A modified CTAB method for high-molecular-weight DNA preparation from deep-sea holothurians

ABSTRACT With the features as global distribution and all depths dwelling, holothurians function an ideal model for the study of deep-sea adaptation. Acquisition of high-molecular-weight DNA is the basic requirement for molecular biology analyses. However, extraction of high-molecular-weight DNA from deep-sea holothurians is difficult by degradation and contamination. Here, we presented a modified CTAB (lysis buffer with cetyl trimethyl ammonium bromide) method and compared its extraction efficiency for deep-sea holothurians with other five common methods, i.e. the Tiangen Biotech kit, Qiagen kit, Omega Bio-tek kit, Sangon Biotech kit, and SDS (lysis buffer with sodium dodecyl sulfate). Based on the results of yield, integrity, and purity, we found that the modified CTAB method is more suitable to extract high-quality DNA for deep-sea holothurians, with a large amount of yield, minimized degradation, and higher purity. This study provides a high-quality DNA of deep-sea holothurians for genome sequencing and further studies.


Introduction
Holothurians are a morphologically diverse, species-rich, ecologically important and economically valued group of marine Echinodermata.They are also the ubiquitous benthic megafauna in the deep sea with global distribution and at all depths (Jamieson et al. 2011;Boulart et al. 2022;Pierrat et al. 2022).In recent decades, more images and specimens of deepsea holothurians have been shown to the world with the development of deep-sea exploration technology, especially Remotely Operated Vehicle (ROV) explorations, and manned submersibles.An increasing number of new holothurian species have been described from the deep sea, including dendrochirotid, elasipodid, persiculid, and apodid holothurians (Thandar 1998;Solís-Marín et al. 2004;Bohn 2005;Rogacheva 2012;Glover et al. 2016;Li et al. 2018;Xiao et al. 2018Xiao et al. , 2019;;Ogawa et al. 2020;Yu et al. 2022).However, the lack of genetic data was a common feature in deep-sea fauna study, this has severely hampered our understanding of DNA-supported taxonomic studies (Glover et al. 2016).As the powerful genetic tool for species identified, cytochrome oxidase Ι (COΙ) barcoding with low amplification success has exacerbated the problem of genetic data lacking in echinoderm species (Hoareau and

2010
).Many studies of deep-sea holothurians focused on their distribution, behavior, morphological description, genetic evolution, and systematic phylogeny based on nucleic acid fragments sequencing, mitogenomes, and a few on transcriptomes and genomes (Jamieson et al. 2011;Mu et al. 2018;Takano et al. 2019;Liao et al. 2020;Sun et al. 2021;Liu et al. 2021;Pierrat et al. 2022;Zhang et al. 2022).The holothurians could provide an opportunity to study the origin and evolution of deep-sea organisms and their adaptation due to their abundance in shallow waters and all kinds of deep-sea ecosystems (Jamieson et al. 2011;Thomas et al. 2020;Boulart et al. 2022;Pierrat et al. 2022).Thus, the extraction of highquality genomic DNA of holothurians is a crucial prerequisite for molecular research within this group of marine organisms.
Many marine invertebrates including holothurians are rich in mucopolysaccharides, collagen, and polyphenolics that are problematic during the isolation of genomic DNA (Sun et al. 2010;Wang et al. 2014;Angthong et al. 2020).To deal with these contamination problems, cationic detergents, such as CTAB are required in specialized protocols to isolate genomic DNA from molluscs, flatworms, cnidarians, and ctenophores (Stefanik et al. 2013).In addition, some commercial kits have been developed specifically to deal with this tissue, such as E.Z.N.A. Mollusc DNA Kit (Omega Bio-Tek, Inc., Georgia, USA) (Stefanik et al. 2013).SDS method has been used to produce large amounts of integral DNA from sea anemones, which avoids problems caused by the tissue consistency, the mucosal tissue, and the symbiotic zooxanthellae (Pinto et al. 2000;Stefanik et al. 2013;Attaran-Fariman and Javid 2015).This method has also been used to extract high-molecular-weight DNA from the somatic tissue of soft corals and could avoid problems associated with the presence of nucleases, pigments, and other secondary metabolites (Lohuis et al. 1990).
Although lyophilization was a viable option for shipping of biological tissues (Straube and Juen 2013;Damsteegt et al. 2016), immediate freezing at low temperatures, especially storage in liquid nitrogen, was still the ideal preservation method of tissue samples which can be obtained high-quality DNA (Hara et al. 2015;Puch-Hau et al. 2019;Soniat et al. 2021).DNA degradation in frozen tissues usually caused by thawing during sample preparation or DNA extraction.Contamination and degradation problems pose difficulties for molecular analyses of deep-sea holothurians, especially genomic analyses that required DNA with a large quantity, high integrity, and relative purity.In the present study, we improved the CTAB method for the genomic DNA extraction from deep-sea holothurians and compared its performance with some commonly used commercial kits (Sangon Biotech, Tiangen Biotech, Qiagen, Omega Bio-tek), and the SDS method from Green and Sambrook (2012).Deep-sea asteroids and shallow-water holothurians were also used for comparison.Our study demonstrates that the modified CTAB method can generate a larger amount of genomic DNA with higher integrity and purity for the deepsea holothurians, and other deep-sea echinoderms.

