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\begin{document}

\title{Relieving efforts in palm-tree tissue sampling for population genetics
analyses.}



\author[1]{Sebastian Espinoza-Ulloa}%
\affil[1]{University of Saskatchewan}%


\vspace{-1em}



  
  \date{May 07, 2024}


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\selectlanguage{english}
\begin{abstract}
The young leaves are the main source of nucleic acids for population
genetic studies in palm-trees; however, the access to this tissue may be
limited by specific features of each species. Using root tissues as an
alternative source of nucleic acids could facilitate the sampling in
large populations. This study tests root tissue viability as an
alternative nucleic acids source (root vs. leaf) and explores different
protocols (tissue storage and DNA purification methods) to obtain
high-quality DNA samples. The results showed no significant differences
in DNA concentration and quality for the comparisons of tissue source
(leaf vs. root) and DNA purification method (manual vs. kit). For tissue
storage method, DNA concentration was significantly higher for root
tissues stored in 70\% and 90\% alcohol solutions versus those obtained
from leaf tissue, however for the quality parameters, no differences
were found. Results showed the effective potential of using root tissue
as an alternative source for nucleic acids, which could facilitate
populations sampling of palm-tree species for future studies and this
methodological alternative could be applied to other plant systems with
similar sampling challenges.%
\end{abstract}%



\sloppy


\textbf{Relieving efforts in palm-tree tissue sampling for population
genetics analyses.}

\textbf{Sebastian A. Espinoza-Ulloa\textsuperscript{1,2}}

\textsuperscript{1} Facultad de Medicina. Pontificia Universidad
Cat\selectlanguage{ngerman}ólica del Ecuador. Avenida 12 De Octubre 1076, EC 170143. Quito,
Ecuador.

\textsuperscript{2} Department of Biology. University of Saskatchewan.
Collaborative Science Research Building, 112 Science Place. S7N 5E2.
Saskatoon, SK. Canada.

Received:\_\_\_\_\_\_; Revised:\_\_\_\_\_\_; Accepted:\_\_\_\_\_\_.

\textbf{ABSTRACT}

\begin{enumerate}
\tightlist
\item
  The young leaves are the main source of nucleic acids for population
  genetic studies in palm-trees; however, the access to this tissue may
  be limited by specific features of each species. Using root tissues as
  an alternative source of nucleic acids could facilitate the sampling
  in large populations.
\item
  This study tests root tissue viability as an alternative nucleic acids
  source (root vs. leaf) and explores different protocols (tissue
  storage and DNA purification methods) to obtain high-quality DNA
  samples.
\item
  The results showed no significant differences in DNA concentration and
  quality for the comparisons of tissue source (leaf vs. root) and DNA
  purification method (manual vs. kit). For tissue storage method, DNA
  concentration was significantly higher for root tissues stored in 70\%
  and 90\% alcohol solutions versus those obtained from leaf tissue,
  however for the quality parameters, no differences were found.
\item
  Results showed the effective potential of using root tissue as an
  alternative source for nucleic acids, which could facilitate
  populations sampling of palm-tree species for future studies and this
  methodological alternative could be applied to other plant systems
  with similar sampling challenges.
\end{enumerate}

\textbf{KEYWORDS:}

Sampling methods, population genetics, palm-tree, root tissue, DNA
methods.

\textbf{INTRODUCTION}

For population genetics, the experimental design for plant tissue
sampling can be affected mainly by individuals' density, the proximity
between individuals, the topography, and the access to the tissue to be
sampled. Additionally, in palm-trees, the sampling of fresh tissue rich
in nucleic acids may be hindered by the specific features of each
species. Traditionally in plants, tissue samples with high
concentrations of nucleic acids are obtained from meristematic tissue of
new shoots or leaves (Edwards \emph{et al} ., 1991; Tai \& Tanksley,
1990; Tapia-Tussell \emph{et al} ., 2005). In palm-trees, new leaves
originate from meristematic tissue located in the center of the crown.
For most species, access to this tissue requires an extraordinary
sampling effort due mainly to stem height, stem modifications (spines or
prominent scars of old leaves) and/or even to high degrees of epiphytism
in addition to habitat factors which also can further increase sampling
efforts. Such increases in sampling effort often lead to modifications
in the experimental design in terms of the number of individuals sampled
or the time designated for it (Lowman \emph{et al} ., 1993).

