Vegetal material

The plant material was selected from a population with a broad genetic base of parthenocarpic plants of bush squash, 46 genotypes from the free recombination of progeny with diallel crossing (method IV of 1989), of seven experimental varieties type round zucchini. This population was developed ex profeso in the Genetic Improvement Program of the Plant Breeding Department of the Autonomous University Chapingo, Mexico.

Molecular analysis

The molecular analysis was carried out in the Plant-Pathogen Interaction Physiology Laboratory (FPP) at the Postgraduate School, Mexico. The genomic DNA of 46 genotypes was extracted from young, healthy and lyophilized leaves with the protocol modified by ^{Rojas et al. (2003)}, known as the combined method: MLO and CTAB 3%.

DNA quality was verified by electrophoresis in 1.2% (w/v) agarose gels stained in a solution of ethidium bromide (10 mg ml^-1) and by means of an ultra-low volume spectrophotometer (NanoDrop ND-1000 Thermo Scientific^® V3.7) the absorbance (260/280) was determined in a range of 1.33 to 1.94, the DNA concentration was 202.3-512.1 ng µL^-1.

The polymerase chain reaction (PCR) was carried out for the ISSR with an initiator (GATA)₄ and for RAMP, four random sequence primers of the Operon series A were used (OPA: 05, 10, 13, 19) combined with (GATA)₄, these primers were synthesized by the Institute of Biotechnology of the National Autonomous University of Mexico (UNAM). The selected initiators are the result of standardization of the protocol and they were the ones that presented polymorphism and marked considerable differences in the banding, among the genotypes.

The volume and concentration of each reagent in the reaction mixture for the PCR was adjusted to 25 μL as the final volume, which contained: 2.5 μL of Buffer [10 X ], 1.5 μL of MgCl2, [30 Mm μL^-1 ], 0.5 μL of Dntp’s [2.5 Mm μL^-1 ], 2 μL of (GATA)₄ [10 pmol μL^-1 ], 0.2 μL of Taq DNA-polymerase (Amplificasa^®) [5 U μL^-1 ], 4 μL of genomic DNA [20 ng μL^-1 ] and 14.05 μL of sterile deionized water, in the case of ISSR, while for RAMP, 2 μL of each random primer was added per reaction (OPA05, OPA10, OPA13 and OPA19) [10 pmol μL^-1 ] and the water was adjusted to 12.05 μL, the combinations of each RAMP were as follows: OPA05+(GATA)₄, OPA10+(GATA)₄, OPA13+(GATA)₄ and OPA19+(GATA)₄. The DNA was placed individually to each tube according to the genotype number of squash to be characterized.

An APOLO^® thermocycler was used (Instrumentation ATC 201, series 00449), thermocycling conditions for ISSR consisted of a pre-denaturation cycle of 1 min at 94 °C, 40 cycles from 40 to 94 °C, 1 min at 40 °C and 1 min at 72 °C, followed by a cycle of 8 min at 72 °C and a period at 4 °C of final extension, while for RAMP the conditions consisted of a pre-denaturation cycle of 2 min at 94 °C, 35 cycles of 1 min at 94 °C, 2 min at 50 °C and 3 min at 72 °C, followed by a 10 min cycle at 72 °C and a final extension period at 4 °C.

The amplification products obtained from the ISSR and RAMP were separated by means of electrophoresis in a 2.5% (w/v) agarose gel at 85 volts, for a period of 4.5 h. The gels were stained with ethidium bromide and the bands were visualized under ultraviolet light in a photodocument (model Gel Doc 2000, BIO RAD^®), the photo of the gel was captured with the program Quantity One 4.0.3. The 1 Kb molecular weight marker (Invitrogen^®) was used on both sides of the agarose gel.

Statistical analysis

The statistical analysis was carried out separately for each of the molecular techniques. In the conglomerate analysis, genetic distances were calculated with the simple mating coefficient, ^{Sokal and Sneath, (1963)} and a similarity dendrogram was obtained by means of the arithmetic average method, unweighted by pairs between groupings, type UPGMA (^{Adams et al., 2000}). The robustness was tested by means of 1 000 resampling in the matrices of genetic distances with α= 0.05, the reliability of the results was verified by means of a ^{Mantel test (1967)}. As parameters of genetic diversity were considered: the percentage of polymorphic loci and the rank of polymorphism in base pairs (bp), the genetic distances of similarity and the range of genetic distances (RDG) between groups and genotypes. The NTSYSpc computer package version 2.21 h was used (Rohlf, 2009).

Analysis of polymorphism

A total of six amplified loci were produced, which represented 100% of the polymorphism generated by the initiator (GATA)₄ used for the ISSR and with a molecular weight range in the amplification of polymorphic loci from 506 to 1 636 bp (Table 1). These results indicate that the squash parthenocarpic genotypes have little genetic differentiation, due to the low number of loci detected with the ISSR; in this regard, ^{Vigoroux et al. (2002)}, mentions that cultivated species have undergone a strong selection pressure directed to genes that control some genomic regions, while the genes that do not have selection pressure maintain similarity with their wild relatives, the selection that the human being has made the species cultivated during its domestication and its genetic improvement have reduced the excess variation especially in the genes for which said species had allelic variation, while other loci experience modification of the diversity according to the natural effect, in this sense Restrepo and Vallejo (2008), in a work with collections of Cucurbita moshata and with polymorphism markers in the length of amplified fragments (AFLP), mention that the levels of genetic variation or higher heterozygosity are due to the distance between populations and to the anthropic exchanges that occurs in this species.