Sample collection
Deep-sea specimens of holothurians and asteroids in this study were collected by the human-occupied vehicle (HOV) Shenhaiyongshi from the deep-sea floor in the South China Sea and the Northwest Pacific Ocean in four dives (Figure 1, A1, Table 1).The arm lengths which start from central disk of asteroids were from 10 cm to 15 cm, and the sizes of deep-sea holothurians were from 15 cm to 30 cm.Specimens were dissected into small pieces of tissues, snap-frozen in liquid nitrogen, and stored at − 80°C until use.Shallow-water specimens of holothurians, with sizes of 10 cm, were collected from the intertidal zone of Sanya (18.2118°N, 109.4758°E).The fresh specimens were desensitized with ice-immersion baths for 3 h and dissected into small pieces of tissues.Then the tissues were frozen immediately in liquid nitrogen and stored at − 80°C until extracted.Detailed information on all the specimens used are shown in Figures 1, A1 and Table 1.

Isolation of DNA
In this study, we set three groups, DA including four deep-sea asteroids, DH including five deep-sea holothurians, and SH including two shallow-water holothurians.DNA extraction was performed by six methods (Figure 2), and the DNA quality was estimated by yield, integrity, and purity.The arms of asteroids were selected for DNA isolation in group DA, while body-wall tissues were chosen in groups DH and SH.
In general, all protocols of DNA isolation methods involve five steps, i.e. tissue lysis, purification, precipitation, rinsing, and elution.Before extraction, we prepared the samples for DNA extraction as follows.The clean tissues were selected and cut into small pieces with a scalpel.Then the tissues were pulverized into fine powder individually, through the use of a pre-chilled mortar and pestle with liquid nitrogen, and saved at − 80°C until the next step.The steps of sample preparation above were carried out carefully on dry ice to avoid DNA degradation.All reagents used in the DNA extraction process are listed in Table A1.The tissue weight of each sample used for DNA isolation was estimated as W t = W bt -W b , where W t is the weight of tissue, W b is the weight of the tube and lysis buffer, W bt is the weight of the tube, lysis buffer, and tissue powder.
The whole genomic DNA was extracted from the tissue powder using four commercial kits [Animals Genomic DNA Quick extraction Kit (Sangon Biotech, Shanghai, China), TIANamp Marine Animals DNA Kit (Tiangen Biotech, Beijing, China), QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), E.Z.N.A. Mollusc DNA Kit (Omega Bio-tek, Inc., Georgia, USA)] according to the manufacturer's instructions.
For the CTAB method, in the step of tissue lysis, CTAB Extraction buffer [100 mM Tris-HCl, pH9.5, 20 mM EDTA, 1.4 M NaCl, 2% (wt/vol) CTAB] was pre-heat at 65°C, pre-mixed with 1% polyvinylpyrrolidone (PVP) and 0.2% βmercaptoethanol (βME) just before extraction (Sun et al. 2010;Xin and Chen 2012;Costa et al. 2015;Jo et al. 2016Jo et al. , 2017)).Proteinase K was added to the lysis buffer until the final concentration of 1 mg mL −1 (Pinto et al. 2000).A low concentration of 20 mM EDTA, was prefer here, due to the similar density between high concentration of 100 mM EDTA and phenol that lead to difficult separation (Green and Sambrook 2012).500 μL of pre-mixed lysis buffer mentioned above was aliquoted into a 1.5 mL micro-centrifuge tube.Then approximately 0.1 to 0.3 g powder was added until the lysis was very viscous but still solubilized.At last, the tubes were incubated for 1 h at 65°C with mixing every 15  min.In the step of phase separation, an equal volume of Phenol/Chloroform/ Isoamyl alcohol (25:24:1) was added and mixed by gently inverting for a few minutes, then tubes were centrifuged at 12 000×g for 5 min at 4°C.The supernatant was pipetted off and transferred to a new tube respectively, an equal volume of Chloroform/Isoamyl alcohol (24:1) was added and mixed by gently inverting for a few minutes, and then tubes were centrifuged again at 12 000×g for 5 min at 4°C.In the step of precipitation, the supernatant was pipetted off and transferred to a new tube, two-volume of ethanol was added, and 3 M sodium acetate (pH5.2) was added until a final concentration of 0.3 M (Lohuis et al. 1990;Green and Sambrook 2012), then mixed by gently inverting and incubated at − 20°C for 1 h.After that, the tubes were centrifuged at 12 000×g for 5 min at 4°C, supernatant was drained off and the DNA pellet was collected.In the step of rinsing, 1 mL pre-chilled 70% (vol/vol) ethanol was added for resuspension and rinsing the DNA pellet, then the tubes were centrifuged at 12 000×g for 5 min at 4°C and 70% ethanol was discarded.The above rinsing step was repeated.Finally, DNA pellet was air-dried for 10 min at room temperature.In the step of dissolution, 100 μL pre-mixed buffer of TE buffer and RNase A, which a final concentration of 0.6 mg mL −1 (Pinto et al. 2000), was added to the sample and incubated at 37°C for 30 min in order to eliminate RNA molecules.
The SDS method was performed by the procedures described by Green and Sambrook (2012).For the tissue lysis, 500 μL pre-mix buffer of lysis buffer and proteinase K with the final concentration of 1 mg mL −1 (Pinto et al. 2000) was added to 1.5 mL micro-centrifuge tube, and about 0.1 g to 0.3 g powder was added until the lysis with powder was very viscous but still solubilized, then the tubes were incubated for 1 h at 56°C with mixing every 15 min.

