enzymes. The modified  CTAB protocol used in this study could be a useful protocol for extraction of high quality DNA not only  for pomegranate but also for other plants rich in polysaccharides, polyphenolices and secondary  metabolites. Using this method, DNA was extracted from 67 accessions of pomegranate. The DNA was  then used for AFLP analysis. To optimize the AFLP protocol, the effects of MgCl2 concentration during  selective amplification, the dilution level of pre-amplified DNA and the cycle number used in the preamplification were studied. After optimization of the reaction conditions, AFLP was used to study  genetic diversity among Iranian pomegranate accessions.    Key words: Pomegranate, DNA extraction, amplified fragment length polymorphism (AFLP), secondary  metabolites.

Comparison of protocols for DNA extraction from long-term preserved formalin fixed tissues

The current study compared the applicability of protocols to extract DNA from formalin fixed heart tissues that have been preserved for more than 50years. Ten methods were tested: a cetyltrimethylammonium bromide (CTAB) standard protocol, seven variants of this standard protocol, and two commercial kits. In the case of younger specimens (fixed in 1951, 1934, or 1914), extracts with DNA concentrations⩾10.0ng/μl were obtained with the standard CTAB protocol, two variants of the standard protocol including prolonged tissue digestion (72h instead of 1–2h), and a commercial kit particularly recommended for DNA extraction from formalin fixed paraffin embedded tissues (FFPE Kit). With the FFPE Kit, DNA could also be extracted from older tissues (fixed in 1893, 1850/1851, or before 1820). In general, the purity of the DNA extracts, assessed from the ratio of the absorbance at 260 and 280nm, was not very high. In spite of their rather low purity, the DNA extracts could, however, be used to amplify a 122-bp sequence and, in most cases, also a 171-bp sequence of the gene coding for human albumin by the polymerase chain reaction (PCR).

First Report of Phytophthora palmivora Causing Root Rot of Cassava in China.

Cassava (Manihot esculenta) is an economically important crop grown widely in South China. Seventy percent of the cassava grown is used for starch and ethanol production and it has become the foundation of local food and bioenergy systems. In November 2010, a new root rot disease was found on cv. HuaNan205 from a cassava plantation in Danzhou, Hainan Province. Disease occurred on 30% or less of the plants. Initially, the upper leaves wilted at noon and recovered in the evening. Eventually, infected plants no longer recovered and the whole plant wilted and died. Root rot symptoms consisting of irregular brown patches occurred on the tuberous roots. Symptomatic root rot tissue was cut into 1-cm pieces, washed in distilled water, and soaked in a solution of 1% sodium hypochlorite for 3 min. A subsection was cut from each sterilized piece, placed on a plate of V8 agar medium, and incubated at 28°C for 7 days. Pathogenicity was established by following Koch's postulates. In July 2011, 10 plants of cassava cv. HuaNan205 were selected from a disease-free plantation in Danzhou. The pathogen was cultivated on V8 agar at 28°C for 14 days. Four holes were established 15 cm from the base of the cassava plants. Five plants were inoculated with 100 mL of the mycelial suspension in each of the four spots and covered by soil. The other five plants were treated with sterile water as control. Plants were maintained for 4 months. All five of the inoculated plants wilted and two died, while the control plants grew normally. Symptoms similar to the original root lesions were observed on tuberous roots of inoculated plants, while only scars formed on tuberous roots of control plants. The pathogen was reisolated from the lesions of inoculated plants. Microscopic examination showed the sporangia as papillate and ovoid with the widest part close to the base. They were easily washed off and each detached sporangium contained a short pedicel 1.2 to 6.9 μm long, average 2.9 μm. Chlamydospores were readily observed on diseased roots and observed in pure cultures on V8 agar. Morphological characteristics of the specimen were similar to the descriptions for Phytophthora palmivora (2). Genomic DNA of this isolate was extracted with a cetyltrimethylammoniumbromide protocol (3) from mycelium and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4 (1). The sequence (GenBank Accession No. HE580279) exactly matched several sequences (e.g., GenBank Accession Nos. HQ237481.1, AY745750, and AY745751) of P. palmivora. To our knowledge, this is the first report of root rot caused by P. palmivora on cassava in China.

