Genotyping of Vanilla planifolia Jacks ex Andrews from the Totonacapan region using Inter-Simple Sequence Repeat markers Genotipagem de Vanilla planifolia Jacks ex Andrews

Vanilla is the only genus of plants in the Orchidaceae family used in the food industry, being the most widely used flavoring worldwide and the second most valuable economically. Cultivation is of great biocultural importance in the Totonacapan region, in the state of Veracruz, as this region is considered its center of origin and distribution. However, little is known regarding the number of genotypes present in this area, as well as the genetic diversity, which is vital for the improvement of this crop. Therefore, the objective of this study was to perform the genotyping of 60 Vanilla planifolia accessions from the Germplasm Bank of The National Institute of Forestry, Agriculture, and Livestock Research (Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias - INIFAP), Experimental Field Ixtacuaco, which were selected and collected in the Totonacapan region. For this purpose, the number of V. planifolia genotypes was determined based on ISSR (Inter-Simple Sequence Repeat) molecular markers. Three DNA extraction protocols from plants and fungi described in the literature were evaluated, and a fourth protocol was adapted with different procedures from the previous protocols, seeking to obtain a high concentration and good quality and integrity. Once quality DNA was obtained, fourteen ISSR primers were evaluated, of which three primers that showed the highest number of amplified bands were selected. Subsequently, the genotyping of the 60 accessions was carried out, resulting in nine genotypes with primer 808 and six genotypes with primers 812 and 856. These results allowed the genotyping of Vanilla planifolia accessions with ISSR markers, which will help evaluate the genetic diversity of this crop in the Totonacapan region in the future.


INTRODUCTION
Vanilla (Vanilla planifolia Jacks ex Andrews) is a member of the orchid family, characterized by being an epiphytic vine with yellowish-green flowers.Traditionally known by the Nahuatl names "Ixtlilxochitl" or "Tlilxóchitll" (black flower), or in Totonac as "Xanath" (hidden flower).It was named "vanilla" by the Spanish, because its fruits resemble sword pods, measuring 15 to 30 cm in length.It is believed that the seeds of the fragrant fruits are dispersed by bats (Soto-Arenas, 2006;Hernández-Hernández, 2011).
Vanilla is the second most expensive spice in the world, only after saffron (Crocus sativus).
However, it is considered the most popular flavor and aroma worldwide.It has an exotic and evocative aroma that can act as an individual flavor or complement other flavors like chocolate (Soto-Arenas, 2006).
Currently, vanilla production relies on vegetative propagation and manual pollination.These conditions lead to a decrease in the genetic variation of cultivars, as a result of the almost nonexistent emergence of individuals from sexual recombination, which is why vanilla is currently listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) under the category of high genetic erosion (CITES, 2019).
Genetic diversity of the species is one form of biodiversity that must be conserved, defined as the hereditary variations that occur within each organism, among individuals of a population, and among populations within a species (Piñero et al., 2008).Currently, it is known that most of the vanilla planting material originated from a limited clonal source.Therefore, the conservation of vanilla genetic resources is of vital importance.In this work, sixty vanilla accessions from the Vanilla Germplasm Bank of INIFAP, Experimental Field Ixtacuaco (CEIXTA), collected from different locations in the Totonacapan region in the state of Veracruz, were analyzed to genetically characterize them using Inter-Simple Sequence Repeat (ISSR) analysis.These results are of great importance for identifying genetically different individuals, which will be used in strategies such as identifying unique DNA polymorphisms associated with potential outstanding agronomic characteristics of the crop in further research.was wrapped in aluminum foil and stored in labeled plastic bags.The collection sites in the state of Veracruz are indicated in Table 1.

Extraction of Genomic DNA
Vanilla leaves were cleaned with 70% ethanol and a sterile paper towel.DNA was extracted from the leaves using the CTAB method (modified from Rodríguez-Quibrera et al., 2010, andSantillán-Mendoza et al., 2018).Vanilla leaves were ground with liquid nitrogen until obtaining a fine powder, avoiding tissue thawing.Approximately 0.5 g of the ground tissue was placed in a 2 Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.1, p. 297-306, jan./mar., 2024 ml microtube, and 1000 μl of lysis (2% CTAB, 0.2% 2-mercaptoethanol) was added.The mixture was incubated at 65°C for 1 hour with vortex agitation every 10 minutes for approximately 10 seconds.Chloroform-isoamyl alcohol 24:1 was added, and the mixture was centrifuged at 13,000 rpm for 15 minutes at 4°C.The recovered supernatant was precipitated with 7.5 M ammonium acetate and isopropyl alcohol for 16 hours.After centrifugation, the supernatant was carefully discarded to avoid disturbing the DNA pellet.The DNA pellet was washed twice with 70% cold ethanol.DNA samples were hydrated with sterile molecular biology-grade water.DNA concentration was determined using a spectrophotometer (NanoDrop 2000c), and quality was evaluated by agarose gel electrophoresis (1% agarose gel stained with ethidium bromide).