Quality evaluation and quantity estimation of DNA
NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the absorbance.The purity and concentration of the isolated DNA samples were calculated by the values of absorbance (A).The purity of extracted DNA was evaluated based on the ratios between the absorbance values for A260/A280 and A260/A230.To compare the efficiency of DNA extraction from different isolation methods, the DNA yield (μg DNA per gram tissue) was calculated.The integrity of the isolation DNA was confirmed by electrophoresis on a 0.6% agarose gel with a stain of SYBR Safe DNA Gel Stain (Invitrogen, USA), and then photographed by Analytik Jena AG ChemStudio (Analytik Jena, Germany).All the isolation DNA samples were stored at − 80°C until used in downstream analyses.The yield of isolated DNA was calculated by the following formula Y ¼ C�V W t , where Y is the yield (μg DNA per gram tissue), C is the concentration (ng μL −1 ), V is the volume (μL) of the final DNA solution that dissolved by TE buffer, and W t is the weight of the tissue powder used.

Data analysis
Experimental data processing statistics and analysis were performed using SPSS 23.0 software.Values were expressed as means ± Standard Error (SE), and significant differences between means were determined at P ≤ 0.05 by using one-way ANOVA and Tukey's honestly significant difference test.

The yield of isolated DNA
As the results shown in Figure 2, we calculated the DNA yields obtained for each sample in each of the six methods.Compared the DNA yields between deep-sea holothurians and deep-sea asteroids, we found that the yields of all deep-sea asteroids were higher than that of deep-sea holothurians in Qiagen (28-64 vs 2-23 μg DNA per gram tissue), SDS (300-381 vs 57-113 μg DNA per gram tissue) and CTAB (321-394 vs 105-182 μg DNA per gram tissue) methods, respectively, except Sangon Biotech, Tiangen Biotech, and Omega Bio-tek methods.Compared the DNA yields between deep-sea holothurians and shallow-water holothurians, we found that the yields of shallow-water holothurians were higher than that of deep-sea holothurians on average.Compared the DNA yields of each sample between the six methods, we found that the DNA yields of individual samples isolated from the methods of SDS and CTAB were significantly higher (P < 0.05) than the commercial kits (Sangon Biotech, Tiangen Biotech, Qiagen, and Omega Bio-tek) on average, and the highest yield was obtained by the CTAB method (all exceeded 100 μg DNA per gram tissue).