Taking into account the problems that arise for several palm-tree
species, regarding the access to young leaves (or meristematic tissue
associated with the crown) and their possible negative implications on
the sampling effort for population genetics studies, it is thus
necessary to explore alternatives that reduce this effort. For instance,
in palms-trees, root tissue would be theoretically an ideal candidate to
obtain high concentrations of nucleic acids, but this tissue has not
been thoroughly tested with regard to DNA extractability, resulting DNA
quality and best extraction and tissue storage protocols. The root apex
is mainly formed by meristematic tissue that is in constant formation
and replacement. Therefore, it would be expected the terminal region of
the root contains high concentrations of nucleic acids mainly from
non-specialized cells (Broschat \& Donselman, 1984; Broschat \&
Donselman, 1990; Jouannic \emph{et al} ., 2011). This study aims to test
the use of root tissues as an alternative source of nucleic acids. It
thus also seeks to establish the best tissue storage and DNA
purification methodologies.

\textbf{MATERIALS AND METHODS}

Four different species of palm-trees (Arecaceae) were used:
1)\emph{Phoenix dactylifera} , 2) \emph{Ceroxylon equinulatum} ,
3)\emph{Ceroxylon ventricosum} and 4) \emph{Prestoea acuminata} . These
species are easily found in the Ecuadorian Andean urban and rural
landscapes (between 1,800 - 2,500 m.a.s.l.). All these species are
conspicuous stem palm-trees that commonly exceed 10 meters in height,
and their roots are placed underground.

For sampling, two individuals were selected per species. The tissue
collection for each individual was carried out 1) climbing along the
stem to obtain young leaf tissue, and 2) using a shovel to dig about 30
cm in the stem base to obtain root tissue (figure 1). Leaf tissue was
stored directly in a single bag with Silica Gel, following the most
commonly used protocol for leaf tissue sampling (control sample). For
root tissue sampling, eight roots per individual were pruned at 5 cm
from the tip (figure 1), and two pruned roots were placed into each
tissue storage treatment (T = 4). The storage treatments for root
tissues were: a) Silica Gel dehydration, b) distilled water, 3) 70\%
ethanol solution (EtOH 70\%), and d) 90\% ethanol solution (EtOH 90\%).
All samples were processed after two weeks of storage.

{[}Figure 1 about here{]}

Each stored tissue underwent two DNA extraction methods: i) DNA
extraction manual method for plant tissues based on Doyle \& Doyle
(1987, with modifications), and ii) commercial method using PureLink®
Plant Total DNA Purification Kit (Invitrogen \selectlanguage{english}). Each extraction started
with 100 mg of macerated tissue. The final DNA extraction solutions were
evaluated using NanoDrop\selectlanguage{english} 1000 Spectrophotometer (Thermo Fisher
Scientific). Three readings for each processed sample were taken.

DNA concentration and quality were compared between tissue sources,
storage treatments, and DNA extraction methods. The concentration was
measured as DNA nanograms contained in one microliter. The quality
parameters are based on the A260/A280 and A260/A230 ratios, where A260
is the absorbance at 260 nm that would be marking the presence of
aromatic bases (nucleotides/DNA/RNA), A280 is the absorbance at 280 nm
that would recognize contaminants such as proteins and phenolic
compounds, and A230 would identify residual contaminants of many organic
compounds such as phenol, TRIzol, salts, among others. In this way, the
A260/A280 ratio would be linked to factors related to the nature and
processing of the tissue, while the A260/A230 ratio would be linked to
the reagents and residues produced by the extraction protocol used
(Matlock, 2015).