Inter-Simple Sequence Repeat (ISSR) Analysis
ISSR analyses were performed by PCR amplification of DNA from the 60 vanilla accessions from the Totonacapan region.PCRs were carried out in a total volume of 25 μl, using 5 μl of 5X Green Go Taq Flexi Buffer (Promega, USA), 2.5 μl of 25 mM MgCl₂, 1 μl of dNTPs (Promega, USA), 1 μl of 10 pmol primer, 0.3 μl of Go Taq Flexi DNA polymerase (5 u/μl) (Promega, USA), 14.2 μl of molecular biology-grade water, and 1 μl of DNA (30 ng/μl).PCR reactions were performed in a MiniAmp plus Thermal Cycler, using the following program: an initial denaturation cycle at 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C for 2 min.The amplification ended with a final extension step at 72°C for 7 min.PCR products were electrophoresed on 2% agarose gels stained with ethidium bromide and run at 90 volts for 100 min.
DNA bands were visualized using a gel documentation system (Gel Doc™ EZ Imager, BioRad).
All agarose gel images were individually processed using the pre-installed software with the Gel Doc™ EZ Imager from BioRad, through which each of the amplified DNA bands from the PCR-ISSR reactions was counted, obtaining the total number of genotypes.

Selection of ISSR primers
Initially, a preliminary assay was conducted using a set of 14 ISSR primers (Table 2)

Genotyping of V. planifolia from Totonacapan
A total of fourteen ISSR primers were analyzed for the sixty accessions of V. planifolia, of which the three that generated the highest number of amplified bands were selected.Primer 808 (Fig. 2) showed a total of nine different genotypes (Table 1).Meanwhile, with primers 812 (Fig. 3) and 856 (Fig. 4), it was possible to identify six different genotypes.Overall, these results preliminarily indicate the existence of genetic diversity in the vanilla accessions from the Totonacapan region, which could be useful for future genetic improvement studies or selection of outstanding genotypes.However, studies using a greater number of ISSR markers, as well as another type of molecular marker (RAPD), and their respective analyses of diversity and population structure, are necessary to clarify the existence of populations originating from sources other than asexual reproduction.
Overall, it is considered that propagation by cuttings and manual pollination has affected the genetic stock of vanilla, causing the genetic diversity of this species to be almost negligible (Azofeifa-Bolaños et al., 2014).However, ISSR analyses comparing banding patterns between samples allowed inferring that not all vanilla (V.planifolia) populations present in the Totonacapan region come from the same genetic background.With primer 808, nine different banding patterns were obtained, and with primers 812 and 856, six different banding patterns were obtained.
Therefore, with the use of ISSRs in this study, it is demonstrated that, in the Totonacapan region, there is the presence of at least nine genotypes, which, in turn, are indicative of the existence of genetic diversity in this species.

CONCLUSIONS
The selected ISSR markers allowed the identification of between six and nine different genotypes among vanilla accessions (V.planifolia).Additionally, the use of ISSRs could lay the groundwork for the generation of specific molecular markers for agriculturally important genotypes.
However, it is important to further explore the search for more polymorphic molecular markers for vanilla, as genetic diversity present in vanilla accessions may be overlooked but not detectable with the ISSRs used in this study, due to their dominant nature.
Vanilla tissue collections were conducted in June 2021.The Vanilla planifolia plants sampled belong to the Vanilla Germplasm Bank of INIFAP, CEIXTA, which were collected from different locations in the Totonacapan region, in the state of Veracruz.The plant material collection consisted of one leaf per individual.Most of the collected tissue was from young leaves, each leaf Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.1, p. 297-306, jan./mar., 2024 designed by the University of British Columbia, which allowed the selection of oligonucleotides 808 [(AG)8C], 812 [(GA)8A], and 856 [(AC)8YA], for the subsequent characterization of the 60 vanilla accessions.These three ISSR markers produced the highest number of amplified DNA bands.The efficiency of the oligonucleotides in detecting different genotypes had already been corroborated in a study where the population structure was analyzed Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.1, p. 297-306, jan./mar., 2024

Table 1 .
Vanilla planifolia accessions from the Totonacapan region preserved in the Germplasm Bank of CEIXTA.