Extraction optimization and amplification of oil palm DNA: leaves at different phases of development
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Ciência Florestal (Online) |
Texto Completo: | https://periodicos.ufsm.br/cienciaflorestal/article/view/39043 |
Resumo: | The objective of this study was to optimize and establish a protocol to enhance the quality and quantity of deoxyribonucleic acid (DNA) extracted from leaves under different development stages and conservation conditions collected from mature plants and seedlings. Furthermore, we aimed to standardize the polymerase chain reaction (PCR) and select an inter simple sequence repeat (ISSR) marker for Elaeis guineensis (oil palm). The modified cetyltrimethylammonium bromide (CTAB) protocol, which used β-mercaptoethanol (0.3%) and polyvinylpyrrolidone (PVP 3%) supplementation in the extraction buffer and 10 % CTAB with 1.4 M NaCl for a 20-min incubation at 65ºC, resulted in improved DNA quality and quantity. However, this protocol presented variable results among samples, probably due to variations in leaf degradation levels and development stages. The two PCR protocols (I and II) for amplifying the DNA, differed mainly in the presence or absence of bovine serum albumin (BSA) and primer concentration. Although, a correlation between PCR amplification and the quality of the DNA extracted from leaves was not established, the addition of BSA (0.075 mg/mL) and the highest primer concentration (0.5 pmol) (protocol II) resulted in more intense and distinguishable bands on gel electrophoresis. DNA quality was essential for a satisfying amplification, considering all the samples. The use of protocol II allowed the selection of five primers: UBC 807, 810, 812, 834, and 848; these were used in the amplification of the DNA extracted from 13 families consisting of one parental plant and eight progenies each. |
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Extraction optimization and amplification of oil palm DNA: leaves at different phases of developmentOtimização da extração e amplificação de DNA de dendezeiro: folhas em diferentes fases de desenvolvimentoElaeis guineensisPCRISSRElaeis guineensisPCRISSRThe objective of this study was to optimize and establish a protocol to enhance the quality and quantity of deoxyribonucleic acid (DNA) extracted from leaves under different development stages and conservation conditions collected from mature plants and seedlings. Furthermore, we aimed to standardize the polymerase chain reaction (PCR) and select an inter simple sequence repeat (ISSR) marker for Elaeis guineensis (oil palm). The modified cetyltrimethylammonium bromide (CTAB) protocol, which used β-mercaptoethanol (0.3%) and polyvinylpyrrolidone (PVP 3%) supplementation in the extraction buffer and 10 % CTAB with 1.4 M NaCl for a 20-min incubation at 65ºC, resulted in improved DNA quality and quantity. However, this protocol presented variable results among samples, probably due to variations in leaf degradation levels and development stages. The two PCR protocols (I and II) for amplifying the DNA, differed mainly in the presence or absence of bovine serum albumin (BSA) and primer concentration. Although, a correlation between PCR amplification and the quality of the DNA extracted from leaves was not established, the addition of BSA (0.075 mg/mL) and the highest primer concentration (0.5 pmol) (protocol II) resulted in more intense and distinguishable bands on gel electrophoresis. DNA quality was essential for a satisfying amplification, considering all the samples. The use of protocol II allowed the selection of five primers: UBC 807, 810, 812, 834, and 848; these were used in the amplification of the DNA extracted from 13 families consisting of one parental plant and eight progenies each.O objetivo deste estudo foi otimizar e estabelecer um protocolo visando elevar a qualidade e quantidade do ácido desoxirribonucleico (DNA) extraído de folhas, em diferentes estágios de desenvolvimento e conservação, coletadas em plantas adultas e plantas jovens. Além disso, almejou-se padronizar a amplificação via reação de polimerase em cadeia (PCR) e selecionar os marcadores de inter sequência simples repetida (ISSR) de Elaeis guineensis (dendezeiro). O protocolo brometo de cetiltrimetilamônio (CTAB) modificado, devido à suplementação com betamercapto etanol (0,3%) e polivinilpirrolidone (PVP) (3%) no tampão de extração e CTAB 10% com 1,4 M de NaCl a 20 min de incubação, a 650C, resultou em melhorias qualitativas e quantitativas na extração do DNA, mas com variações entre amostras, provavelmente, devido às variações na degradação e estágio de desenvolvimento das folhas. Foram utilizados dois protocolos de PCR (I e II) para a amplificação do DNA, os quais diferiram principalmente com relação à presença de albumina de soro bovino (BSA) e concentração de primer. Não foi realizada a correlação entre amplificação via PCR e qualidade de DNA, obtidos de folhas danificadas ou sadias, mas se observou que a adição de BSA (0,075 mg/mL) e o aumento na concentração de primer (0,5 pcmol) (protocolo II) resultou em bandas mais intensas e distinguíveis, porém se ressalta que a qualidade do DNA foi essencial para uma boa amplificação, considerando-se todas as amostras. A amplificação do DNA com o protocolo II possibilitou a seleção de cinco primers: UBC 807, 810, 812, 834 e 848; os quais foram utilizados para a amplificação de13 famílias, compostas de uma planta parental e oito progênies cada.Universidade Federal