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

Onkar Nath,Stephen J Fletcher,Rimjhim Agarwal,Robert J Henry,Neena Mitter,Alice Hayward,Lindsay M Shaw,Agnelo Furtado

doi:10.3390/plants11030242

Onkar Nath, Stephen J Fletcher + Show 6 more

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https://doi.org/10.3390/plants11030242

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Abstract

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.

Highlights

Deciphering molecular and cellular mechanisms using common molecular biology techniques, such as polymerase chain reaction (PCR), quantitative polymerase chain reaction, RNA-Seq (RNA sequencing), cDNA library synthesis, gene cloning and northern blot analysis requires the use of high-quality nucleic acids
DNA was extracted from the leaf tissue of 40 avocado cultivars representing the three horticultural groups of avocadoes, West Indian, Guatemalan and Mexican, which are known to vary widely in their traits and morphologies
The reported DNA extraction protocol was conceptualized based on previous extraction protocols by Singh et al [33], Zou et al [34], Xia et al [35] and Barbier et al [29] and optimised for avocado

Summary

Introduction

Deciphering molecular and cellular mechanisms using common molecular biology techniques, such as polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), RNA-Seq (RNA sequencing), cDNA (complementary DNA) library synthesis, gene cloning and northern blot analysis requires the use of high-quality nucleic acids. Long-read sequencing techniques require high-molecular weight, contaminantfree DNA for the generation of long contigs and full-length transcripts [1,2]. Avocado comprises three major horticultural groups, namely, Mexican, Guatemalan and. West Indian, corresponding to the three geographical areas of highland Mexico, highland. A Mexican and Guatemalan hybrid, remains the most important commercial variety [8]. With the rapid expansion of the avocado industry, advances in molecular biology and genomic resources are important for the improvement of varieties

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