Improved method for genomic DNA extraction for Opuntia Mill. (Cactaceae)

Background Genomic DNA extracted from species of Cactaceae is often contaminated with significant amounts of mucilage and pectin. Pectin is one of the main components of cellular walls, whereas mucilage is a complex polysaccharide with a ramified structure. Thus, pectin- and mucilage-free extraction of DNA is a key step for further downstream PCR-based analyses. Results We tested our DNA extraction method on cladode tissue (juvenile, adult, and herbaria exemplars) of 17 species of Opuntia Mill., which are characterized by a large quantity of pectin and mucilage. Conclusion We developed a method for the extraction of gDNA free of inhibitory compounds common in species of Opuntia Mill., such as pectin and mucilage. Compared to previously extraction protocols, our method produced higher yields of high-quality genomic DNA.


Background
Present-day DNA-based molecular studies are useful tools with a wide-range of applications in different biological disciplines. Molecular studies, especially in species with similar morphologies, can be used to characterize and differentiate species [1,2]. Such studies have used molecular techniques involving PCR amplification of DNA [3,4] to successfully solve taxonomic and phylogenetic controversies [5]. More specifically, DNA analyses have been used at different taxonomic levels, from communities of bacteria, fungi, yeast, plants and animals, to the cloning of specific genes [6]. High-quality DNA extraction is a necessary first step to conduct molecular studies. This can be performed using conventional methods or commercial kits specifically designed for particular types of samples. Most commercial kits efficiently capture DNA using extraction columns and resins, but the cost of these kits limits their application to large numbers of samples [7].
In these species, pectin is the main component of the cellular wall and its composition often varies among species (e.g., Opuntia), location and environments. The main molecular components of pectin are α-(1 → 4) chains linked to d-galacturonic acid interspersed by the insertion of (1 → 2) residues linked to adjacent or alternate residues of l-rhamnopyranosyl. The lineal segments are predominantly composed of homogalacturone [16].
Mucilage is an organic component present in large cells (idioblasts) in the chlorenchyma and adjacent water-retaining parenchymal cells [17,18]. Mucilage is composed of complex polysaccharides with ramified structures [16] containing varying proportions of different sugars (e.g., l-arabinose, pyranose, furanose, d-galactose, l-rhamnose and d-xylose) and galacturonic acid. The primary structure of the molecule consists of lineal repetitive chains of 1,4-β-d-galacturonic acid and α-1,2-l-rhamnose with a trisaccharide of β-1,6-d-glucose with a lateral chain joined to O-4-l-residues of rhamnose [19,20]. Mucilage is found throughout all body parts, including flowers [11]. In most species of cacti, mucilage is secreted in response to wounds and during the DNA extraction process. More specifically, during the DNA extraction process mucilage appears as soon as the tissue is pulverized, which significantly hinders the efficiency of the extraction and purification [21].
Generally, extraction and purification of high-quality genomic DNA (gDNA) is hindered by the presence of pectin that precipitates alongside DNA [22], thus reducing the quality and yield of the extraction process [23]. Although efficient DNA extraction is crucial for downstream PCR-based analyses, there are relatively few studies focusing on gDNA extraction efficiency in species of cacti [11,13,22,[24][25][26][27]. In this context, the aim of the present study was to develop a simple and cost-effective method to obtain large yields of high-quality gDNA from cladode tissue of Opuntia species.

Methods
We obtained tissues samples from the national Opuntia collection of the Botanical Garden at Instituto de Biología, Universidad Nacional Autónoma de México. and mix vigorously for 20 s. 6. Centrifuge for 60 min at 12,000 g (4 °C). 7. Transfer the aqueous phase to a 1.5 mL eppendorf tube. 8. Add 400 µl of isopropyl alcohol previously cooled to − 20 °C. Mix gently for 1 min. 9. Centrifuge for 25 min at 10,000 g. Discard the supernatant. 10. Add 500 µl HPLC-grade water to the DNA pellet to dissolve the pectin (evident as a gelatinous sub-stance). Do not mix and discard the disolved pectin with a micropipette. 11. Resuspend the pellet in 1 mL of ethanol (70) previously cooled to − 20 °C. 12. Centrifuge for 5 min at 10,000 g. Discard the supernatant. 13. Air-dry pellet at room temperature for 40 min. 14. Resuspend the pellet in 50 µl of HPLC-grade water. 15. Heat to 60 °C for 15 min.

