Collection of maize varieties

Samples were collected by field trips to rural community from the states of Puebla, Jalisco, Michoacán and Oaxaca. Each sample weighted 1000 g of composite seed and was obtained from participate producer which were randomly chosen. In this study, a total of 215 maize local varieties were obtained, 53 out of 215 seed samples were collected from Jalisco, 96 samples from Michoacán, 46 samples from Oaxaca and 20 samples from Puebla State (Table 1). Each sampling site was geo-referenced in terms of altitude, latitude and longitude. Seed from each sample was placed for conservation in the maize germplasm bank of the Universidad Autonoma Agraria Antonio Narro.

DNA extraction, PCR amplification and detection of transgenes in vegetal material

Seeds from each collected variety were planted and germinated to obtain foliar tissue for GMO-DNA analyses. Polystyrene trays were filled with sterile forest soil and substrate (peat-moos) in a 6:4 ratio. After, a group of 10 seeds of each maize variety were planted. At 14 days after plantlet emergence, leaf tissue from 10 germinated seeds was cutting in mass and stored at 4 °C. For DNA isolation was used the method proposed by ^{Graham et al. (1995)}. DNA integrity was determined by electrophoresis in 1.5% agarose gel and DNA quality was estimated in an Epoch Microplate Spectrophotometer with the Gen5 1.11 software. A Polymerase Chain Reaction (PCR) was performed with each DNA sample composite to amplify eight accompanying sequences (Table 2) and nine transgenic events (Table 3), in this case, specific primers and specific amplification temperatures for each of these sequences were used. Most of the published studies analyzed one or a few transgenic sequences and, in some cases, only a few samples of maize varieties and most of the studies were concentrated in Oaxaca (^{Mercer and Wainwright 2008}). The PCR amplified segments were visualized using agarose gel electrophoresis (1.5%). The molecular markers of 50bp and 100bp DNA ladder (Invitrogen) were used as reference for determination of amplified segments size. In addition, the different amplified segments were sequenced to corroborate the identity of the amplified sequence.

Determination the level of introgression of transgenes into maize varieties

The level of introgression of transgenic event and accompanying sequence into local maize varieties which were planted as not biotech crops was determined in percent, in respect to varieties with presence of GMO into its genome vs total number of analyzed maize varieties in each Mexican State.

Statistical analysis

The PCR amplified bands of transgenic inserts were coded as absent (0) and present (1), then these values were summarized and employed for all percentage calculations. In addition, categorical analyses (tables SxR) were performed using the SAS software (9.0 version).

Collection of maize seed

Each collection site of maize varieties was georeferenced on a map (Figure 1). From 215 maize seed samples, 24.65% were from Jalisco, 44.65% from Michoacán, 21.39% from Oaxaca and 9.3% from Puebla.

Figure 1 Geo-referential distribution for the collection sites of maize varieties in the Jalisco, Michoacán, Oaxaca and Puebla States. 

Detection of transgenic sequences in plant tissue

In this study, it was only detected the presence of the cry1Ab transgenic event in all the maize varieties collected in the Mexican Central Region, and the ntpII accompanying sequence only was detected in Michoacan and Jalisco; which suggest that local maize varieties from these Mexican States are contaminated with transgene insert.

Estimate of maize contamination frequencies

Only the number of varieties positive for the transgenes cry1Ab and ntpII presence were included in the statistical analysis. In this analysis about frequency of these transgenic sequences present in the maize varieties were detected highly significant differences (P < = 0.01) (Table 4), the frequency rate for the cry1Ab gene was 41%, while the frequency rate for the ntpII sequence was 9.7%, in this analysis Q value was 56.2661 which was strongly significant and indicated that both frequencies are different. One explanation may be that during transformation of plants with the cry1Ab gene, different marker genes are utilized which may be different to ntpII.

The frequency of transgenic events presents in to the maize varieties from Puebla, Oaxaca, Jalisco and Michoacán, are shown in Table 5. The present study focused on detect transgenic events and accompanying sequences into maize varieties by PCR and using seeds collected of local maize varieties from Puebla, Jalisco, Michoacán and Oaxaca States, allowed to detecting the presence of the cry1Ab transgenic gene and the presence of the ntpII accompanying sequence in the maize varieties (Table 5). The localities with presence of the cry1Ab transgenic event and accompanying sequence ntpII in the maize populations are described in Table 6.

