Procedures for DNA Extraction from Opium Poppy (Papaver somniferum L.) and Poppy Seed-Containing Products

Several commonly used extraction procedures and commercial kits were compared for extraction of DNA from opium poppy (Papaver somniferum L.) seeds, ground seeds, pollen grains, poppy seed filling from a bakery product, and poppy oil. The newly developed extraction protocol was much simpler, reduced the cost and time required for DNA extraction from the native and ground seeds, and pollen grains. The quality of extracted DNA by newly developed protocol was better or comparable to the most efficient ones. After being extended by a simple purification step on a silica membrane column, the newly developed protocol was also very effective in extracting of poppy DNA from poppy seed filling. DNA extracted from this poppy matrix was amplifiable by PCR analysis. DNA extracted from cold-pressed poppy oil and suitable for amplifications was obtained only by methods developed previously for olive oil. Extracted poppy DNA from all tested matrices was analysed by PCR using primers flanking a microsatellite locus (156 bp) and two different fragments of the reference tubulin gene (553 bp and 96 bp). The long fragment of the reference gene was amplified in DNA extracted from native seeds, ground seeds, and pollen grains. Poppy DNA extracted from the filling of bakery product was confirmed only by amplification of short fragments (96 bp and 156 bp). DNA extracted from cold-pressed poppy oil was determined also only by amplification of these two short fragments.


Introduction
Extraction of nucleic acids from various matrices is the first and crucial step in analysis of biological materials generally. Methods of DNA extraction have evolved over time [1], but still contain several basic and necessary steps such as cell disruption, removal of undesirable molecules (lipids, proteins, polyphenols, and others), and purification. In addition to the cell wall disruption, the chemical diversity of metabolites contained in the plant cell is a major complication in the DNA isolation process. There is no available universal protocol for extraction of DNA which would be applicable independently of plant species, plant tissues, and plant matrix [2,3]. Generally, extraction of DNA from young, fast-growing, and healthy tissues is much easier. However, it is often necessary to extract DNA from plant tissues rich in polysaccharides, lipids, secondary metabolites, or even from very complex matrices (processed seeds, oils, foods, feeds). This is also the case of oilseeds where extraction of DNA is considered more demanding than from vegetative plant tissues (e.g., young leaves). Lipids usually prevent the action of solvents during removal of polysaccharides and phenolic

DNA Extraction from Pollen Grains
Three methods used for isolation of DNA from 0.1 g of pollen grains included DNeasy ® Plant Maxi Kit, QIAamp DNA Stool Mini Kit. The third was the newly developed protocol (described above). An efficient mechanical homogenization of pollen grains was particularly important.

DNA Extraction from Poppy Seed Filling
DNA was extracted from 0.5-2.0 g of filling of the bakery product using methods: Bayer BioScience N.V. [25], Monsanto Company [26], QIAamp DNA Stool Mini Kit, and newly developed protocol. Extracted DNA was purified through the silica membrane spin-columns [28].

Qualitative and Quantitative Analysis of Extracted DNA
Integrity of the extracted DNA from different poppy matrices was assessed by agarose gel electrophoresis. Parameters of extracted DNA were tested by UV spectrophotometry (NanoDrop ND-1000 spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA) as well as by electrophoresis in 0.8% agarose gel stained with ethidium bromide.