The integrity of isolated DNA
The integrity of the isolated DNA was checked by agarose gel electrophoresis (Figure 3).DNA was successfully isolated by the modified CTAB and other five methods for the deep-sea and shallow-water holothurians, and deep-sea asteroids.However, the DNA samples extracted by Sangon Biotech, Tiangen Biotech, Qiagen, Omega Bio-tek, and SDS presented obvious signs of degradation (Figure 3(a-e)), which indicated the DNA samples were degraded into small strands.Contrarily, the DNA samples extracted by the CTAB method presented no signs of degradation (Figure 3(f)), which indicated the high integrity of the DNA samples.

The purity of isolated DNA
A highly pure DNA relied on absorbance ratios as A260/A280 between 1.8 and 2.0, and A260/A230 > 2, lower or higher ratios indicated the DNA presented contaminations by polysaccharides, proteins, and chemical residues (Xin and Chen 2012;Stefanik et al. 2013).Spectral ratio (A260/A280 and A260/A230) measurements of isolated DNA exhibited purity (Tables 2 and 3).For the deep-sea samples, the A260/A280 ratios of the deep-sea holothurians (1.50-2.05)were generally lower than that of the deep-sea asteroids from the same dive (1.72-2.11),and their A260/A230 ratios (0.51-2.37) were significantly lower (P < 0.05) than that of the deep-sea asteroids (0.87-2.30) in average.For the holothurians, the A260/A280 ratios of the deep-sea samples were slightly higher than that of the shallow-water species (1.25-1.89), the A260/A230 ratios of the deep-sea samples were also slightly higher than that of the shallow-water species (0.37-2.36).For the six DNA isolation methods, the A260/A280 ratios of the Qiagen method were significantly higher (P < 0.05) than the other five methods, then the values of A260/ Figure 3. Electrophoresis of DNA on a 0.6% (wt/vol) agarose gel in 6 × loading buffer (TaKara), 5 μL of DNA was loaded in each lane followed the order as DA01, DA02, DA03, DA04, DH01, DH02, DH03, DH04, DH05, SH01, and SH02.And 5 μL of DNA maker as Quick-Load 1 kb Extend DNA Ladder (New England) in the last lane.
A280 ratios were followed by Omega Bio-tek, Tiangen Biotech, CTAB, Sangon Biotech and SDS on average.The A260/A230 ratios of DNA extracted by Omega Bio-tek were significantly higher (P < 0.05) than other five methods, then the values of A260/A230 ratios were followed by Qiagen, CTAB, Tiangen Biotech, SDS and Sangon Biotech in average.

