Extracting DNA of nematodes communities from Argentine Pampas agricultural soils

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INTRODUCTION
Nematodes are an evolutionarily successful group of organisms and represent an important part of the soil microfauna that affect the soil microflora as they occupy positions at primary, secondary and/or tertiary consumer level in soil food webs (Moore and de Ruiter 1991).Despite the relevance of nematodes, the current adoption of this groups as a bioindicator is limited mainly due to difficulties in obtaining fine-scale taxonomic resolution (genus level or below) based on morphological traits, in addition to often being limited by systematic expertise (Yeates 2003).
Molecular assays utilizing the conserved 18S rDNA [small subunit: SSU] gene have been developed as an alternative to study the diversity of nematodes as a monitoring tool (Chen et al. 2010).Molecular analysis of nematode soil community DNA involves recovery and purification of nucleic acids, followed by PCR amplification.However, the key step is the efficient extraction of high-quality DNA that is representative of the nematode community and its variations within EDUARDO A. MONDINO et al. the environment.When DNA extraction is done on samples collected from soil it is difficult to completely remove the humus with a routine total DNA extraction procedure which can inhibit the activity of Taq DNA polymerase in the PCR reaction (Steffan et al. 1988).Although DNA extraction kits from soil samples are available and successful, the few milligrams of soil that are required for DNA extraction may not be representative of the environmental diversity of nematodes in the sample.Our objective was to asses a reliable protocol for DNA extraction of nematodes communities from agricultural environmental samples.

STUDY AREA, SOIL COLLECTION AND NEMATODE EXTRACTION
Fifteen surface soil samples selected to obtain a wide range in agricultural management and climatic seasons were used in the development of the method (Table I).Soil samples were taken with a soil sampler (2.5 inner diameter x 20 cm long) from the top 20 cm from a long-term crop-pasture rotation experiment at the INTA-FCA, UNMdP Balcarce station, Argentina (37°45' S, 58°18' W; 130 m above sea level; 870-mm mean annual rainfall; 13.7°C mean annual temperature).Soil was a complex of Typic Argiudoll and Petrocalcic Paleudoll soils, pH 5.7 in water, loamy texture, 33.1 cmol kg -1 cation exchange capacity, and 5.0 mg kg -1 Bray and Kurtz P, organic matter content of 62.0 g kg -1 ).
Field-crop management conditions where the soil was collected in winter, summer and spring (w, u and s, respectively) were: Pasture (P), Croppasture rotation with conventional tillage -CT-(Cpct), Crop-pasture rotation with no tillage -NT-(Cpnt), Crop rotation (30 years) with CT (Cct), Crop rotation (30 years) with NT (Cnt) (Table I).Eight sub-samples were collected from each experimental unit (5 x 25m).These were thoroughly mixed to obtain a single composite sample of approximately 1kg.Nematodes were extracted from 100g of fresh soil from each sample by the centrifugal-flotation method (Caveness and Jensen 1955) and placed in 25ml containers (4°C until use 24h later).

DNA EXTRACTION OF THE SOIL NEMATODE COMMUNITY
Each sample was homogenized with a magnetic shaker (2 min) and immediately 4 subsamples of 5 ml each were collected.Nematodes of each subsample were individually collected (at least 20 nematodes) with the help of an "L"-shaped needle and placed in 1.5 ml Eppendorf tubes with 100 μl of milliQ autoclaved water and stored at -20 °C until use.Each subsample was processed according to one of four DNA extraction methods as described.
SODIUM DODECYL SULFATE.Nematodes were crushed with a sterile micropestle in an ice bath in 500 μL of SDS (1% sodium dodecyl sulfate), 50 mM EDTA, 100 mM NaCl, 100 μg K proteinase ml -1 (Invitrogen), 1% β-mercaptoethanol (Sigma) and 100 mM Tris-HCl pH 8.5 following Donn et al. (2008).The material was frozen in liquid nitrogen, followed by thawing and heating to 60 °C for 30 min.The supernatant was transferred to a fresh tube, and subjected to phenol:chloroform:isoamyl alcohol (25:24:1, pH=8) extraction, with a subsequent chloroform extraction.The DNA was then precipitated (cold ethanol and 1/10 volume sodium acetate) and the resulting pellets were washed twice with 70% cold ethanol, dried, resuspended in 50 μl PCR-quality water and stored at -20 °C until use.
CHELEX RESIN.Nematodes were crushed in 240 μl TE buffer with 160 μl of 20% Chelex 100 (Bio-Rad Laboratories, Hercules, Calif.), with a sterile micropestle in an ice bath as a modification of the method described by Stock (2009)  The DNA concentration was quantified in all extracts by measuring absorbance at 260 nm with the Epoch (Biotech) spectrometer.The quality of the extract was also assessed by the 260/280 nm absorbance ratio (Sambrook et al. 1989).All experimental samples were subjected to PCR before any purification, to establish whether a purification step was essential for the successful amplification of gDNA.
When the PCR amplification failed, the extracts were purified with the Wizard ® SV Gel and PCR Clean-Up System and each purified DNA sample was then subjected to PCR as described above.Agarose gels were stained with GelRed ® and visualized with UV transillumination.