DNA from Mature Seeds
Poppy seeds are specific commercial commodity used in the food industry particularly in some regions of the world. However, the food quality and related price of seeds vary considerably for different P. somniferum L. cultivars. Unfortunately, it is likely that premium quality seeds (sweet taste, blue colour) of some poppy cultivars are intentionally handled while trading. They are usually exchanged with low-quality seeds or mixed with them, whether intentionally or not. Therefore, different protocols for extraction of total DNA from poppy seeds and poppy containing products were tested. DNA analysis should be used to determine poppy seed cultivar origin. In addition to the six extraction protocols and four commercial kits tested (Table 2), the modified extraction procedure ("newly developed protocol") was proposed in this study. It is based on the results and experiences obtained during testing of ten extraction procedures.
Spectrophotometric analysis as well as gel electrophoresis of DNA from seeds revealed significant differences between used extraction protocols, both in quantity and quality of obtained DNA. The qualitative parameters of DNA were primarily important ( Table 2). The protocol of Dellaporta et al. [24] and its modification by incorporation of CTAB showed that mechanical homogenization of seeds directly in the extraction buffer, even without the use of liquid nitrogen, did not lead to deterioration in quality or amount of DNA (Table 2, Figure 1a). It may be concluded that the need to use liquid nitrogen during mechanical homogenization of poppy seeds is not necessary for prevention of degradation of extracted DNA [34][35][36]. Quality of extracted DNA varied according to the extraction procedure. Procedures according to Sangwan et al. [21], Bayer BioScience N.V. [  *-Homogenization of seeds with liquid nitrogen; Bayer BioScience N.V. [25] (1)-0.2 g of seeds extracted in volume of extraction buffer for 0.2 g seeds; Bayer BioScience N.V. [25] (2)-DNA extracted from 0.2 g of seeds extracted in volume of extraction buffer for 0.5 g of seeds.
Foods 2020, 9, x FOR PEER REVIEW 6 of 15 Amplifications were successful from DNA extracted from mature native seeds by almost all of used protocols and the relevant fragments were generated ( Figure 2). The only exception was DNA extracted by the Plant DNAzol ® Reagent. DNA extracted by PowerSoil DNA Isolation Kit was probably highly degraded considering that 553 bp fragment of reference tubulin gene was not amplified, but a 156 bp length microsatellite marker was generated ( Figure 2).
The only one currently available protocol developed for extraction of DNA from poppy seeds [21] did not provide high quality of DNA within this study (Table 2, Figure 1a). The newly developed protocol has been proven as effective. Compared to the original protocol [25], extraction steps were rearranged, time intervals between steps were changed, and some chemicals/enzymes were eliminated. Both absorbance parameters (A260/280 and A260/230) as well as electrophoretic profile of DNA predicted very good quality and quantity (Table 2, Figure 1a) that should be suitable for amplification by PCR ( Figure 2).

DNA from Ground Seeds
Ground poppy seeds are commonly available in food stores. The sensory values (especially taste and smell) and related varietal origin of high-quality seeds may be easily masked in ground seeds by various additives, mainly by sugar. Analytical testing and confirmation of the poppy seeds varietal origin is necessary in such cases. DNA from ground poppy seeds was extracted by two protocols [25,26], two commercial kits, as well as newly developed protocol. The QIAamp DNA Stool Mini Kit and DNeasy ® Plant Maxi Kit produced DNA with the A260/280 and A260/230 ratios furthest from optimal values ( Table 2). Both spectrophotometric ratios of DNA extracted by Monsanto Company [26] protocol indicated high contamination of DNA with proteins, organic solvents, and secondary metabolites, and also very low concentration ( Table 2). The yield of DNA was significantly different between tested protocols, but at the same amount of loaded DNA (25 ng/μL) the electrophoretic profiles of all DNA samples were appropriate (Figure 1b). The highest quality and concentration of DNA has been extracted by protocols Bayer Biocience N.V. [25] with changed However, the success in amplification of extracted DNA is not guaranteed only by purity, but also by concentration and structural integrity of DNA [37,38]. Although values A 260 of DNA extracted by protocols Murray and Thomson [27], Sangwan et al. [21], Plant DNAzol ® Reagent, and PowerSoil DNA Isolation Kit were high, DNA was not observed in agarose gel ( Figure 1a, Table 2). There were probably only limited amounts of poppy DNA and absorbance values have been increased by the presence of RNA and other contaminants. DNA extracted by these protocols also had very low quality. Significant RNA contamination was reported only for the original CTAB method [27] and the QIAamp DNA Stool Mini Kit due to absence of RNase A treatment ( Figure 1a).
Amplifications were successful from DNA extracted from mature native seeds by almost all of used protocols and the relevant fragments were generated ( Figure 2). The only exception was DNA extracted by the Plant DNAzol ® Reagent. DNA extracted by PowerSoil DNA Isolation Kit was probably highly degraded considering that 553 bp fragment of reference tubulin gene was not amplified, but a 156 bp length microsatellite marker was generated ( Figure 2).
The only one currently available protocol developed for extraction of DNA from poppy seeds [21] did not provide high quality of DNA within this study (Table 2, Figure 1a). The newly developed protocol has been proven as effective. Compared to the original protocol [25], extraction steps were rearranged, time intervals between steps were changed, and some chemicals/enzymes were eliminated. Both absorbance parameters (A 260/280 and A 260/230 ) as well as electrophoretic profile of DNA predicted very good quality and quantity (Table 2, Figure 1a) that should be suitable for amplification by PCR (Figure 2).