An Improved CTAB–Ammonium Acetate Method for Total RNA Isolation from Cotton

Cotton is an important economic crop. Genetic, developmental and molecular studies of cotton require high-quality total RNA from different tissues. Due to the richness in polyphenols and polysaccharides, the Trizol-based methods and other commercial kits are unsuitable for RNA isolation from cotton. Available methods are generally laborious and time-consuming. To develop an easy, simple and rapid cetyltrimethylammonium bromide (CTAB)-ammonium acetate protocol that takes less time and obtains high yield and quality of RNA from polysaccharide- and polyphenol-rich cotton tissues. Based on the original CTAB protocol, we used phenol-chloroform and chloroform-isoamyl alcohol to remove proteins, polysaccharides and polyphenols, and ammonium acetate to precipitate RNA, reducing the incubation time prior to RNA precipitation. After adding ammonium acetate to precipitate RNA, all centrifugation steps (14000 × g) were carried out at 4°C to avoid degradation. The procedure took only 1.5 h and was suitable for different cotton tissues. The A(260) : A(280) ratios ranged from 1.80 to 1.85 with clear 28 s and 18 s ribosomal RNA bands in 1.2% agarose gel. The isolated RNA was usable for downstream molecular studies, such as reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. The CTAB-ammonium acetate method is easy, rapid, low-cost and effective for high-quality RNA isolation from polysaccharide- and polyphenol-rich cotton tissues.

Polyphenolics free DNA isolation and optimization of PCR-RAPD for fennel (Foeniculum vulgare Mill.) from mature and young leaves

Molecular analysis of fennel ( Foeniculum vulgare Mill.) strictly relies on high yield, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide (CTAB) protocol introducing several modifications such as inclusion of variable percentage of polyvinylpyrrolidone (PVP, 2 - 6%), different molarity of sodium chloride (NaCl, 1.5 - 4 M), activated charcoal (1 to 2%), and pre-heated buffer (65°C) with and without liquid N2 extraction for the mature leaves. In contrast, the CTAB protocol without any additional anti-oxidants using liquid N 2 extraction was performed for the fresh young leaf tissue. Optimization of polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD) conditions included 10X PCR buffer compositions such as (NH 4 ) 2 SO 4 with 0.1% (v/v) Tween 20 over KCl buffer with and without 0.8% (v/v) Nonidet P40 and an ‘optimized’ buffer which contains KCl and (NH 4 ) 2 SO 4 , MgCl 2 (2.5 mM), Taq enzyme (1 to 1.5 U), annealing temperature of 37 and 42°C and PCR reaction volume of 10 and 25 μl. The results show that DNA isolated from fresh young leaves were superior in quality and quantity over mature (stored) leaves and was amenable to optimized PCR-RAPD conditions. Keywords: Activated charcoal, cetyl trimethyl ammonium bromide (CTAB)-DNA, Foeniculum vulgare , NaCl, polymerase chain reaction-random amplification of polymorphic DNA (PCR-RAPD), PCR buffer, polyphenolics, polyvinylpyrrolidone (PVP)

Optimization of DNA Extraction for RAPD and ISSR Analysis of Arbutus unedo L. Leaves

Genetic analysis of plants relies on high yields of pure DNA. For the strawberry tree (Arbutus unedo) this represents a great challenge since leaves can accumulate large amounts of polysaccharides, polyphenols and secondary metabolites, which co-purify with DNA. For this specie, standard protocols do not produce efficient yields of high-quality amplifiable DNA. Here, we present for the first time an improved leaf-tissue protocol, based on the standard cetyl trimethyl ammonium bromide protocol, which yields large amounts of high-quality amplifiable DNA. Key steps in the optimized protocol are the addition of antioxidant compounds—namely polyvinyl pyrrolidone (PVP), 1,4-dithiothreitol (DTT) and 2-mercaptoethanol, in the extraction buffer; the increasing of CTAB (3%, w/v) and sodium chloride (2M) concentration; and an extraction with organic solvents (phenol and chloroform) with the incubation of samples on ice. Increasing the temperature for cell lyses to 70 °C also improved both DNA quality and yield. The yield of DNA extracted was 200.0 ± 78.0 μg/μL and the purity, evaluated by the ratio A260/A280, was 1.80 ± 0.021, indicative of minimal levels of contaminating metabolites. The quality of the DNA isolated was confirmed by random amplification polymorphism DNA and by inter-simple sequence repeat amplification, proving that the DNA can be amplified via PCR.