scholarly journals AVALIAÇÃO DE DOIS MÉTODOS DE TRITURAÇÃO FOLIAR DE Acianthera ciliata (Orchidaceae) PARA EXTRAÇÃO DE DNA

Nativa ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 97
Author(s):  
Eliane Cristina Moreno de Pedri ◽  
Catiane Dos Santos Braga ◽  
Carlos Alberto Da Cunha Oliveira ◽  
Auana Vicente Tiago ◽  
Ana Aparecida Bandini Rossi
Keyword(s):  
Genetic Diversity ◽  
Molecular Markers ◽  
Liquid Nitrogen ◽  
Dna Extraction ◽  
Plant Species ◽  
Genomic Dna ◽  
Extraction Buffer ◽  
Foliar Tissue ◽  
Genomic Regions ◽  
Issr Primers

O estabelecimento de protocolo de extração de DNA de espécies vegetais é uma técnica empregada para a obtenção de um DNA puro e de qualidade. Diante disso, objetivou-se neste estudo padronizar um protocolo para a extração de DNA da espécie Acianthera ciliata, visando posteriores estudos de diversidade genética. Foram testados dois métodos de trituração do tecido foliar, sendo eles: Tampão STE e nitrogênio líquido. Para cada método de trituração foram testadas duas concentrações de β-mercaptoetanol (0% e 2%). Os dois métodos utilizados, foram eficientes na extração do DNA genômico de A. ciliata. As amostras extraídas com 0% de β-mercaptoetanol, para os dois métodos, STE e nitrogênio líquido, apresentaram menor quantidade de DNA quando comparado com as amostras extraídas com 2% de β-mercaptoetanol. Os dois primers testados amplificaram regiões do genoma de A. ciliata. Para a extração de DNA de A. ciliata indica-se a utilização de CTAB 5% no tampão de extração e β-mercaptoetanol a 2%. Os iniciadores ISSR foram eficientes na amplificação e são recomendados para estudos de diversidade genética de A. ciliata.Palavras-chave: diversidade genética; CTAB; marcadores moleculares; orquídeas. EVALUATION OF TWO MACERATION METHODS IN Acianthera ciliata (Orchidaceae) LEAVES FOR DNA EXTRACTION ABSTRACT: The establishment of DNA extraction protocol for plant species is a technique employed to obtain pure and good quality DNA. In this study, we standardized a protocol for the extraction of DNA of the species Acianthera ciliata, aiming studies of genetic diversity subsequently. Two maceration methods for foliar tissue were tested, and they were STE buffer and liquid nitrogen. Two concentrations of β-mercaptoethanol (0% and 2%) were tested for each method. The two methods used were efficient for genomic DNA extraction of A. ciliata. In both methods the samples extracted using 0% of β-mercaptoethanol, they presented lesser amount of DNA than the samples extracted using 2% of β-mercaptoethanol. The two tested primers amplified genomic regions of A. ciliata. For the DNA extraction of A. ciliata, we indicated the use of CTAB 5% in the extraction buffer as well as β-mercaptoethanol to 2%. The ISSR primers were efficient in amplification and thus they are indicated for studies of genetic diversity of A. ciliata.Keywords: genetic diversity; CTAB; molecular markers; orchids.

scholarly journals Selection of ISSR Primers to Study the Genetic Diversity of Myrciaria floribunda O. Berg

2020 ◽  
pp. 23-28
Author(s):  
Luiz Sergio Costa Duarte Filho ◽  
Danielson Ramos Ribeiro ◽  
Allison Vieira da Silva
Keyword(s):  
Genetic Diversity ◽  
Molecular Markers ◽  
South America ◽  
Central America ◽  
Dna Extraction ◽  
Endemic Species ◽  
Germplasm Bank ◽  
Slow Growing ◽  
Selection Of ◽  
Issr Primers

