Resumen
The objective of this project is to optimise the methodology for the extraction of High
Molecular Weight (HMW) DNA from plants collected in the wild. HMW DNA extraction has been
optimised for model plant species such as Arabidopsis thaliana and Oryza sativa, but it still presents
many challenges for non-model plants. Most plants are rich in polysaccharides and secondary
metabolites like polyphenols and tannins which impair DNA extraction. An alternative is to isolate
nuclei first, and then extract the DNA from the nuclei. Although several of these methods have been
proved to be effective with species like Gossypium hirsutum, Alopecurus myosuroides, and Fragaria x
ananassa, they are still limited to the use of young material that has not undergone any stresses1
. This
is unrealistic for samples collected in the wild. Nevertheless, there are an extensive number of
protocols focused on the isolation of nuclei for the genome size estimation of recalcitrant species that
could be helpful to overcome this problem.
We propose to apply a battery from five to ten different protocols of nuclei isolation3
(Galbraith's, LB01, Otto's, Tris-MgCl2, and TissueRuptor disruption with HB buffer or LN2 disruption)
and HMW DNA extraction (standard/modified CTAB, SILEX, and Nanobind plant nuclei kit) to five to
ten different plants species collected in the wild with different traits that make them recalcitrant for
DNA extractions (e.g., succulence, lignification, coloration¿). Samples of wild plants will be collected
in El Saler (Valencia). Potential species to be tested are: Pistacia lentiscus, Phillyrea angustifolia or
Sarcocornia fruticosa. Three different sample conservation methods will be added also to the different
extraction methodologies (ambient and cold temperature fresh, and ambient temperature with silica
gel desiccant). Solanum lycopersicum samples will be used as a control in all cases, as our laboratory
has a wide experience extracting HMW DNA from it. Each extraction will be repeated twice for a total
between 150 and 600 independent extractions.
The efficiency of nuclei isolation will be assessed by flow cytometry. Once the DNA has been
extracted, the quality of the DNA will be evaluated by three different methodologies: Spectrometric
(e.g., Nanodrop), Fluorometric (e.g., Qubit), and DNA fragment size analysis (e.g., using a Tapestation).
To identify the most important factors associated to the nuclei isolation as well as the DNA extraction,
we will analyse the different variables (e.g., sample traits, sample conservation, nuclei isolation buffer
components, HMW DNA extraction protocol¿) using a machine learning methodology such as random
forest and/or gradient boosting with cross validation. Once we have identified the best methodology
as well as the most important factors influencing the quality of the HMW DNA extraction, we will apply
it to 3-5 new samples to test the methodology. The final HMW DNA will be sent for sequencing to the
SCSIE-Secció de Genòmica at the Universitat de València for PacBio HiFi sequencing. Oxford Nanopore
sequencing will be performed in our laboratory with a MinION system.
The timeline for this project is: Sample collection (August-September, 2023), Nuclei isolation
and HMW DNA extractions (September-November, 2023; one methodology per week), analysis of the
results and DNA sequencing submission (December, 2023).