Discussion
Degradation and low yield are common that have plagued us in DNA isolation experiments from deep-sea holothurians.Overcoming these problems in the preparation of high-molecular-weight DNA from deep-sea organisms is the key step for successful genome sequencing and downstream genetic and molecular biological studies.
To prevent the degradation of DNA in deep-sea holothurians, the fresh sample should be dissected and frozen in liquid nitrogen as soon as possible, to protect the cell the from damage to autolytic enzymes (Sun et al. 2010).In addition, low temperatures could reduce the biochemical activities in organism tissue that avoid problems of DNA degradation associated with secondary metabolism (Lohuis et al. 1990), therefore, the whole DNA extraction procedure should be performed on ice.For tissue lysis, many previous studies suggested that half to 3 h was suitable for digestion with proteinase K, and incubation for a long time would cause DNA degradation by over-digestion (Blin and Stafford 1976;Stefanik et al. 2013;Jo et al. 2016Jo et al. , 2017)).In this study, we incubated the tissue powder for 1 h based on our pre-experiment, which contributed to the highest integrity of DNA in the modified CTAB method.
The concentration of DNA samples was usually determined by NanoDrop spectrophotometer (Pinto et al. 2000;Straube and Juen 2013;Stefanik et al. 2013;Attaran-Fariman and Javid 2015;Puch-Hau et al. 2019) or Qubit fluorometer (Soniat et al. 2021).In the concentration measurement, the fluorometer has higher sensitivity than the spectrophotometer (Green and Sambrook 2012), and the Qubit fluorometer could also avoid the influence of contaminants which usually happened in NanoDrop spectrophotometer, but only NanoDrop  spectrophotometer was available in our lab.In order to reduce the effect of contaminants, proteinase K was added to break down the DNA-dinding proteins (Stefanik et al. 2013), RNase was used to eliminate any RNA molecules (Pinto et al. 2000;Stefanik et al. 2013;Attaran-Fariman and Javid 2015;Angthong et al. 2020), then we could obtain the DNA calculation that was close to actual values.
Though commercial kits, without extra work for preparing the relevant buffers, were more convenient than traditional methods (such as SDS and CTAB), the DNA obtained by kits with low throughput, low yield, or costly usually prohibited many downstream applications (Xin and Chen 2012).The required quantity of isolated DNA is significantly different on purpose for the following molecular analyses.For PCR amplification, 1-20 ng quantity and 50 ng/µl concentration of DNA were required (Pinto et al. 2000;Puch-Hau et al. 2019).DNA isolated by all the methods in our study has achieved the basic requirements for PCR.For nextgeneration sequencing, a large amount (≥10 µg for one sequencing) of DNA with long strands (≥40 kb), high concentration (≥50 ng/µl), and relative purity was needed.CTAB, which has the main advantages of high yield and high integrity, was well suited for whole genome sequencing.The sample preparation step was critical for achieving a high yield for whole genome sequencing during DNA isolation.In our experiment, crushing and grinding tissues completely improved the digestion efficiency.Additionally, the powder was added into the lysis until saturated but not exceeded also could improve the yield of DNA, 0.1 g to 0.3 g powder was an appropriated amount in this CTAB lysis based on our preexperiment.According to our results (Figure 2), 0.5 g tissue powder of holothutians body-wall could be easily obtained about 50 µg high-quality genomic DNA, which was sufficiently required for whole genome sequencing (Xin and Chen 2012).Isopropyl alcohol and ethanol were commonly used as precipitating solutions (Pinto et al. 2000;Green and Sambrook 2012;Jo et al. 2016Jo et al. , 2017)).Previous studies have confirmed that the addition of sodium ions and ethanol to the DNA solution could increase the yield (Lohuis et al. 1990;Green and Sambrook 2012;Stefanik et al. 2013;Attaran-Fariman and Javid 2015).In this study, we added salt (sodium acetate) and ethanol for DNA precipitation from an aqueous solution.
Moreover, the ethanol could increase the DNA pellet adhered to the tube wall, and reduce the wastage in rinsing (Green and Sambrook 2012).These modifications in the CTAB method generated a high yield of DNA from deep-sea holothurians as shown in this study.On the contrary, the yield of DNA extracted by the commercial kits was limited, such as Tiangen Biotech, Qiagen, and Omega Bio-tek (Figure 2), the less yield may be caused by the fixed DNA-binding capacity of a filter membrane (Stefanik et al. 2013).For downstream applications, the DNA binding capacity of the filter membrane should be considered for DNA extracted by commercial kits.Purity is also a major factor that affects DNA quality.Considering the accumulation of extracellular mucopolysaccharides, tissues were pre-washed by PBS before lysis to remove mucopolysaccharides and other impurities (Sun et al. 2010;Jo et al. 2017).During tissue lysis, EDTA at low concentration is recommended in lysis buffer, which permits easier separation of the phenolic and aqueous phases while maintaining a high degree of protection against degradation of the DNA nucleases and heavy metals (Green and Sambrook 2012).Therefore, the lysis buffer of CTAB was prepared with 20 mM EDTA in this study following Green and Sambrook (2012).Residue such as ethanol or isopropanol severely affects DNA purity (Sun et al. 2010;Green and Sambrook 2012).Ethanol was used in this study for DNA precipitation and rising, which could be removed by air-dried and avoiding chemical residues.In this study, the spectral ratios (A260/A280 and A260/A230) of DNA extracted by the Qiagen, Omega Bio-tek, and Tiangen Biotech resulted in higher purity than the other methods, probably because the contaminations could flow through the filter membranes and be discarded.Except for commercial kits with filter membranes, CTAB-extracted DNA had a relatively higher purity that achieved the requirement of the following analysis (Tables 2 and 3).The A260/A280 ratios of deep-sea holothurians from CTAB were 1.70 to 1.90 indicating low mucopolysaccharides (Sun et al. 2010;Aboul-Maaty and Oraby 2019).The A260/A230 ratios of that were 0.84 to 1.72 signify chemical residues of DNA isolation reagents.It is necessary to purify the low-purity DNA by the purification step as Angthong et al. (2020) described and Qiagen Genomic-tip kit described before the library preparation of whole genomic sequencing.
The time taken to perform in steps of lysis incubation, precipitation, and DNA pellets dissolution varied from 30 min to a few days (Blin and Stafford 1976;Lohuis et al. 1990;Pinto et al. 2000;Sun et al. 2010;Stefanik et al. 2013;Wang et al. 2014;Attaran-Fariman and Javid 2015;Jo et al. 2016).The whole procedure of DNA extraction usually lasts an extended period; therefore, less time consumption is also needed, especially for a large number of samples.In this study, to save time in incubation, we used proteinase K to increase the digestion of tissues and pulverized the tissue as a powder to digest rapidly.Previous studies also suggested that pre-wash to remove contaminations could significantly increase digestive efficiency, or else the contaminations like extracellular mucopolysaccharides and impurities on the body surface would form a flocculent mass and are not digested even after extending incubated time (Sun et al. 2010).In particular, cost was the main consideration.Tiangen Biotech kit is relatively less cost than kits of Omega Biotek and Qiagen.Although it cost more than Sangon Biotech kit, but produced stable quality of DNA than the later.Thus, Tiangen Biotech was an optional method which offered greater convenience and relatively less consumption for PCR analysis.Traditional methods of CTAB and SDS here were less expensive than commercial kits.With the requirements for whole genome sequencing, CTAB would be a feasible DNA isolated method, which generated large amount of DNA with high quality.Subsequently, the quality of the sequencing data will be serve as an assessment of the DNA performs by the modified CTAB method in further work.Furthermore, some previous studies [e.g.Sun et al. (2010) and Puch-Hau et al. (2019)] have investigated the isolated effects from different tissues of holothurians, and found that longitudinal muscle bands and intestine could obtain high-quality DNA compared with tube feet, body wall, respiratory tree, gonad, and cloaca.However, deep-sea samples are difficult to collect, especially for holothurians, since evisceration is a defensive strategy shared by holothurians that would lead to viscera loss during the collection, and gonad are not available due to growth stages and season (Jo et al. 2017;Zhang et al. 2017).Without the viscera or gonad, body wall of deep-sea holothurians was the main tissue left.Consequently, the body wall of deep-sea holothurians is the feasible tissue for DNA isolation (Sun et al. 2010;Jo et al. 2017;Zhang et al. 2017).