DNA from Ground Seeds
Ground poppy seeds are commonly available in food stores. The sensory values (especially taste and smell) and related varietal origin of high-quality seeds may be easily masked in ground seeds by various additives, mainly by sugar. Analytical testing and confirmation of the poppy seeds varietal origin is necessary in such cases. DNA from ground poppy seeds was extracted by two protocols [25,26], two commercial kits, as well as newly developed protocol. The QIAamp DNA Stool Mini Kit and DNeasy ® Plant Maxi Kit produced DNA with the A 260/280 and A 260/230 ratios furthest from optimal values ( Table 2). Both spectrophotometric ratios of DNA extracted by Monsanto Company [26] protocol indicated high contamination of DNA with proteins, organic solvents, and secondary metabolites, and also very low concentration ( Table 2). The yield of DNA was significantly different between tested protocols, but at the same amount of loaded DNA (25 ng/µL) the electrophoretic profiles of all DNA samples were appropriate (Figure 1b). The highest quality and concentration of DNA has been extracted by protocols Bayer Biocience N.V. [25] with changed ratio of sample-extraction buffer (w/v) and the newly developed protocol (Table 2, Figure 1b). Both protocols contained SDS in extraction buffer. It is suggested that SDS-based DNA extractions could be more appropriate for oily plant matrices like ground poppy seeds. The SDS-containing method modified for ground raw soybean seeds had the highest yield of DNA in comparison with the CTAB method and two commercial kits [39]. A lower amount of DNA yielded the CTAB method also from soybean flour [40]. Amplifications of DNA from ground poppy seeds using primers flanking microsatellite marker psSSR69 and longer fragment of gene for tubulin beta-7 chain resulted in production of both the 156 and 553 bp amplicons in DNA extracted by all used protocols (Figure 3).

DNA from Poppy Pollen Grains
DNA was extracted by two commercial kits and by newly developed protocol ( Table 2). Homogenization by pestle and mortar in liquid nitrogen was efficient for disruption of pollen exine with high structural integrity. Both ratios A 260/280 and A 260/230 confirmed that the best quality had DNA extracted by newly developed protocol (Table 2). This simple protocol produced also very high amount of DNA. On the opposite, the QIAamp DNA Stool Mini Kit and DNeasy ® Plant Mini Kit extracted the least amount of DNA (Figure 4a). Amplifications of DNA from poppy pollen grains were basically without any complications. All primer pairs were able to amplify relevant amplicons ( Figure 4). The genomic DNA is well protected inside the pollen grain therefore, a large fragment of the reference gene (553 bp) was simply amplified (Figure 4b). Amplifications of both shorter, the 156 bp microsatellite marker and 96 bp fragment of reference gene were also easily feasible (Figure 4c,d). Amplification of 156 bp microsatellite psSSR69 (a) and 553 bp fragment of gene for tubulin beta-7 chain (b) in DNA extracted from poppy seeds by: Dellaporta et al. [24] with and without liquid N2 (lanes 1 and 2), Dellaporta et al. [24] with CTAB with and without liquid N2 (lanes 3 and 4), Bayer BioScience N.V. [25]

DNA from Poppy Pollen Grains
DNA was extracted by two commercial kits and by newly developed protocol ( Table 2). Homogenization by pestle and mortar in liquid nitrogen was efficient for disruption of pollen exine  Extraction of DNA from pollen grains is needed in different applications including monitoring of pollen grains transfer from transgenic opium poppy plants to the environment [41], detection of pollen species in food (e.g., in honey) for the prevention of allergens [42], forensic palynology [43] and others.