Finally, as a positive-negative test, a PCR test was performed with two
microsatellite loci developed for \emph{Oenocarpus bataua}
(\emph{Ob01}and \emph{OB11} ; Montufar \emph{et al} ., 2007) that had
previously been tested and had positive results for all species used in
this study. The amplification parameters for these loci were the same
described in the source paper.

\textbf{RESULTS}

For all the samples used, positive results were obtained for DNA
extraction. The results for the concentration showed relatively high
values for all samples, getting more than 95\% of the samples above
100~ng/\selectlanguage{greek}µ\selectlanguage{english}l. The leaf tissue mean DNA concentration was 603.71~ng/\selectlanguage{greek}µ\selectlanguage{english}l,
while for root tissue was 599.12~ng/\selectlanguage{greek}µ\selectlanguage{english}l. For quality, the average for
leaf was 2.06 for A260/280 ratio and 2.08 for A260/230 ratio, while for
root was 1.91 and 2.03 respectively (Supp. table 1).

Regarding the storage treatments, the leaf DNA concentration (603.71
ng/\selectlanguage{greek}µ\selectlanguage{english}l) was overcome by all liquid storage treatments for roots: EtOH
90\% (822.57 ng/\selectlanguage{greek}µ\selectlanguage{english}l), EtOH 70\% (692.84 ng/\selectlanguage{greek}µ\selectlanguage{english}l), and distilled water
(631.34 ng/\selectlanguage{greek}µ\selectlanguage{english}l; Supp. table 1; figure 2). On the other hand, for quality,
the highest values are for distilled water storage treatment, where the
A260/280 ratio was 1.97 and the A260/230 ratio was 2.09, while in leaf
they were 2.06 and 2.07 respectively (Supp. table 1; figure 2).

{[}Figure 2 about here{]}

The statistical comparison by ANOVA found no significant differences for
DNA concentration between leaf and root tissues (\emph{p} = 0.927; Supp.
table 2). However, the analysis showed significant differences for DNA
concentration between species and storage treatments, also the
comparison between extraction methods is at the edge of significance
(\emph{p} = 0.050; Supp. table 2). Regarding the A260/280 ratio, there
was a significant difference between species, while for tissues the
probability was very close to the significance limit (\emph{p} = 0.053).
Finally, for the A260/230 ratio no significant differences were found
for all comparisons carried out (Supp. table 2).

As a final result, although no statistical tests were performed, the PCR
test for all samples obtained was positive, showing that the DNA
solutions obtained did not contain important reaction inhibitors.

\textbf{DISCUSSION}

Tissues with high cell division rates are the best option in order to
get a DNA sample of good concentration and quality, mainly due to the
high amount of cells and high replication activity. In plants, this kind
of tissues are found in germ buds (meristems), which give rise to newly
forming plant organs. In palm-trees, meristematic tissues, in general,
are only found in the crown and roots (Arif \emph{et al} ., 2010;
Broschat \& Donselman, 1990). Traditionally, tissue sampling for DNA
extraction is obtained from young leaves (that only are found in the
crown); however, access can sometimes be quite complicated which could
affect negatively the number of sampled individuals (Ihase \emph{et al}
., 2016). Moreover, easier access to a meristematic tissue for sampling
palm-trees would be found in their roots, where is expected to get
similar DNA concentration and quality as is obtained from leaves
(Broschat \& Donselman, 1984; Jouannic \emph{et al} ., 2011). The
results of this study have shown that obtain DNA from root tissue is not
only viable, but that the DNA concentration can be higher and its
quality similar to what was obtained from leaf tissue as traditional
methodology.

Regarding the source tissue, no differences for DNA concentrations were
found between the overall root results with leaf tissue results.
However, for quality, the A260/280 ratio borders the limit of
significance. This almost significant difference may be explained by the
higher amount of contaminants (mainly proteins) linked to the root
tissue. In most monocotyledons, the roots are constantly growing and to
maintain their structure underground, the tissues need to be constituted
by higher concentrations of lignin (and other related proteins) compared
to the leaves (Abiven \emph{et al} ., 2011; Hans-Walter \& Piechulla,
2011; Merewitz \emph{et al} ., 2011). For this reason, it would be
expected that the DNA extraction quality from the root is somewhat lower
than that was obtained from the leaf tissue. However, the quality could
be improved with standardized storage condition and adjusting the
extraction protocol.