Conclusions
Here, we presented the first study to compare the efficiency of a modified CTAB method and several commonly used methods for DNA extraction from deep-sea holothurians.The CTAB method obtained a large amount of high-molecular-weight DNA with high integrity that is suitable for subsequent high-throughput sequencing, while the rest five methods only obtained less quantity with severe shearing.DNA extracted by commercial kits with filter membrane resulted in high purity with a limited quantity that only can be applied to small nucleic acid fragments sequencing.Overall, the modified CTAB method is a preferable method for DNA isolation from deep-sea holothurians, which could obtain large amounts of pure and integral DNA with high molecular weight.The highquality DNA, obtained by the CTAB method carried out in this study will provide a solid molecular basis for further studies on the genomic mechanism of holothurian evolution and deep-sea adaptation.

Figure 1 .
Figure 1.In situ photo of live samples (photos of deep-sea fauna were support by IDSSE Deep-sea Scientific Research Image and Video Database).DA01 and DH01 were collected from Xisha area of South China Sea.DA02, DH02 and DH03 were collected from Mid sea mount of South China Sea.DA03 and DH04 were collected from the connecting area between the Mariana Trench and Yap Trench.DA04 and DH05 were collected from the Mariana Island Arc District.SH01 and SH02 were collected from Sanya, Hainan Island.

Figure 2 .
Figure 2. The DNA yields of samples (μg DNA per gram tissue).Values are expressed as mean ± Standard Error (SE), n = 3 replicates.Different letters indicate significant difference among different methods for DNA isolation within each sample according to one -way ANOVA (P < 0.05) by SPSS 23.0.

Table 1 .
Taxonomy, collected sites, depth, and date of samples.

Table 2 .
Spectral ratios (A260/A280) of the DNA samples were determined by measuring UV absorbance on a NanoDrop spectrophotometer (Thermo Scientific).