Integrity of the extracted DNA
We analyzed the integrity of extracted gDNA from 17 species of Opuntia by electrophoresis (1 h with a 87 V cm −3 current) using 1.5 agarose gels prepared with TAE buffer (Tris Acetate-EDTA) and stained with Gel red (Biotium, USA). DNA bands were visualized under UV light with an Infinity 3000 transilluminator (Vilber Lourmat, Germany), which confirmed the presence of intact high quality gDNA without conspicuous contamination by proteins or other compounds ( Fig. 1).

Evaluation of gDNA concentration
We determined gDNA concentration with a spectrophotometry analysis using a NanoDrop 8000 (Thermo, USA) and with a fluorometry analysis using Quant-iT ™ PicoGreen ® dsDNA Assay Kit (Invitrogen ™ ) according to the manufacturer's instructions.

Evaluation of the quality of gDNA
We assessed the purity of all the gDNA samples by spectrophotometry with a Nanodrop 8000 (Thermo, USA) ( Table 1).

Sequence assembly
DNA sequences were visualized, edited and assembled using BioEdit vers. 7.0.5 [36]. For each gene, consensus sequences were compared with those deposited in Gen-Bank using the BLASTN 2.2.19 search algorithm [37].

Comparison with previous methods
Our protocol was compared with two previous methods [11,13] using 17 species of Opuntia.  Only one species (Opuntia ficus-indica) was shared with the protocol of Mondragón et al. [11].

Results
The list of the 17 species of Opuntia studied is shown in Table 1.
Our new extraction method allowed us to obtain high quality gDNA from young and mature cladodes using standard protocols using CTAB (Cetyl Trimethyl Ammonium Bromide), which efficiently extracts polysaccharides from leaf tissue. The Agarose gel electrophoresis showed the presence of large quantities of gDNA free of contaminants (Fig. 1). Accordingly, the large amount of gDNA was confirmed with two different methods (i.e., spectrophotometry and fluorimetry). These analyses yielded a mean gDNA ratio (PicoGreen concentration/ Nanodrop concentration) of 0.80-0.98 ng/µl for all of the samples tested (Table 1). We obtained reliable absorbance readings from the spectrophotometric analysis.
The estimation of the A 260 /A 280 absorbance ratio is a common way to measure DNA purity. Nucleic acids have a maximum absorbance at a wavelength of 260 nm, thus absorbance at this wavelength is directly proportional to DNA concentration. On the other hand, proteins show a maximum absorbance at 280 nm wavelength (mainly resulting from tryptophan residues), thus absorbance readings at 280 nm measure the concentration of proteins in the sample. Depending on the base composition of DNA, reading for the A 260 /A 280 ratio between 1.6 and 1.9 are indicative of high-quality DNA. In addition, absorbance readings at 230 nm wavelength measure the concentration of salts, carbohydrates and other contaminants, so the A 260 /A 230 absorbance ratio should also be considered. Both A 260 /A 280 and A 260 /A 230 absorbance ratios are typically used to determine the purity of DNA samples that were extracted using biological, organic and inorganic compounds. Sambrook et al. [8] suggested that when measuring pure double-stranded DNA, the A 260 / A 280 and A 260 /A 230 absorbance ratios should ideally be in the range of 1.6-1.9 and 2.0-2.2, respectively. Accordingly, our absorbance analysis for all samples yielded values for A 260 /A 280 and A 260 /A 230 within the ideal range (Table 1), which is indicative of high quality of the extracted gDNA.
PCRs of rbcL, cox3 and ITS regions were successful for all samples (Fig. 2). DNA sequencing for all three regions was successful (Fig. 3), which allowed us to construct high-quality consensus sequences for all three regions.
In order to complement sequence quality assessment, we decided to assess the identity of sequences, at least preliminary, with a basic BLAST search. It has been documented that BLAST is not the proper mean for taxonomical identification, but it provides an easy way to broadly verify if the sequence belongs to the sample (e.g., verifying a potential contamination).
We conducted a BLAST search for each of the 17 sequences and the first hit on each search was recorded ( Table 3). All the searches hit in sequences of Opuntia, but only five ITS sequences matched with the corresponding species. The other loci (rbcL and cox3) matched on Opuntia as well, but with non-corresponding species.
BLAST results on rbcL and cox3 are due to the fact that those loci have very low variability at species level. Sequence variability was not enough for proper species identity, but sufficient for genera identity.
On the other hand, ITS is a loci with larger variability at species level. We found five searches that matched with the corresponding species. At four searches, the corresponding species were not available in GenBank, and no correct match was possible, but the search hit in Opuntia. The remaining searches on the ITS sequences did not match on the correct species, but did match in Opuntia. This result is due to two main reasons: 1) the BLAST search is not designed for species match, even if the species are available in the database, and in consequence it is not a suitable tool for specimens identification; and 2) because in most cases our sequences are longer (including ITS1 and 2 as well as 5.8S region) than those available in GenBank; this extra length may induce some errors.