DNA from Poppy Seed Filling
DNA was extracted by two procedures, one commercial kit, and the newly developed protocol ( Table 3). The purification step using the silica membrane spin-columns [28] was added to protocols Monsanto Company [26] and newly developed one. Both ratios A 260/280 and A 260/230 confirmed that DNA extracted using almost all extraction protocols had these values out of the optimal range (Table 3). Undamaged high molecular weight DNA extracted from poppy seed filling from the bakery product was not visualizable in agarose gel (data not shown). This reflects fragmentation of poppy DNA to very short fragments due to high degradation during baking. This is common for DNA extracted from a matrix that has undergone processing by high temperature [29] and a combination of grinding, mechanical manipulation, and thermal treatment [44]. However, the objective quality and usability of DNA extracted can only be revealed by its amplification. Complex food matrices contain a variety of PCR inhibitors [45]. Other effects of the matrix include degradation, fragmentation, and restricted extractability of DNA, as well as presence of DNA from different organisms [46]. Baking temperature around 200 • C used in processing of bakery goods containing poppy seed filling substantially reduces the size of extracted DNA. Moreover, higher moisture content inside the product, in this case in poppy filling, contributes to greater degradation of DNA [9]. Amplifications of poppy DNA extracted from filling of the baked product were more difficult. As expected, primer pair designed for amplification of 553 bp fragment of reference gene was not able to generate amplicon (data not shown). The Bayer BioScience N.V. method [25] and the QIAamp DNA Stool Mini Kit provided DNA with quality allowing amplification of the 156 bp microsatellite and short (96 bp) fragment of reference gene ( Figure 5). Both these methods were effective also without the need of purification in columns. DNA extracted by the Monsanto Company method [26] and newly developed protocol was amplifiable only if the purification step in the silica membrane column [28] was added ( Figure 5). Columns were able to bind impurities and inhibitors of polymerase chain reaction from primary DNA extracts.
Foods 2020, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/foods Complex food matrices contain a variety of PCR inhibitors [45]. Other effects of the matrix include degradation, fragmentation, and restricted extractability of DNA, as well as presence of DNA from different organisms [46]. Baking temperature around 200 °C used in processing of bakery goods containing poppy seed filling substantially reduces the size of extracted DNA. Moreover, higher moisture content inside the product, in this case in poppy filling, contributes to greater degradation of DNA [9]. Amplifications of poppy DNA extracted from filling of the baked product were more difficult. As expected, primer pair designed for amplification of 553 bp fragment of reference gene was not able to generate amplicon (data not shown). The Bayer BioScience N.V. method [25] and the QIAamp DNA Stool Mini Kit provided DNA with quality allowing amplification of the 156 bp microsatellite and short (96 bp) fragment of reference gene ( Figure 5). Both these methods were effective also without the need of purification in columns. DNA extracted by the Monsanto Company method [26] and newly developed protocol was amplifiable only if the purification step in the silica membrane column [28] was added ( Figure 5). Columns were able to bind impurities and inhibitors of polymerase chain reaction from primary DNA extracts.

DNA from Poppy Oil
Oil from poppy seeds is mainly used for culinary and pharmaceutical purposes, but also for production of cosmetics, paints and varnishes. Cold-pressed oil is quite expensive, so it can sometimes be adulterated by much cheaper vegetable oils (e.g., from rapeseed, sunflower, oil palm). Techniques of analytical chemistry are developing for distinguishing between cheaper oils (e.g., sunflower, oilseed rape) and poppy oil [17]. However, chemical analysis may not be unambiguous [31] due to variation in chemical composition of vegetable oils among growing areas and seasons. Alternative approaches are based on the DNA analysis and require extraction of DNA from oil. Such protocols were developed mainly for olive oil. Four of such methods [4,[29][30][31], the QIAamp DNA Stool Mini Kit as well as Bayer BioScience N.V. [25], Monsanto Company [26], newly developed protocols were tested for different volumes of poppy seed oil. Bayer BioScience N.V. [25], Monsanto Company [26] and newly developed protocol were unable to extract detectable and usable DNA (data not shown). DNA extracted by other protocols had also both absorbance parameters (A260/280, A260/230) far from the optimal values (Table 3); however, DNA was amplifiable by PCR ( Figure 6).