de Santa Maria2020-09-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://periodicos.ufsm.br/cienciaflorestal/article/view/3904310.5902/1980509839043Ciência Florestal; Vol. 30 No. 3 (2020); 916-926Ciência Florestal; v. 30 n. 3 (2020); 916-9261980-50980103-9954reponame:Ciência Florestal (Online)instname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMporhttps://periodicos.ufsm.br/cienciaflorestal/article/view/39043/33594Copyright (c) 2020 Ciência Florestalinfo:eu-repo/semantics/openAccessBomfim, João Pedro de AndradeLima, Laiana Pinheiro deMelo, Claúsio Antônio Ferreira deCôrrea, Ronan XavierGaiotto, Fernanda AmatoBarbosa, Antônia Marlene Magalhães2020-09-02T20:52:06Zoai:ojs.pkp.sfu.ca:article/39043Revistahttp://www.ufsm.br/cienciaflorestal/ONGhttps://old.scielo.br/oai/scielo-oai.php||cienciaflorestal@ufsm.br|| cienciaflorestal@gmail.com|| cf@smail.ufsm.br1980-50980103-9954opendoar:2020-09-02T20:52:06Ciência Florestal (Online) - Universidade Federal de Santa Maria (UFSM)false |
dc.title.none.fl_str_mv |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development Otimização da extração e amplificação de DNA de dendezeiro: folhas em diferentes fases de desenvolvimento |
title |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development |
spellingShingle |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development Bomfim, João Pedro de Andrade Elaeis guineensis PCR ISSR Elaeis guineensis PCR ISSR |
title_short |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development |
title_full |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development |
title_fullStr |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development |
title_full_unstemmed |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development |
title_sort |
Extraction optimization and amplification of oil palm DNA: leaves at different phases of development |
author |
Bomfim, João Pedro de Andrade |
author_facet |
Bomfim, João Pedro de Andrade Lima, Laiana Pinheiro de Melo, Claúsio Antônio Ferreira de Côrrea, Ronan Xavier Gaiotto, Fernanda Amato Barbosa, Antônia Marlene Magalhães |
author_role |
author |
author2 |
Lima, Laiana Pinheiro de Melo, Claúsio Antônio Ferreira de Côrrea, Ronan Xavier Gaiotto, Fernanda Amato Barbosa, Antônia Marlene Magalhães |
author2_role |
author author author author author |
dc.contributor.author.fl_str_mv |
Bomfim, João Pedro de Andrade Lima, Laiana Pinheiro de Melo, Claúsio Antônio Ferreira de Côrrea, Ronan Xavier Gaiotto, Fernanda Amato Barbosa, Antônia Marlene Magalhães |
dc.subject.por.fl_str_mv |
Elaeis guineensis PCR ISSR Elaeis guineensis PCR ISSR |
topic |
Elaeis guineensis PCR ISSR Elaeis guineensis PCR ISSR |
description |
The objective of this study was to optimize and establish a protocol to enhance the quality and quantity of deoxyribonucleic acid (DNA) extracted from leaves under different development stages and conservation conditions collected from mature plants and seedlings. Furthermore, we aimed to standardize the polymerase chain reaction (PCR) and select an inter simple sequence repeat (ISSR) marker for Elaeis guineensis (oil palm). The modified cetyltrimethylammonium bromide (CTAB) protocol, which used β-mercaptoethanol (0.3%) and polyvinylpyrrolidone (PVP 3%) supplementation in the extraction buffer and 10 % CTAB with 1.4 M NaCl for a 20-min incubation at 65ºC, resulted in improved DNA quality and quantity. However, this protocol presented variable results among samples, probably due to variations in leaf degradation levels and development stages. The two PCR protocols (I and II) for amplifying the DNA, differed mainly in the presence or absence of bovine serum albumin (BSA) and primer concentration. Although, a correlation between PCR amplification and the quality of the DNA extracted from leaves was not established, the addition of BSA (0.075 mg/mL) and the highest primer concentration (0.5 pmol) (protocol II) resulted in more intense and distinguishable bands on gel electrophoresis. DNA quality was essential for a satisfying amplification, considering all the samples. The use of protocol II allowed the selection of five primers: UBC 807, 810, 812, 834, and 848; these were used in the amplification of the DNA extracted from 13 families consisting of one parental plant and eight progenies each. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-09-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://periodicos.ufsm.br/cienciaflorestal/article/view/39043 10.5902/1980509839043 |
url |
https://periodicos.ufsm.br/cienciaflorestal/article/view/39043 |
identifier_str_mv |
10.5902/1980509839043 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
https://periodicos.ufsm.br/cienciaflorestal/article/view/39043/33594 |
dc.rights.driver.fl_str_mv |
Copyright (c) 2020 Ciência Florestal info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Copyright (c) 2020 Ciência Florestal |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria |
dc.source.none.fl_str_mv |
Ciência Florestal; Vol. 30 No. 3 (2020); 916-926 Ciência Florestal; v. 30 n. 3 (2020); 916-926 1980-5098 0103-9954 reponame:Ciência Florestal (Online) instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Ciência Florestal (Online) |
collection |
Ciência Florestal (Online) |
repository.name.fl_str_mv |
Ciência Florestal (Online) - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
||cienciaflorestal@ufsm.br|| cienciaflorestal@gmail.com|| cf@smail.ufsm.br |
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1799944135074906112 |