Comparison with previous methods
We replicated the protocols of Mondragón-Jacobo et al. [11] and Griffith and Porter [13] using the same 17 species of Opuntia (Table 4). We confirmed that our method got better performance (quality and quantity of gDNA), and that it has some advantages over other protocols (Table 5). In addition, our protocol is the cheapest one and considered as a micro-method due to the amounts of reagents and tissue involved.

Discussion
Several gDNA extraction protocols were developed recently, but few of these have been focused on the elimination of pectin and polysaccharides. These two compounds are among the most difficult contaminants to separate from the DNA [38] and significantly interfere with the activity of DNA polymerases. Therefore, the elimination of these compounds during the extraction of gDNA favors the efficiency of PCR amplification [39]. Pectin and mucilage (polysaccharides) are two of the main tissue components tissue in Opuntia. More specifically, pectin is the main component of the middle layer of cell walls and mucilage is one of the principal components of the parenchyma. Mondragón-Jacobo et al. [11] developed a DNA extraction method for several cacti species (e.g., Cleistocactus spp., Echinocereus spp., Nopalea spp., Opuntia spp., Stenocereus spp.). The amount of tissue used in this extraction protocol is species-dependent due to varying mucilage content among species. Griffith and Porter [13] extracted DNA from epidermal cells from several species of Austrocylindropuntia, Brasilopuntia, Consolea, Cumulopuntia, Cylindropuntia, Grusonia, Maihueniopsis, Miqueliopuntia, Nopalea, Opuntia, Pereskiopsis, Pterocactus, Tephrocactus and Tunilla. In recent years, Mihalte et al. [25] showed that the protocol of Pop et al. [30] yielded sufficient amounts of DNA from small amounts of tissue for species of Rebutia, Mediolobivia, Sulcorebutia and Aylostera. Accordingly, Yu et al. [26] introduced a protocol, similar to that of Pop et al. [30], for reliable DNA extraction from Hylocereus spp. Montiel et al. [27] used root tissue from Opuntia to extract DNA due to the difficulties encountered during extraction from cladode tissue. Wong et al. [22] developed a method to extract DNA from Hylocereus spp. Out of these studies, only those of De la Cruz et al. [10], Mondragón-Jacobo et al. [11], Griffith and Porter [13], Montiel et al. [27] and Fehlberg et al. [40] tested extraction efficiency on species of Opuntia.
Our improved gDNA extraction method is based on the protocols of Mondragón-Jacobo et al. [11] and Griffith and Porter [13]. We developed this method for the extraction of DNA from Opuntia cladodes, which contain large quantities of mucilage and pectin [20]. More specifically, improvements in the method involved changes to centrifugation and incubation steps (e.g., increased times and temperatures), the addition of water to remove pectin and the elimination of various reactive agents, such as polyvinylpyrrolidone (PVP), β-mercaptoethanol and protein and RNA degrading enzymes. The increased centrifugation times allowed for a better separation of gDNA from fiber cells and non-soluble cellular components, such as proteins. As pectin is watersoluble, the addition of water permitted the extraction of this compound, forming a gelatinous substance over the precipitated gDNA [41,42].
Generally, polyvinylpyrrolidone (PVP) is used to suppress polyphenolic oxidation during the extraction process [43]. However, PVP was not used because the main issue associated with DNA extraction from Opuntia samples is the presence of pectin and mucilage, and not of phenolic compounds.