DNA from Poppy Oil
Oil from poppy seeds is mainly used for culinary and pharmaceutical purposes, but also for production of cosmetics, paints and varnishes. Cold-pressed oil is quite expensive, so it can sometimes be adulterated by much cheaper vegetable oils (e.g., from rapeseed, sunflower, oil palm). Techniques of analytical chemistry are developing for distinguishing between cheaper oils (e.g., sunflower, oilseed rape) and poppy oil [17]. However, chemical analysis may not be unambiguous [31] due to variation in chemical composition of vegetable oils among growing areas and seasons. Alternative approaches are based on the DNA analysis and require extraction of DNA from oil. Such protocols were developed mainly for olive oil. Four of such methods [4,[29][30][31], the QIAamp DNA Stool Mini Kit as well as Bayer BioScience N.V. [25], Monsanto Company [26], newly developed protocols were tested for different volumes of poppy seed oil. Bayer BioScience N.V. [25], Monsanto Company [26] and newly developed protocol were unable to extract detectable and usable DNA (data not shown). DNA extracted by other protocols had also both absorbance parameters (A 260/280 , A 260/230 ) far from the optimal values (Table 3); however, DNA was amplifiable by PCR ( Figure 6). DNA in cold-pressed vegetable oil has undergone a process of significant degradation, caused by DNA nucleases released during crushing and malaxation of oily plant material. This will certainly happen when pressing oil from poppy seeds as well. If enzymatic mixtures of proteases are applied during this process, the DNA is prevented to damage and could be extracted with high integrity and concentration, similarly as from vegetative tissues [47]. However, this cannot be ensured in the already pressed oil. Another significant complication in the extraction of DNA is the time since pressing and conditions of the oil storage before the DNA extraction. After a relatively short time interval, a significant decreasing of quality of extracted DNA was observed due to oxidation damage [48]. Following the assumed high degradation, DNA has not even been electrophoretically controlled and only its amplifications revealed the potential utility of the extracted DNA. Statistical analysis did not reveal relationship between concentration, A 260 /A 280 ratio, and the ability to undergo amplification by PCR [49].
concentration, similarly as from vegetative tissues [47]. However, this cannot be ensured in the already pressed oil. Another significant complication in the extraction of DNA is the time since pressing and conditions of the oil storage before the DNA extraction. After a relatively short time interval, a significant decreasing of quality of extracted DNA was observed due to oxidation damage [48]. Following the assumed high degradation, DNA has not even been electrophoretically controlled and only its amplifications revealed the potential utility of the extracted DNA. Statistical analysis did not reveal relationship between concentration, A260/A280 ratio, and the ability to undergo amplification by PCR [49].
Four extraction protocols [4,[29][30][31] and the QIAamp DNA Stool Mini Kit provided different results ( Figure 6). In addition, DNA extraction was also tested from different starting volumes of poppy seed oil. Extraction protocol developed for authentication of olive oils [30] was efficient either from 3 mL or 6 mL samples of poppy oil. Poppy DNA obtained by this protocol, from both the oily and water phases were amplifiable and provided templates for relevant amplicons. Other used DNA extraction protocols were also developed for olive oil, but based on the CTAB in extraction buffer [4,31]. The resulting poppy DNA behaved unreliably in the PCR reaction. Convincing and reliable amplifications were obtained from DNA extracted by another protocol, modified for olive oil [29] containing guanidine thiocyanate in extraction buffer. The capability of tested QIAamp DNA Stool Mini Kit for DNA extraction from poppy oil has been demonstrated in low oil volumes (0.2-1 mL). The quality and quantity of DNA extracted from native or processed poppy seeds strongly depended on the character of poppy matrix entering the extraction procedure as well as level of its processing. Amplifications of obtained DNA were also influenced by many factors, especially by the presence of contaminants and inhibitors. Positioning of used primers for PCR analysis considered the expected length of extracted DNA fragments depended on the expected disruption of DNA during processing (baking, pressing) of poppy seed matrix. DNA extracted from different poppy seed matrices by different extraction protocols was amplified using primer pairs flanking the 553-, 156-, and 96 bp fragments, respectively ( Table 1). The presence of the longest 553 bp fragment was detected by PCR in poppy DNA extracted from native seeds and ground seeds, but not from processed poppy seed matrices (filling of the bakery product, oil). Both types of poppy seed processing (baking, pressing) reduced the effective concentration of poppy DNA fragments capable Four extraction protocols [4,[29][30][31] and the QIAamp DNA Stool Mini Kit provided different results ( Figure 6). In addition, DNA extraction was also tested from different starting volumes of poppy seed oil. Extraction protocol developed for authentication of olive oils [30] was efficient either from 3 mL or 6 mL samples of poppy oil. Poppy DNA obtained by this protocol, from both the oily and water phases were amplifiable and provided templates for relevant amplicons. Other used DNA extraction protocols were also developed for olive oil, but based on the CTAB in extraction buffer [4,31]. The resulting poppy DNA behaved unreliably in the PCR reaction. Convincing and reliable amplifications were obtained from DNA extracted by another protocol, modified for olive oil [29] containing guanidine thiocyanate in extraction buffer. The capability of tested QIAamp DNA Stool Mini Kit for DNA extraction from poppy oil has been demonstrated in low oil volumes (0.2-1 mL).
The quality and quantity of DNA extracted from native or processed poppy seeds strongly depended on the character of poppy matrix entering the extraction procedure as well as level of its processing. Amplifications of obtained DNA were also influenced by many factors, especially by the presence of contaminants and inhibitors. Positioning of used primers for PCR analysis considered the expected length of extracted DNA fragments depended on the expected disruption of DNA during processing (baking, pressing) of poppy seed matrix. DNA extracted from different poppy seed matrices by different extraction protocols was amplified using primer pairs flanking the 553-, 156-, and 96 bp fragments, respectively ( Table 1). The presence of the longest 553 bp fragment was detected by PCR in poppy DNA extracted from native seeds and ground seeds, but not from processed poppy seed matrices (filling of the bakery product, oil). Both types of poppy seed processing (baking, pressing) reduced the effective concentration of poppy DNA fragments capable of amplification of fragments longer than 100 bp, as was detected in maize cornmeal [50]. DNA from heat-processed and other highly degraded plant matrices should be amplified only in short DNA sequences. This is the strategy also in analysis of DNA from genetically modified organisms in processed foods [9,51]. Analysis of highly degraded DNA by PCR is more advantageous in DNA regions higher in GC content because their stability during heat treatment of the analysed matrices is higher [51].
Specific morphological characteristics, extreme heterogeneity and variation in chemical composition of plant cells cause many problems in DNA extraction. Although numerous protocols for plant DNA extraction have been published, none is found to be universally applicable [52]. Newly developed DNA extraction protocols are usually modifications of already existing protocols. The extraction protocol developed in our study demonstrated a relatively high degree of universality, with respect to poppy matrices. Compared to other DNA extraction protocols, it was quite universal. In comparison with the Bayer BioScience N.V. [25] protocol, from which the most steps were taken, it was approximately one third shorter in time. A significant reduction in time was achieved by adjusting the centrifugation steps. 2-mercaptoethanol ME was added to the first extraction buffer. Some steps during the extraction procedure were eliminated. Along with purification on silica membrane columns, the newly developed extraction protocol was highly efficient and represents a simple and inexpensive alternative to commercial DNA extraction kits. Extraction of DNA from oil required specific extraction protocols that were developed specifically for this type of matrix only.