RESULTS AND DISCUSSION
The disruption of nematodes in TE buffer, in SDS, and in Chelex Resin methods, failed in the subsequent PCR amplification, even though the DNA samples were purified with the Wizard® kit.The validity of using molecular techniques for environmental studies depends on obtaining representative extracts of nucleic acids from an environmental sample.The DNA extraction protocols used for soils usually include from one to all three of the following basic elements: physical disruption, chemical lysis, and enzymatic lysis (Sambrook et al. 1989).In our experiment, we performed the same physical disruption of nematodes but we changed the buffer for chemical extraction and/or enzymatic lysis.Nucleic acid extraction methods are often inefficient because of coextraction of enzymatic inhibitors from the soil together with the DNA.
Even though direct lysis techniques have been used frequently because they yield more DNA, in our study this did not provide the expected successful result.The major drawback of direct lysis methods is that more substances PCR-inhibitory of the Taq polimerase are extracted along with the DNA (Leff et al. 1995).Although we obtained DNA product after direct physical disruption in TE buffer we found no visible PCR products.The efficacy of diverse chemical lysis components remains largely unknown.Donn et al. (2008) reported that DNA extraction of nematodes from environmental samples with lysis mixtures containing the detergent SDS yielded both quantifiable DNA and PCR product.However, in our experiment, the SDS procedure resulted in DNA product but failed to obtain a nematode DNA extract that could be amplified by PCR.We made modifications to the basic chemical lysis techniques, which included high-temperature (90 °C to boiling) incubation (Stock 2009) and incorporation of the chelating agent Chelex resin, to inhibit nucleases and 695 DNA EXTRACTION METHOD OF NEMATODES disperse soil particles (Jacobsen and Rasmussen 1992).However, this method also completely failed and no PCR products were obtained after direct amplification or amplification after purification.
The CTAB/phenol chloroform extraction was the only DNA extraction method that provided positive PCR amplicons of 18S rDNA.The PCR amplifications using the primer pairs NEMF1/S3 and NS1/NS4 resulted in expected fragments of about 700 bp and 1,000 bp, respectively (Figs. 1  and 2).The NEMF1 primer, targets the variable V3 and V5 regions of the 18S rDNA and is widely used in molecular-biodiversity studies of nematodes; S3 is a "universal" primer for eukaryotes that targets with highly a conserved region of the 18S rDNA (Waite et al. 2003).The NS1 and NS4 hybridized with highly-conserved regions of the eukaryote 18S rDNA molecule (White et al. 1990).
CTAB is known to help remove humus materials from most soils that contain high concentrations of humus acids which inhibit PCR enzymes.It has been reported that phenol/chloroform helps deproteinize the homogenate and remove a good deal of the humus and pigmented components  of soil (Schneegurt et al. 2003).In our experiment, the DNA extracts obtained by the other (TE, SDS and Chelex) methods were highly pigmented (data not shown), but the extract obtained by the CTAB method combined with phenol/chloroform/isoamyl alcohol was clear and free of pigments.
The concentration of DNA of extracts from the CTAB/phenol chloroform method, ranged from about 20 ng µl -1 to 80 ng µl -1 (Table I).This could probably be associated with both, the variable abundance of nematodes communities in a sample and to the different contribution of DNA associated to the size of nematodes in each condition/station of sample collection.This wide range of DNA yield confirms the efficiency of the CTAB/phenol chloroform extraction method for a variable abundance of DNA in environmental samples.The highest purity of the extracted DNA was recorded in samples in which extraction was performed by the CTAB/phenol chloroform method (with values of absorbance A260/A280 ratio, close and higher to 1,5, Table I).While other tested methods of extracting DNA resulted in high yield, the quality of the extractions was lower than those obtained by the CTAB method (data not shown).Although the CTAB in the chemical lysis buffer and the proteinase K in the enzymatic lysis, combined by the phenol, chloroform and isoamyl alcohol extraction, was the most time-consuming method, it yielded both quantifiable DNA and amplifiable by PCR reaction.Used primers resulted in the expected amplicon`s sizes and served to test the success of the DNA extraction methods used.
In conclusion, we developed a simple and relatively affordable extraction method (CTAB combined with proteinase K and phenol-chloroformisoamyl alcohol) for the extraction of DNA of nematodes communities from agricultural soils.The proposed protocol was successful for all analyzed samples of nematodes which were representative of different agricultural management and environmental stations.This provides the opportunity for studying nematodes as indicators of disturbances associated with agricultural management.After the successful extraction of DNA from nematodes communities, fingerprinting strategies (such as DGGE, SSCP, t-RFLP, etc.) or cloning-sequencing, may help define the role of the communities of nematodes as bioindicators of edaphic disturbances.

Figure 2
Figure 2 -A representative sample of 18S rDNA PCR product by using the NS1/NS4 primer pair for each habitat and season.P = pasture; Cpnt = Crop-pasture rotation with no-tillage; Cpct = Crop-pasture rotation with conventional tillage; Cnt = Continuous cropping with no-tillage, Cct = Continuous cropping with conventional tillage; w = winter; s = spring; u = summer; M= 1 kb ladder (Invitrogen).
. The samples were then subjected to two cycles of DNA EXTRACTION METHOD OF NEMATODES