Introduction

Fruit growing in Mexico has 63 commercial species and 220 species with food potential, some species are not yet reflected in the statistics as they are grown locally in home gardens or are collected (^{Borys and Borys, 2001}), among which are those of the family Sapotaceae; of this, caimito (C. cainito L.) is native to Central America and the Caribbean Islands and the Antilles. In the state of Morelos, 1.7 ha of caimito have been reported in the municipalities of Coatlán del Río and Tetecala de la Reforma (^{Álvarez-Vargas et al., 2006}; SIACON, 2018).

Chrysophyllum L. (Sapotaceae) species are propagated widely as ornamental plants due to their colorful foliage density and edible fruits (El-Harwary et al., 2019). They are fleshy fruits, globose or rounded berry type, the pulp is fleshy, it stands out for its exquisite flavor and nutritional value, since the fruits are rich in carbohydrates, calcium and phosphorus, they can be consumed as fresh fruit, in juices or salads, they are also used for ornamental purposes for their appearance and golden color of the abaxial of the leaves (^{Morton, 1987}; ^{Pennington, 1990}; ^{Rojas-Rodríguez and Torres-Córdoba, 2012}).

It is for the above mentioned that ^{Azurdia et al. (1995}) suggests the importance of caimito as an alternative source for the diversification of agriculture as well as for fruit exports. There is little knowledge about the techniques of sexual and asexual propagation of this fruit tree, as well as about its cultivation in general, with conditions and practices that ensure a good production of high-quality fruits. To achieve this, it is necessary to produce suitable rootstocks in order to perpetuate the selected varieties or types of this plant (^{Álvarez et al., 2004}).

In this regard, ^{Crane and Balerdi (2019}) mention that the caimito can be propagated by seed, cuttings and grafting, trees that originate from seed bear fruit in 5 to 10 years, while vegetatively propagated trees can begin fruit production in 1 to 2 years, and in grafting, it has been reported that caimito produces slow-growing dwarf trees. Asexual propagation limits the number of generations that have passed since their first cultivation by humans and the degree of evolution that has occurred under human selection in these taxa (^{Zohary and Spiegel-Roy, 1975}; ^{Clement, 1989}). When the caimito is propagated by seed, it generates a wide genetic diversity, so it is advisable to multiply the trees selected for their productive capacity and quality of their fruits through grafting (^{Avilan et al., 1992}).

If different cultivated varieties are selected by contrasting phenotypes, the phenotypic variance will be greater in cultivated plants (^{Parker et al., 2010}). The knowledge of the genetic variability of the species will allow the development of programs oriented to the conservation of germplasm in situ or ex situ, certification programs, as well as provide producers with a wide selection of plants (^{Azurdia et al., 1997}; ^{Carrara, 2004}; ^{Nascimento et al., 2008}). There are different methods to characterize, identify plant species, evaluate their diversity and phylogeny, those based on morphology are the most used (^{Bayuelo-Jiménez et al., 2006}).

The first characterization studies of sapotaceous used morphological, phenological, and agronomic characters; however, classification based on these characters can be confusing because they are affected by environmental factors. The use of isoenzymes may be more useful; in this regard, they were applied to study in situ 246 types of M. sapota and 287 of C. cainito in Nicaragua (^{Benavides, 1998}; ^{Weaver, 1993}) by morphological characterization, registered a new species of caimito called Micropholis chrysophylloides in Puerto Rico; however, these can be affected by the environment and the phenological stage of the plant.

A large number of methods to identify and analyze molecular markers have also been generated, among which those based on the polymerase chain reaction (PCR) stand out, which allow amplifying specific or random sequences of DNA and require small initial amounts of it (^{Orona et al., 2006}), they are not affected by the environment and can be analyzed at any stage of development of the crop, RAPD markers are simple, efficient, economical, fast and efficiently discriminate between genotypes (^{Yasmin et al., 2006}).

Markers such as cytogenetic and molecular allow analyzing the differences between chromosomes, proteins or DNA (^{Avise, 2004}). Another technique used is the sequencing of the ndhF chloroplast gene to study the phylogeny in the family (^{Anderberg and Swensony, 2003}). Simple sequence repeats (SSRs) were used for the analysis of the variability of the chloroplast DNA in Manilkara hubery (^{Rennó et al., 2008}). RAPDs were used to determine the effect of environmental conditions on the genotype of Manilkara sapota (^{Heaton et al., 1999}) and to determine the spatial genetic structure of Chrysophyllum sanguinolentum (^{Degen et al.,2001}), as well as to study the genetic diversity of Vitellaria paradoxa (^{Fontaine et al., 2004}).