Introgression of transgenes into maize varieties

According to the presence of cry1Ab into the genome of the sampled maize varieties from these four Mexican states, it was observed that maize varieties grown as non-biotech crops from Puebla State have the highest levels (70%) of contamination o introgression of transgenes inserts into their genomes, followed by maize variety samples from Oaxaca with 52.17%, Michoacán maize samples with 44.79% and Jalisco maize samples with 15.09%, respectively. While, the presence of accompanying sequence ntpII was only detected in the Michoacán and Jalisco maize samples in 89.47 and 10.52%, respectively (Figure 2).

Figure 2 Level of introgression (Percentage) of the transgene event cry1Ab in Mexican maize varieties from Puebla, Oaxaca, Jalisco and Michoacán states. 

Collection of maize seed

Although some studies have confirmed the contamination of native maize by transgenic events, these studies considered a low number of samples, for example, ^{De-Ita (2012)} indicates the use of samples from 22 localities, while The National Commission for the Knowledge and Use of Biodiversity (Conabio) confirmed the presence of transgenes from 3 to 10% in 15 locations. Another study for the determination of transgenes carried out by ^{Serratos-Hernández et al. 2007} is based on a sample of 25 communities. The present study considered a greater number of localities (215) from 4 Mexican States, which allows to have a better perspective of the contamination of native maize by transgenic segments.

Detection of transgenic sequences in plant tissue

These results confirm the introgression of transgenes into maize varieties as indicated by (^{Quist and Chapela 2001}, ^{Cleveland et al. 2005}, ^{Serratos-Hernández et al. 2007}, ^{Mercer and Wainwright 2008}, ^{Piñeyro et al. 2009}, ^{Orozco-Ramírez et al. 2016}, ^{Rendón-Aguilar et al 2019}) who reported the presence of transgenic DNA in native varieties of maize grown in remote mountains of Oaxaca, this place is part of the center of origin and diversification of the crop in Mexico.

Estimate of maize contamination frequencies

The high detection rate of the cry1Ab transgenic event in the maize varieties populations implies that protective measures must be taken in the short term to produce maize varieties seeds free of these transgenic segments. This detection is controversial because in Mexico, it is not allowed to grow commercially maize transgenic cultivars, only at an experimental level (^{García and Toscana 2016}, ^{Ortega-Villegas et al. 2018}, ^{Lopez-Hernandez 2020}), precisely, for preservation of biodiversity of the native maize, because Mexico is considered a center of origin and diversity of this commodity (^{Lohn et al. 2021}).

Introgression of transgenes into maize varieties

This level of introgression of transgenes into the tested maize varieties could be considered very high, as it is determined in a specific way, what was found for each state (Figure 2), and assuming that the states with the lowest experimental seeding (Puebla and Oaxaca) are those with the greater genetic contamination, having thus that 70% of the varieties collected in Puebla state presented residues of the cry1Ab gene; while, 52% of the maize collected in Oaxaca were positive for the cry1Ab gene. On the other hand, in these states, not varieties were detected contaminated with accompanying sequences.

The cry1Ab gene was detected in 15% of the maize varieties collected in Jalisco, and 4 out of 53 samples collected were found to be introgressed with the accompanying ntpII sequence. While 44% of the varieties collected in Michoacán were observed to have the cry1Ab gene, and 17.7% of the samples collected in this state showed the accompanying ntpII sequence. The presence of transgenic events in maize varieties could have an cultural, economic, and social impact, on continuity of indigenous and peasant peoples as refer (^{Sánchez and Romero 2018}, ^{Ibarrola-Rivas et al. 2020}), who point out the importance of this area on maize conservation, it is in this territory where the milpa takes place, and at the same time, the milpa is part of the territory; and with the cultural and natural characteristics of the territory, particular milpa are configured. Therefore, the contamination of maize or its substitution by transgenic varieties is not limited to changes in maize as a source of food, but also implies negative repercussions for farmers in the milpa and in the territory, increasing the risk to their cultural continuity. Milpa is defined as agroecosystem conformed by polyculture, which is a dynamic space of genetic resources (^{Ortiz et al. 2014}, ^{González and Reyes 2014}, ^{Fonteyne et al. 2023}). ^{CCA (2004)} mentioned that in Mexico, maize is not only a commercial commodity, it is the base of Mexican diet and constitutes an integral expression of the relation between nature and culture, from this relation depends the subsistence of a great part of the Mexican rural population, and through this relation, the social tissue and people of these communities and diversity conservation are fortified.