The longer time of incubation at higher temperatures results in a more efficient denaturation of the proteins and enzymes found in tissue samples of Opuntia. Therefore, the extra step of incubation with proteinases is not needed.
The Β-mercaptoethanol inhibits the activity of DNAs and RNAs and thus protects gDNA from degradation. However, we do not use this compound in our extraction protocol because EDTA (contained in CTAB) forms a molecular complex with Mg2 + ions that prevents the functioning of DNAs [8]. In turn, we do not use RNAse because we included a final drying step for 40 min, followed by 15 min at 60 °C, that allows for the efficient degradation of RNA.
Ribonucleases (RNAses) are abundant in all biological and most of these are fairly stable and difficult to inactivate even when extraction reagents and materials have been autoclaved. Thus, when extracting RNA from biological samples RNAses should be eliminated rapidly with denaturing compounds [8]. The presence of RNA in the samples is controlled with the fluorimetry analysis using the Quant-iT ™ PicoGreen ® Kit (Invitrogen ™ ), which is an ultra-sensitive method for quantifying double-stranded DNA. The determination of absorbance at 260 nm wavelength is the commonly used technique for measuring the overall concentration of nucleic acids. However, absorbance measures have the main disadvantage of confounding the absorbance contribution of single-stranded nucleic acids, thus being unable to distinguish between DNA and RNA.     The purity of the extracted gDNA was confirmed by spectrophotometry. Generally, a higher A 260 /A 280 value is indicative of RNA contamination, whereas lower values are indicative of protein contamination. On the other hand, lower A 260 /A 230 values indicate the presence of phenolic compounds and carbohydrates, whereas higher values are usually associated with calibration errors [44]. The A 260 /A 280 and A 260 /A 230 ratios for dsDNA ideally range from 1.6 to 1.9 and from 2.0 to 2.2, respectively [8]. Our analyses showed A 260 /A 280 and A 260 /A 230 within these ideal ranges (Table 1), which confirm the purity of the gDNA samples. Through the improvement of DNA extraction protocols, we were able to improve the overall yield and purity of gDNA (1500-9147 ng/μl, Table 1) extracted from different species of Opuntia. In addition, with these changes, the extraction protocol becomes cheaper and the use of toxic reagents is diminished.
When we compared our method with other two previous protocols [11,13], we observed that the necessary amount of tissue in these two protocols is huge. Also, both methods need a great amount of expensive chemical reagents, making them impractical. With our new protocol, we obtained a higher DNA performance with high molecular weight (1500 ng/μl), and an average of the ratio A 260 /A 280 of 1.8.
Our protocol is a good alternative to these methods, since it requires milligrams of tissue and small volumes of reagents, facilitating the handling of a large number of samples. In short, our method is cheaper, quick and simple, and it does not need to carry out additional purification.

Conclusion
In this study, we developed a method of DNA extraction that yields high-quality gDNA free of inhibitory organic compounds common in species of Opuntia, such as pectin and mucilage. This improved method allowed us to obtained higher yields of gDNA of excellent quality. Our method works in other species of cacti (e.g., Nopalxochia [45]); it will be interesting to test it in other Cactaceae and succulent plants such as Crassulaceae. Finally, we are demonstrating that the addition of RNAses is not necessary to remove RNA from the genomic DNA samples. The use of RNAse is replaced by a heat treatment to remove the RNA making the protocol cheaper.