In Pouteria sapota, AFLPs have also been used to study the genetic variation of cultivated selections (^{Carrara, 2004}). ^{Majourhat et al. (2008}) used RAPDs and SSRs to characterize morphotypes of Argania spinosa and observed that RAPDs had high polymorphism and more information than SSRs. There are currently many germplasm collections with genotypes of high agronomic value that could be used as parents in genetic improvement programs; however, in many cases their degree of diversity and the relationship between materials are unknown, which makes it difficult to use them (^{Becerra and Paredes, 2000}).

Molecular markers are widely used to implement crop breeding and management programs (^{Xin-hua et al., 2007}). This method is beneficial for ecologists and conservationists by allowing the identification of samples (^{Hebert and Gregory, 2005}). ^{González-Hernández et al. (2012}) mention that, although the sapodilla (Sapotaceae) is an economically important species in Mexico, there is no information that allows generating appropriate programs for its improvement and crop management.

Due to the limited information about caimito in terms of molecular characterization that allows identifying the trees with better characteristics that could be used to conserve them and eventually integrate them into a program of long-term genetic improvement or vegetative propagation that allows having caimito orchards established in a homogeneous way and with quality fruits, the objective of this research was to molecularly characterize previously selected caimito trees in Coatlán del Río and Tetecala de la Reforma, Morelos, Mexico, using the RAPDs technique, in order to determine if there is a genetic differentiation of trees that allows the identification and selection of outstanding germplasm; through molecular markers.

Materials and methods

Thirteen caimito trees were selected based on morphological and organoleptic characteristics such as fruit flavor, higher yield, size and quality, through interviews with the producers conducted by ^{Álvarez-Vargas et al. (2006}) in Coatlán del Río and Tetecala de la Reforma, Morelos, Mexico. The characteristics for which they were selected are because the caimito fruits are red, opaque and dark, there is great variation in the hue and chromaticity of the fruits, probably due to the wide variety in colors and their intensity. The dimensions of the fruit, such as length and diameter, show a high variation with ranges of 60.3 and 54.1 cm, respectively.

Sixty percent of the total weight of the fruit corresponds to the peel, 3% to the seed and 37% to the pulp, the peel of the fruit is composed of a thin and hard epicarp, as well as part of the mesocarp that at commercial maturity forms a resistant peel, the other part of the mesocarp is purple or white, aqueous and sweet and represents the pulp of the fruit. The great variation in these characters makes it possible to search for materials with a higher proportion of pulp, to increase their potential use as fresh or processed fruit. The caimito can have up to eight seeds, as well as seedless fruits.

Caimito fruits have great variation in chemical characteristics such as soluble solids, total sugars. The average values of soluble solids are 10.1 Brix and indicate that the fruits provide a good amount of energy to those who consume them, some fruits reach values of 185.9 mg g^-1 of total sugars. Currently, few quality studies have been carried out to determine the characteristics of the fruit that are attractive to the consumer; however, large fruits with few seeds and a high concentration of total sugars are preferred (^{Álvarez-Vargas et al., 2006}). The trees were 1, 2, 3, 4, 5, 9, 10, 13, 14, 15, 17,18 and 19.

Results and discussion

The selected primers were OPB (01, 04, 05, 06, 07, 08, 09, 10, 11 and 12), SAP-01 and SAP-04, for presenting the largest number of amplified products, with brighter and better-defined bands. These primers generated 190 RAPD fragments ranging from 254 to 3 407 bps. Ninety-seven percent of the fragments were polymorphic and allowed characterizing 9 of the 13 trees studied (1, 3, 5, 10, 13, 14, 15, 17 and 19) with 71 marker fragments. In trees 2, 4, 9 and 18, no marker fragments that would allow their characterization with the primers used were obtained (Table 2). The primers that generated the largest number of markers were OPB-06 and OPB-10, the latter allowed identifying a greater number of trees (6) along with SAP-01 and SAP-04 (Figure 1). In contrast, primer OPB-11 produced only threes fragments which characterized two trees. No single primer allowed to characterize all plants.

Figure 1 Banding pattern generated by primer SAP-04 for 13 caimito trees in the state of Morelos, Mexico. 1kb: molecular weight marker 1 kb DNA Ladder. 