Regarding the root storage treatments, the general comparison showed
significant differences for DNA concentration, while for both quality
ratios there were no differences. This would be explained by the degree
of stabilization and fixation of nucleic acids that would result from
the different treatments used. Within treatments, the most satisfactory
outcomes for DNA concentration were yielded by both ethanol solutions
(70\% and 90\%). However, a specific comparison between leaf tissue and
root tissue stored in ethanol 90\%, showed a significant higher DNA
concentration recovered from the root. Ethanol is a tissue fixative,
since it dehydrates the tissue violently (exchanging the water from the
sample) allowing to maintain the cellular structure and precipitate the
nucleic acids, in consequence, higher concentration of ethanol allows to
recover more DNA (Bressan \emph{et al} ., 2014). On the other hand, the
least effective result was that obtained with Silica Gel. Silica Gel
salt works by dehydrating the tissue to fix the cells and all their
components, and it has been a great tool for the fixation of leaf
tissues in plants. However, its low effectiveness in root DNA extraction
could be explained by the speed of tissue dehydration. Leaf tissue is
arranged in layers forming a single almost regular structure, while the
root tissue sample is an irregular and cylindrical structure. Therefore,
the root sample will take more time for dehydration, which would allow
the breakdown of nucleic acids (Arif \emph{et al} ., 2010;
Bressan\emph{et al} ., 2014; Jouannic \emph{et al} ., 2011; Tai \&
Tanksley, 1990).

Finally, the comparison between species demonstrated significant
differences for concentration, as well as for quality (A260/280), which
would be explained by the intrinsic characteristics of each species.
Tissues of each species will differ in their shape, composition and
cellular structure; therefore, it is understandable that a specific
quantity and quality of DNA is recovered from each species according to
the specific nature of their tissues (Abiven \emph{et al} ., 2011;
Tapia-Tussell \emph{et al} ., 2005).

In conclusion, this short experiment proved that it is possible to
obtain a ``good'' DNA sample from palm-tree root tissue. This fact could
facilitate palm-tree population sampling for large-scale studies by
reducing the sampling efforts. Additionally, the storage treatment
experiments provide a basis for development of an effective,
standardized protocol to obtain best DNA possible for further processing
(even for genomic analyses) despite of species or DNA extraction
(purification) method.

\textbf{FIGURES}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/377650/articles/494289-relieving-efforts-in-palm-tree-tissue-sampling-for-population-genetics-analyses}

Figure 1. A) For root sampling, the soil in the base of the palm-tree
stem was removed digging about 30 cm to expose the roots. B) The tissue
sampling consisted of prune the final 5 cm of the root and store it in
the different treatments.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 1. A) DNA concentration (ng/ul) means and their 95\% confidence
intervals for each tissue-treatment. B) Quality ratios, A260/280
(squares-lines) and A260/230 (circles-dots), for each tissue-treatment.
Tissue-treatments are set as follows: 1) Leaf-Silica Gel (n = 48), 2)
Root-Silica Gel (n = 48), 3) Root-Distillated Water (n = 48), 4)
Root-EtOH70\% (n = 48) and 5) Root-EtOH90\% (n = 48).

\textbf{ACKNOWLEDGMENTS}

This research was supported by the Pontificia Universidad Catolica del
Ecuador (PUCE). I would like to say thank you to this institution, and
specifically to Ph. D. Rommel Montufar, director of Molecular Ecology
Laboratory where this research was carried out. I thank my lab colleague
Jalil Maiguashca who provided insight and expertise that assisted the
development of this research. I thank my colleague Jana Ebersbach for
helping me with the checking and formatting of this document.

\textbf{DISCLOSURE STATEMENTS}

None. There is not a conflict of interest.

\textbf{DATA ACCESSIBILITY}

The data set as well as the detailed statistics output is available at:
https://github.com/Sebastardito/Palm-tree\_Root\_Sampling.git

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