Conclusions
Protocols tested for extraction of DNA from native and ground poppy seeds, pollen grains, poppy seed filling from the bakery product, and poppy oil have been differently effective and suitable depending on individual poppy seed matrices or products. DNA from seeds, ground seeds and pollen grains extracted by almost all extraction procedures had quantity and quality sufficient for PCR analysis of short microsatellite marker (156 bp) and also long fragment of the reference gene (553 bp). The best of these protocols have been tested for DNA extraction from the poppy seed filling from the bakery product. It has been very useful to use silica membrane columns for purification of the extracted DNA. Purified DNA was then amplifiable. Poppy DNA extracted from thermally processed poppy seed filling from the baking product did not amplify long fragment (553 bp) of the reference gene. However, primers designed for amplification of shorter fragment of the reference gene (96 bp) as well as for the microsatellite marker (156 bp) provided the appropriate amplicons. The new extraction protocol developed within this study has proven to be universally applicable to poppy seeds, pollen, and poppy seed containing products. It can be used for various control purposes in poppy breeding programmes, production and distribution of elite poppy seeds for crop production, control of poppy seeds identity as an interesting market commodity, control of products containing poppy seeds during food production. Protocols tested for extraction of poppy DNA from cold-pressed poppy oil were originally developed or modified for olive oil. The most of them [29,30] were effective, and extracted DNA was amplified using primers for the microsatellite marker and the short fragment of the reference gene.