The methodology used allowed characterizing nine trees, the 14 was the one that showed the greatest presence of markers (30 fragments) that were produced with six primers. This indicates that the DNA of this tree had a greater number of particular DNA sequences that were complementary to primers OPB (05, 07, 10, 12), SAP-01 and SAP-04. Another tree that showed a greater number of marker bands was tree 15, with 15 marker fragments, generated by oligonucleotides OPB (06, 08, 09 and 11).

In contrast, trees 2, 4, 9 and 18 did not produce any markers, which means that the DNA molecule of these plants did not present particular DNA sequences that identified them with the use of the primers used. The organoleptic characteristics of the fruits are not reported; however, the tasting of the fruits was carried out, in flavor, the fruits of trees 3, 4, 10, 14 and 15 stood out; however, there were no markers for all these trees (Table 3).

The RAPD methodology allowed the characterization of 72.9%of the caimito trees studied, this because they amplify in a wide region of the genome; however, the possibility of finding molecular markers for all trees could have been higher if more primers had been used, in this regard, (^{Vinicius et al., 2014}), when conducting a study to find what the best marker for the identification of Sapotaceae species was, mention and conclude the importance of molecular markers, which they consider essential to adopt conservation strategies and an appropriate management plan for a given species, as they are highly discriminatory and useful for the molecular identification of the Sapotaceae specie.

The RAPDs in the present research presented a high discriminative percentage, which allowed detecting the genetic diversity for the selection of outstanding trees, promising to selection, management and conservation programs that could be efficiently implemented, molecular markers serve as a parameter to classify and allow identifying the plant (mother) material of interest to propagate.

The total and polymorphic RAPD fragments obtained in the characterization of the caimito trees were greater than those obtained by ^{Fontaine et al. (2004}), who observed a total of 67 polymorphic and 15 monomorphic RAPD loci ranging from 1 670 to 280 bp, in 179 individuals of the sapotaceous Vitellaria paradoxa, with the primers OPB-07, OPB-11, OPN-15, OPR-15, OPW-9, OPW-12, OPW-13, OPX-3, OPX-6, OPX-11, OPY-6, OPY-13, OPY-20, OPW-5, OPW-19. In this research, the number of bands ranged from 12 to 21, while Fontaine et al. (2004) only identified 1 to 6 bands, which is attributed to the difference in species.

As for the number of marker fragments, in this research, 58 different ones were obtained with the use of 12 primers, while ^{Heaton et al. (1999}), when studying sapodilla using RAPDs with 80 primers, only obtained 28 different bands, this indicates the usefulness of the oligos used in this research. In this research, 185 polymorphic fragments were obtained, while ^{Degen et al. (2001}), using the same methodology to study 68 individuals of Chrysophyllum sanguinolentum with 11 primers (OPE-02, OPE-05, OPE-07, OPY-04, OPY-06, OPY-07, OPY-10, OPY-13, OPY-14, OPY-15, OPY-16), obtained only 48 polymorphic fragments. From the above described, it can be said that the number of amplified fragments varies according to the species to be studied, as well as the sequence of indicators.

The cluster analysis obtained (Figure 2) shows that at a level of 0.71 of genetic distance, four groups form: 1) trees 1, 4, 13, 18 and 19; 2) trees 2, 5, 3, 10, 9 and 17; and 3) tree 15 and 4) tree 14. The group that integrated more trees was the 2, the greatest genetic similarity between trees also occurred in this group. Plants two and five had the lowest genetic distance (0.92), in contrast, trees 14 and 15 were the most distant from the other trees, at the same time they were the ones that showed the highest number of marker bands and they also stood out for flavor.

Figure 2 Dendrogram of thirteen caimito trees, built with the UPGMA method (average linkage). The distance coefficient refers to genetic distance. Dotted line: reference for group formation. 

Conclusions

The information generated on the molecular characterization of the caimito is one of the first references of DNA characteristics, for trees with outstanding fruit characters, which can be useful to start the process of selection and registration of varieties of this fruit species in Mexico. The RAPDs were useful for the characterization of nine caimito trees, achieving the molecular characterization of more than 72.9% of the plants studied. Trees 14 and 15 showed the highest number of marker bands and share the least genetic similarity with the other trees.

The species with potential for the conservation of germplasm in situ or ex situ are trees 15 and 15, which can enter a conservation and certification program to provide other producers with plants with the outstanding characteristics for the cultivation of caimito.