The species Myrciaria floridunda O. Berg, popularly known as cambuí, belongs to the Myrtaceae family. Cambui is a native, non-endemic species that occur in diverse environments in Central America and South America. They are slow-growing plants with a shrub or sub-shrub habit. The fruits, the product of interest of the species, are small, spherical berries orange or red in colorturning to wine colorwhen they are ripe. The exploitation of the species is still mostly extractivist, carried out by traditional local families who, in times of fruiting of the species, leverage their income by selling fruits at fairs. The fruits can be eaten fresh, in the form of jellies, liquor or wine. To study the genetic diversity of the species using ISSR-type molecular markers, it is necessary to first isolate DNA in sufficient quality and quantity. Here, leaves for DNA extraction were collected from the active germplasm bank of the Federal University of Alagoas, Brazil. The DNA of the species was extracted using CTAB detergent methodology with modifications adapted to the species. Twelve ISSR primers were tested on DNA from two cambui genotypes. Of the twelve primers, eight were selected due to their polymorphism index above 50%, namely: UFAL-2, UFAL-3, UFAL-5, UFAL-6, UFAL-7, UFAL-8, UFAL-9 and UFAL-10.

scholarly journals Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)

1997 ◽  
Vol 20 (3) ◽  
pp. 421-423 ◽  
Author(s):  
Ana Maria Waldschmidt ◽  
Tânia Maria Fernandes Salomão ◽  
Everaldo Gonçalves de Barros ◽  
Lúcio de Antônio Oliveira Campos
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Stingless Bee ◽  
The Other ◽  
Melipona Quadrifasciata ◽  
Other Hand ◽  
Extraction Buffer

The objective of the present study was to test three different procedures for DNA extraction of Melipona quadrifasciata based on existing methods for DNA extraction of Apis, plants and fungi. These methods differ in the concentrations of specific substances in the extraction buffer. The results demonstrate that the method used for Apis is not adequate for DNA extraction from M. quadrifasciata. On the other hand, with minor modifications this method and the methods for plants and fungi were adequate for DNA extraction of this stingless bee, both for adults and larvae

scholarly journals Genetic Diversity Analysis between Different Varieties of Chickpea (Cicer arietinum L.) Using SSR Markers

2019 ◽  
Vol 7 (2) ◽  
pp. 236-242
Author(s):  
Summy Yadav ◽  
Vaidehi Shah ◽  
Bhavya Mod
Keyword(s):  
Genetic Diversity ◽  
Ssr Markers ◽  
Genomic Dna ◽  
Physiological Data ◽  
Diversity Analysis ◽  
Hierarchical Tree ◽  
Cicer Arietinum L ◽  
Equal Distance ◽  
Genomic Regions ◽  
Simple Sequence

The paper aims at evaluating genetic diversity among genomes of chickpea comprising of 5 different varieties with the help of simple sequence repeats (SSR) molecular markers. Genomic DNA isolated from all varieties was checked with 15 different SSR markers specific for ENDPOINT PCR using PCR based techniques. Amplification bands with different markers enabled identification of the genomic regions responsible for Drought Tolerance in chickpea. All 15 SSR markers chosen gave monomorphic bands.  A hierarchical tree was also constructed using UPGMA Dendogram for figuring out the exact genetic distance of cultivars using band amplification data. It depicted GUJ-1 and GUJ-2 are closest of all cultivars. GUJ-5 is at the center having GUJ-3 and UJJAVAL at an almost equal distance but GUJ-5 and GUJ-3 are more related. Physiological data also supported this genetic evidence. Int. J. Appl. Sci. Biotechnol. Vol 7(2): 236-242

scholarly journals Characterization of genetic diversity of cactus species (Opuntia spp.) in Morocco by morphological traits and molecular markers

2017 ◽  
Vol 5 (2) ◽  
pp. 149-159 ◽  
Author(s):  
Y. El Kharrassi ◽  
M.A. Mazri ◽  
M.H. Sedra ◽  
A. Mabrouk ◽  
B . Nasser ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Molecular Markers ◽  
Morphological Traits ◽  
Polymorphic Information Content ◽  
Random Amplified Polymorphic Dna ◽  
Rapd Marker ◽  
Group Method ◽  
Rapd Primer ◽  
Opuntia Spp ◽  
Issr Primers