Milpa is not only the space where maize, and other plant species are sown, but it is also a space for linguistic, cultural, symbolic, spiritual, social, and economical activities and food (^{Agapito-Tenfen et al. 2017}, ^{Ibarrola-Rivas et al. 2020}), is more than a farming technique, it is an agroecosystem in which farmers of Mesoamerican origin cultivate dozens of comestible and medicinal herbs, along with fruit and timber trees. For these communities, the most important thing is that the diversity of the maize germplasm can be lost. Year after year, the farmers keep the best seeds for planting in the following season, passing the knowledge of selection of them, the preparation of the land and the accumulation of information on the weather, among other elements, to the next generation (^{Sánchez and Romero 2018}). Maize varieties genetic diversity offers specialized genotypes which have demonstrated capacity of adaptation to different environments, pest and disease resistance, and satisfy divers demands for culinary, artisanal or industrial uses; this diversity is a key factor for the Mexican food security (^{WHO 2015}).

It is considered that maize is a continuous process of domestication, since it depends completely on the farmer, who through selection has favored the survival, and reproduction of phenotypes that have advantageous characteristics to be used mainly as food for humans, and to date represent a total of 64 native maize races reported for Mexico (^{Santillán-Fernández et al. 2021}). It is also convenient to clarify that natural selection operates during the process of domestication. The domestication process results in Mexico being a center of maize genetic diversity (^{Santillán-Fernández et al. 2021}), and is the Mexicans’ responsibility to preserve this great maize genetic diversity, in addition to conducting selection programs to gather more, and better characters in the plants that will serve as progenitors to the next generation. Agricultural and cultural practices have been very important during the whole process of maize domestication, since each variety have adapted to the specific environment that includes the cultivation method (^{Guzzon et al. 2021}).

Maize is currently important in the diet of Mexicans and for many of them, it is their main source of protein and energy, which can be considered as cheap diet (^{Erenstein et al. 2022}); with an average consumption of 350 g per capita per day in 600 different presentations of maize. Therefore, it has been pointed out that maize requires a special protection regime for genetically modified organisms that can negatively modify the nutritional qualities of this cereal (^{Brookes and Barfoot, 2016}). The genetic contamination by transgenes of the natural ecosystems of Mexico has been documented. Since 2001, there have been isolated reports that indicate the unintentional presence of GM maize (^{Quist and Chapela 2001}, ^{Serratos-Hernández et al. 2007}, ^{Mercer and Wainwright 2008}, ^{Piñeyro et al. 2009}) and there is only low work in Mexico in which the absence of GMOs was reported in maize (^{Cleveland et al. 2005}, ^{Rendón-Aguilar et al 2019}). Most of the published studies analyzed one or a few transgenic sequences and, in some cases, only a few samples of maize varieties and most of the studies were concentrated in Oaxaca (^{Mercer and Wainwright 2008}). On the other hand, there are reports that maize shipments imported for consumption have a large percentage of transgenic residues, in addition, the grain is viable. From 29 imported shipments of maize for consumption were analyzed for presence of transgenic residues, all the samples presented at least one transgenic sequence, either an event or an accompanying sequence (^{Carvajal et al. 2017}). So, there is a risk that grains fall during transport, germinate on the sides of roads or railroads, or that farmers take these grains as seed and when they are planted in regions with high genetic diversity of maize, pollen can transfer transgenic genes to the native maize. It is proposed that the information obtained in this work be integrated into the National Program for the Monitoring of Genetically Modified Organisms, which will allow a diagnosis of the current situation in Mexico on the introgression of transgenes to the native maize and will serve as the basis for taking the actions necessary in matters of Biosafety.