Research Article A simple and inexpensive modified CTAB and silica particles-based method for recalcitrant plant genomic DNA isolation

Huanca‐Mamani, Wilson; Choque-Ayaviri, E.; Cárdenas-Ninasivincha, S.

doi:10.4238/gmr18510

Cited by 3 publications

(1 citation statement)

References 23 publications

(29 reference statements)

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“…CTAB lysis buffers with high salt concentrations have been developed and regularly used for recalcitrant tree species, as most commercial kits produce very low yields, if any, in avocado [ 36 , 37 , 38 ]. We therefore used CTAB as a lysing agent in our buffer.…”

Section: Resultsmentioning

confidence: 99%

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado

Nath

Fletcher

Hayward

et al. 2022

Plants

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High-quality DNA and RNA forms the basis of genomic and genetic investigations. The extraction of DNA and RNA from woody trees, like avocado (Persea americana Mill.), is challenging due to compounds which interact with nucleic acids and influence separation. Previously reported methods of DNA and RNA extraction from avocado have issues of low yield, quality and applicability across different cultivars and tissue types. In the current study, methods have been optimised for high-quality DNA extraction from 40 avocado cultivars and RNA extraction from multiple tissue types, including roots, stem, leaves, flowers and fruits. The method is based on the modification of the cetyltrimethylammonium bromide buffer, centred around the specific optimisation of chemicals, such as sodium dodecyl sulphate, polyvinylpyrrolidone, sodium sulphite, polyethylene glycol and β-mercaptoethanol. The DNA extraction method yielded high-molecular weight DNA from the leaf tissue of 40 avocado cultivars belonging to Mexican, Guatemalan and West Indian avocado horticultural groups. The method was further optimised for RNA extraction from different avocado plant parts, enabling extraction using amounts as low as ~10 mg of starting material. The DNA and RNA extracted was successfully used for long- and short-read sequencing and gene expression analysis. The methods developed may also be applicable to other recalcitrant plant species.

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Section: Resultsmentioning

confidence: 99%

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado

Nath

Fletcher

Hayward

et al. 2022

Plants

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A DNA Extraction Method for Nondestructive Testing and Evaluation of Cotton Seeds (Gossypium L.)

Karaca,

Ince

2023

Biochem Genet

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Kernels of cotton provide lint and linter for textiles, oil and protein for food and feed. Cotton seed is formed following fertilization between an ovule and a pollen grain. The seed coat is maternal in origin, whereas the embryo and attached cotyledonary leaves are hybrids of parental lines. The extraction of genomic DNA from an ungerminated whole, a portion or mixed seeds are prerequisite in genetic and genomic studies of cotton. As far as our knowledge, there is only one method of nondescriptive DNA extraction from ungerminated cotton seeds without affecting the seed germination capability, but it has technical di culties and requires special equipment. Furthermore, the amount of DNA extracted using the published method is low and, therefore, it is only suitable for routine marker assisted selection studies. In this study, a modi ed CTAB DNA isolation protocol referred to as the CTAB-LiCl DNA extraction method was developed for single whole cotton seed, a portion of cotton seed and bulked cotton seeds. The CTAB-LiCl DNA extraction method was evaluated in ninety-six individuals of six different cotton cultivars along with two genetic standards of cotton, TM-1 (G. hirsutum L.), Pima 3-79 (G. barbadense L.), and several other plant species of different plant genera. Results revealed that this method produced high quality and amounts of DNA as con rmed by spectrophotometry, agarose gel, restriction enzyme digestion, polymerase chain reaction, and library production for next generation sequencing studies of whole genome bisul te sequencing. It does not require the use of liquid nitrogen, RNase, proteinase K, or betamercaptoethanol and can be completed in approximately 2 h. Small tissues of the chalaza ends of ungerminated cotton seeds could be used to obtain high quality and quantity of DNA ranging from 14 µg to 28 µg without affecting the seeds' germination ability, allowing marker-assisted selection before planting and owering.

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A DNA extraction method for nondestructive testing and evaluation of cotton seeds (Gossypium L.)

Karaca

İnce

2023

Preprint

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Kernels of cotton provide lint and linter for textiles, oil and protein for food and feed. Cotton seed is formed following fertilization between an ovule and a pollen grain. The seed coat is maternal in origin, whereas the embryo and attached cotyledonary leaves are hybrids of parental lines. The extraction of genomic DNA from an ungerminated whole, a portion or mixed seeds are prerequisite in genetic and genomic studies of cotton. As far as our knowledge, there is only one method of nondescriptive DNA extraction from ungerminated cotton seeds without affecting the seed germination capability, but it has technical difficulties and requires special equipment. Furthermore, the amount of DNA extracted using the published method is low and, therefore, it is only suitable for routine marker assisted selection studies. In this study, a modified CTAB DNA isolation protocol referred to as the CTAB-LiCl DNA extraction method was developed for single whole cotton seed, a portion of cotton seed and bulked cotton seeds. The CTAB-LiCl DNA extraction method was evaluated in ninety-six individuals of six different cotton cultivars along with two genetic standards of cotton, TM-1 (G. hirsutum L.), Pima 3–79 (G. barbadense L.), and several other plant species of different plant genera. Results revealed that this method produced high quality and amounts of DNA as confirmed by spectrophotometry, agarose gel, restriction enzyme digestion, polymerase chain reaction, and library production for next generation sequencing studies of whole genome bisulfite sequencing. It does not require the use of liquid nitrogen, RNase, proteinase K, or beta-mercaptoethanol and can be completed in approximately 2 h. Small tissues of the chalaza ends of ungerminated cotton seeds could be used to obtain high quality and quantity of DNA ranging from 14 µg to 28 µg without affecting the seeds’ germination ability, allowing marker-assisted selection before planting and flowering.

show abstract

Research Article A simple and inexpensive modified CTAB and silica particles-based method for recalcitrant plant genomic DNA isolation

Cited by 3 publications

References 23 publications

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado

A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado

A DNA Extraction Method for Nondestructive Testing and Evaluation of Cotton Seeds (Gossypium L.)

A DNA extraction method for nondestructive testing and evaluation of cotton seeds (Gossypium L.)

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