The genetic diversity within and among 124 accessions of Opuntia spp. collected from different regions of Morocco was assessed using morphological descriptors and molecular markers. Based on 10 morphological traits, the accessions were separated into 3 main clusters; each cluster was containing accessions from different regions and species. Polymerase chain reaction (PCR) was then performed on 22 accessions from different regions and species, with 10 inter-simple sequence repeat (ISSR) primers and one random amplified polymorphic DNA (RAPD) primer. ISSR primers produced 66 bands overall, 64 (96.9 %) of which were polymorphic while 6 bands were generated by the RAPD marker, all polymorphic. The polymorphic information content (PIC) values ranged from 0.62 to 0.97, with an average of 0.82. The dendrogram of genetic differences generated using the unweighted pair-group method using arithmetic averages (UPGMA) method showed 7 different clusters at a similarity of 0.76, which was confirmed by the principal component analysis (PCA). The main conclusion of our work is the high genetic similarity between Opuntia ficus indica and Opuntia megacantha species in Morocco. Our results will be useful for plant breeding and genetic resource conservation programs.

scholarly journals Comparative Analysis of Different DNA Extraction Methods and Preliminary Analysis of Genetic Diversity of Hedera nepalensis K. Koch. in Vietnam Based on GBSSI Marker

2019 ◽  
Vol 35 (1) ◽  
Author(s):  
Dinh Doan Long ◽  
Nguyen Xuan Bach ◽  
Nguyen Thi Thu Thao ◽  
Pham Thi Hong Nhung ◽  
Do Thi Le Hang ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Dna Extraction ◽  
Genomic Dna ◽  
Preliminary Analysis ◽  
Herbarium Specimens ◽  
Arnold Arboretum ◽  
Sprague Dawley ◽  
National Academy Of Sciences ◽  
Manihot Esculenta Crantz ◽  
Spacer Length

Though the ivy (Hedera nepalensis K. Koch.) has long been utilized in traditional medicine, its genome information is very limited. For plants, an effective method of DNA extraction is a very important step which greatly affects subsequent genetic analyses. In this study, four different methods of DNA extraction from dry leaves were used. A comparison of different protocols resulted in the yield of extracted DNA that ranged from 10.5 to 437.4 ng/μl and with a purity ranged from 1.8 to 2.2. Based on the PCR results of GBSSI gene, Gene JET Plant Genomic DNA Purification Mini Kit is the most optimal extraction method for Vietnam ivy’s dry leaves. A preliminary analysis of the phylogenetic tree based on the GBSSI marker showed that ivy growing in a number of northern mountainous provinces of Vietnam belonged to the H. nepalensis K. Koch species. The high - quality total DNA will allow us to amplify different DNA markers, providing valuable genetic information to preserve and develop medicinal resources in Vietnam. Keywords GBSSI, Hedera nepalensis K. Koch, DNA extraction. References [1] J. Ackerfield, J. Wen, A morphometric analysis of Hedera L. (the ivy genus, Araliaceae) and its taxonomic implications, Adansonia Sér. 24 (2002) 187-212.[2] U.S. National Plant Germplasm System, Taxon: Hedera nepalensis K. Koch, https://npgsweb.ars -grin.gov/gringlobal/taxonomydetail.aspx?id= 18567, 2019 (accessed 21 March 2019). [3] L. Jafri, S. Saleem, T.P. Kondrytuk, I.Q. Haq, N. Ullah, J.M. Pezzuto, B. Mirza, Hedera nepalensis K. Koch: A Novel Source of Natural Cancer Chemopreventive and Anticancerous Compounds, Phytotherapy Reserch. 30 (2016) 447-453.[4] S. Saleem, L. Jafri, I. Haq, L.C. Chang, D. Calderwood, B.D. Green, B. Mirza, Plants Fagonia cretica L. and Hedera nepalensis K. Koch contain natural compounds with potent dipeptidyl peptidase-4 (DPP-4) inhibitory activity, Journal of Ethnopharmacology. 156 (2014) 26-32.[5] W.J. Hashmi, H. Ismail, F. Mehmood, B. Mirza, Neuroprotective, antidiabetic and antioxidant effect of Hedera nepalensis and lupeol against STZ+ AlCl3 induced rats model, DARU: Journal of faculty of Pharmacy, Tehran University of Medical Sciences. 26 (2018) 179-190.[6] H. Ismail, A. Rasheed, I.U. Haq, L. Jafri, N. Ullah, E. Dilshad, M. Sajd, B. Mirza, Five indigenous plants of Pakistan with Antinociceptive, anti-inflammatory, antidepressant, and anticoagulant properties in Sprague Dawley rats, Evidence-based Complementary and alternative medicine 2017 (2017) 1-10.[7] A. Mitchell, J. Wen. Phylogenetic utility and evidence for multiple copies of granule-bound starch synthase I (GBSSI) in Araliaceae, Taxon 53 (2004) 29-44.[8] M.A. Saghai-Maroof, K.M. Soliman, R.A. Jorgensen, R.W.L. Allard, Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics, Proceeding of the National Academy of Sciences of the USA. 81 (1984) 8014-8018.[9] M. Elias, G.S. Mühlen, D. McKey, A.C. Roa, J. Tohme, Genetic diversity of traditional South American landraces of cassava (Manihot esculenta Crantz): an analysis using microsatellites, Economic Botany. 58 (2004) 242-256.[10] B.D. Lade, A.S. Patil, H.M. Paikrao, Efficient genomic DNA extraction protocol from medicinal rich Passiflora foetida containing high level of polysaccharide and polyphenol, Springerplus. 3 (2014) 1-7.[11] J.H. Cota-Sánchez, K. Remarchuk, K. Ubayasena, Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue, Plant Molecular Biology Reporter. 24 (2006) 161.[12] J. Zhang, J.M. Stewart, Economical and rapid method for extracting cotton genomic DNA, Journal of Cotton Science. 4 (2000) 193-201.[13] T. Li, H. Pan, Y. Feng, H. Li, Y. Zhao, Bioactivity-guided isolation of anticancer constituents from Hedera nepalensis K. Koch, South African Journal of Botany. 100 (2015) 87-93.[14] L. Jafri, S. Saleem, N. Ullah, B. Mirza, In vitro assessment of antioxidant potential and determination of polyphenolic compounds of Hedera nepalensis K. Koch, Arabian Journal of Chemistry. 10 (2017) S3699-S3706.[15] B. Ahmad, N. Munir, S. Bashir, S. Azam, I. Khan, M. Ayub, Biological screening of Hedera nepalensis, Journal of Medicinal Plants Research. 6 (2012) 5250-5257.[16] K.H.E. Koch, Hortus Dendrologicus, F. Schneider & Co., Berlin, 1985, pp 250.[17] A. Rehder, New species, varieties and combinations from the herbarim and the collections of the Arnold arboretum, Journal of the Arnold Arboretum. 4 (1923) 250.  

scholarly journals Preservation and maceration of amazon açai leaflet tissue to obtain genomic DNA

2019 ◽  
Vol 35 (4) ◽  
Author(s):  
Hellen Sandra Freires da Silva Azêvedo ◽  
Polinar Bandeira Rufino ◽  
José Marlo Araújo de Azevedo ◽  
Luciélio Manoel da Silva ◽  
Lúcia Helena de Oliveira Wadt ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Silica Gel ◽  
Genomic Dna ◽  
Leaf Tissue ◽  
Dna Concentration ◽  
Randomized Design ◽  
Molecular Studies ◽  
Tissue Maceration ◽  
Genomic Regions ◽  
Transport Buffer

The objective of this study was to test the efficiency of preservation and maceration methods for Euterpe precatoria leaflet tissue to obtain genomic DNA for molecular studies. The leaflets of E. precatoria were collected in an experimental field at Embrapa Acre, Brazil. The study was conducted in a completely randomized design with 10 replicates, in a 12 × 2 factorial structure, with 12 storage treatments (fresh; lyophiliser 3 days; refrigerator 3, 5, and 7 days; silica gel 7, 10, 20, and 30 days; and transport buffer 3, 5, and 7 days) and two leaf tissue maceration methods (liquid nitrogen and the TissueLyser®). Statistically significant differences in the obtained DNA concentration were found between the maceration and storage treatments. The TissueLyser® macerator produced higher DNA concentrations when compared to liquid nitrogen. For the storage treatments, five groups were formed based on DNA concentration when macerated with the TissueLyser® and two groups when macerated with liquid nitrogen. The DNA concentrations ranged from 285.00 ng/µL (7 days in transport buffer) to 702.00 ng/µL (30 days in silica gel) when the leaflets were macerated with liquid nitrogen, and from 572.73 ng/µL (30 days in silica gel) to 2,850.00 ng/µL (3 days in lyophiliser) using the TissueLyser® macerator. The DNA purity (A260/A280 nm) varied from 1.30 to 1.70 when the leaflets were macerated with liquid nitrogen and from 1.30 to 1.90 with the TissueLyser® macerator. Despite the variations in leaf tissue preservation and DNA concentration, all treatments were effective for DNA isolation and it was possible to amplify genomic regions of microsatellite markers by PCR. It was concluded that leaflets of E. precatoria stored in a lyophiliser and processed with an automatic macerator resulted in satisfactory DNA for molecular studies.

scholarly journals Development of a High Quality, Rapid, Efficient and Economical DNA Extraction Protocol from Climate Resilient Pearl Millet Crop Without Liquid Nitrogen

2020 ◽  
pp. 85-94
Author(s):  
Supriya Ambawat ◽  
Subaran Singh ◽  
C. Tara Satyavathi ◽  
Rajbala Meena ◽  
R. C. Meena ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Dna Extraction ◽  
Pearl Millet ◽  
Genomic Dna ◽  
Sodium Dodecyl ◽  
Cost Effective ◽  
Proteinase K ◽  
High Yield ◽  
Pcr Analysis ◽  
Ammonium Bromide

Extraction of good quality genomic deoxyribonucleic acid (DNA) from plants is a major prerequisite for molecular biology experiments. An efficient genomic DNA protocol must be simple, fast and cost effective with high yield and purity. Presence of polyphenols, polysaccharides and secondary metabolites in some plants hamper with DNA extraction making it a very laborious, difficult and time consuming procedure. Here, we portrayed a modified protocol based on the cetyl trimethyl ammonium bromide (CTAB) method to isolate DNA from climate resilient pearl millet leaf tissues having higher amount of polysaccharides. It also excludes the use of expensive chemicals and equipments like proteinase K, liquid nitrogen and tissue lyser. This method includes extraction of DNA using a buffer (pH 8.0) containing 200 mM Tris-HCl, 20 mM ethylenediamine tetracetic acid (EDTA),1.4 M NaCl, 2% CTAB, 2% sodium dodecyl sulphate (SDS) and 1.0 % β-mercaptoethanol followed by purification of DNA with phenol, chloroform, isoamyl alcohol and finally precipitation of DNA by sodium acetate and isopropanol. Good quality genomic DNA with sharp and clear bands was obtained from 48 pearl millet genotypes using this protocol. The yield of DNA varied from 105.2 to 328.3 ng/μl. The purity of DNA sample ranged from 1.74 to 1.95 based on the absorbance at A260/A280 ratio indicating that it’s free from ribonucleic acid (RNA) and protein contamination. PCR analysis using simple sequence repeat (SSR) primers exhibited consistent and reliable amplification products ranging from 150 to 650 bp.This study reveals a fast, simple, efficient, specific, reproducible, reliable and cost effective method for extraction of DNA from small to large number of plant samples amenable to PCR amplification and could be stored for longer duration.

scholarly journals Plant Genomic DNA Extraction for Selected Herbs and Sequencing their Internal Transcribed Spacer Regions Amplified by Specific Primers

2016 ◽  
Vol 11 (10) ◽  
pp. 1934578X1601101
Author(s):  
Farah Izana Abdullah ◽  
Lee Suan Chua ◽  
Zaidah Rahmat ◽  
Azman Abd Samad ◽  
Alina Wagiran
Keyword(s):  
Dna Extraction ◽  
Plant Species ◽  
Dna Sequences ◽  
Incubation Time ◽  
Internal Transcribed Spacer ◽  
Genomic Dna ◽  
Molecular Technique ◽  
Hexadecyltrimethylammonium Bromide ◽  
Genomic Dna Extraction ◽  
Dna Concentration

This study was focused on plant genomic DNA extraction and sequencing from five commonly used medicinal herbs, namely Impatiens balsamina, Ficus deltoidea, Centella asiatica, Andrographis paniculata and Orthosiphon aristatus. This molecular technique is another highly reliable alternative for plant species identification besides phytochemical profiling. Three cetyl hexadecyltrimethylammonium bromide (CTAB) based methods with slight modification on incubation time, salt content and other additives were used for DNA extraction. The CTAB method of Doyle and Doyle produced higher DNA concentration from I. balsamina, most probably due to the presence of ammonium acetate in the washing buffer and longer incubation time (2 h). The CTAB based method was suitable for A. paniculata because a high DNA concentration of acceptable quality was obtained for all the modified methods. However, O. aristatus was likely to have a lower DNA concentration (33–87 μg/g) and quality, probably due to the high concentration of phenolic compounds, particularly rosmarinic acid. The extracted genomic DNA was effectively amplified by a polymerase chain reaction using a universal primer of internal transcribed spacer (ITS), particularly AB101 and AB102 at the optimum annealing temperature of 48°C. The DNA sequences were analyzed by phenetic analysis and it was found that they have high similarity with the nucleotide sequences of ITS regions for similar plant species in the GenBank database of the National Center for Biotechnology Information.

Genetic Diversity of Solidago canadensis Correlates Negatively with the Number of Buds and Positively with Species Number of Plant Community

2014 ◽  
Vol 955-959 ◽  
pp. 720-727
Author(s):  
Jing Jing Pan ◽  
Qian Qian Hu ◽  
Lin Yue Xiong ◽  
Ying Liang Wu ◽  
Zhi Ru Zhu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Plant Species ◽  
Plant Community ◽  
Genomic Dna ◽  
Diversity Index ◽  
Gene Diversity ◽  
Significant Negative Correlation ◽  
Species Number ◽  
Solidago Canadensis ◽  
Generative Organs

The genetic diversities of 5 populations of Solidago canadensis were studied using intersimple sequence repeat markers method (ISSR). Genomic DNA was extracted by a modified NaOH method from samples collected in Zejiang province, China. Meanwhile, the generative organs (buds and rhizomes) of S. canadensis and plant species number in its grown site were investigated. Our results indicated that among 5 populations, the polymorphic percentages ranged from 78.08 to 91.03. Neis gene diversity index and Shannon diversity index were more than 0.25 and 0.38 respectively. The 5 populations displayed some genetic differentiations (Gst=0.3208), showing Jiaxing population and Zhoushan population in one group, while Hangzhou population, Wenzhou population and Quzhou population in another group. We found that the number of buds varied greatly among the 5 populations, genetic diversity of S. canadensis displayed significant negative correlation with the number of buds and significant positive correlation with plant species number in its grown site. S. canadensis population has higher genetic diversity index and less buds, when grown with more other species, than with less other species. The result implied that increased competition from other species of plant community could reduce fecundity of S. canadensis.

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