^{Pradeep (2002)} mentions that ISSRs frequently amplify 25 to 50 bands in a single reaction and allow the detection of polymorphism among individuals of the same population, evaluation of genetic diversity, as well as the distinction and identification of intraspecific varieties (particularly in species with economic importance), among others.

For RAMP, 34 loci of 37 amplified totals were polymorphic representing 91.8% and with an average of 8.5 loci per initiator. The amplification range was between 298 and 3 054 bp. The combinations in which less genetic variability was identified among the genotypes was with the combinations OPA10+(GATA)₄ and OPA13+(GATA)₄ with 25% and 8.3% monomorphic loci, respectively (Table 1), ^{Wu et al. (1994)} reported between 10 and 20 polymorphism per initiator in acrylamide gels in Arabidopsis ecotypes using the RAMP technique, which exceeded the expectations of the use of this technique with respect to other alternatives, in addition to which it mainly favors the amplification of microsatellites, without excluding the amplification of RAPD.

^{Valadez-Moctezuma et al. (2005)}, mention that with the RAMP technique it is possible to identify new profiles that share some fragments, from the profiles obtained independently with the RAPD and microsatellite techniques, also indicate that the RAMP analyzes provide a new alternative to analyze and make studies of genetic diversity practically in any genome; these results support this research because they corroborate the differences found between the ISSR polymorphism, with respect to RAMP, in the squash parthenocarpic genotypes, so the RAMP technique turned out to be very informative.

Cluster analysis

The consensus tree or dendrogram of genetic distances of similarity, obtained from the robustness of the clusters by 1 000 resampling generated five groups with ISSR and four groups with RAMP. These groups were defined mainly by the genetic variability that exists between the genotypes (Figure 1 and 2), ^{Méndez et al. (2010)} in a previous work characterized the same partenocarpal genotypes, with morphological and agronomic variables, in which they found three groupings.

Figure 1 Average dendrogram constructed from 1 000 combinations of resampling obtained by the simple mating coefficient (SM) of the ISSR (GATA)₄ amplified products for 46 squash parthenocarpic genotypes.  

The groups obtained in the dendrogram for ISSR show genotypes 3, 15, 16, 20, 25, 28, 33, 38, 47, 51, 82, 84, 97, 103, 106 and 107 in group I, with a RDG from 0.781 to 1, group II is composed of genotypes 5, 13, 14, 21, 30, 35, 45, 48, 62, 63, 70, 90, 91 and 105 (RDG from 0.802 to 1), while that group III is composed of genotypes 19, 26, 29, 41, 44, 56, 57, 87, 93 and 108 (RGD: 0.849 to 1), group IV is made up of genotypes 23, 69 and 88 with similarity genetics of 1, while group V is composed of genotypes 73, 83 and 96, this group was found at greater genetic distance with respect to the other groups (0.469), the RDG among the genotypes was 0.761 to 0.849 (Figure 1).

These results show agreement with ^{Méndez et al. (2010)}, so that the groups formed with ISSR presented the following characteristics: group I is defined by plants with long internodes, round fruits of intermediate weight, thin skin, a lot of seed and small, in groups II and V, predominate plants with short internodes, elongated fruits with low weight, thin skin, little seed and small. The group IV presents plants with intermediate internodes, round, heavy fruits, thick rind, a lot of seed and large.

While, with the results obtained with RAMP the groups were formed as follows: group I, genotypes 3, 5, 35, 47, 48 and 56, RDG from 0.729 to 0.919, group II conformed by genotypes 14, 16 , 28, 29, 30, 33, 62, 69, 83, 91, 93, 96 and 97, RDG from 0.720 to 0.945, group III composed of 13, 15, 19, 20, 21, 23, 25, 26, 38 , 41, 44, 45, 73, 84, 88, 90, 106, 107 and 108, RDG from 0.782 to 0.974 and group IV with genotypes 51, 57, 63, 70, 82, 87, 103 and 105, the latter group contains the most genetically related genotypes (RDG: 0.8352 to 1) (Figure 2). The morphoagronomic characteristics reported by ^{Méndez et al. (2010)} for these genotypes, are defined as follows: for groups I and II, plants with short internodes, elongated fruits with low weight, thin skin, little seed and small predominate. In group III, plants with long internodes prevail, round fruits of intermediate weight, thin peel, a lot of seed and small.

Figure 2 General dendrogram constructed from 1 000 combinations of resampling, using the simple mating coefficient (SM) of RAMP amplified products (OPA05, 10, 13 and 19)+(GATA)₄) for 46 parthenocarpic squash genotypes.  

Group III and IV (ISSR and RAMP, respectively), did not show a predominance of some morphological and agronomic characteristics among the genotypes. In this sense, ^{Demey et al. (2008)}, showed that the characterizations generated by morphological descriptors of those made by molecular markers, are usually independent responding in each case to different rules and evolutionary pressures.

The groups formed in RAMP presented greater stability than the groupings that the analysis of the ISSR showed, in addition there was a greater number of concordances with the groups presented by ^{Méndez et al. (2010)}, so the relationships found with the molecular markers used in this work indicate the genetic condition in which squash genotypes are found for their future use in the improvement and search for parthenocarpy. However, ^{Sánchez et al. (2000)}, mention that studies with molecular markers are not associated with phenotypic differentiation and the way in which empirical and scientific improvement has been made.

^{Demey et al. (2008)} in maize works, have suggested to combine information of different nature for diversity analysis because it allows to analyze all the variability and obtain a more precise analysis, better distribution and definition of the classification of the populations and the resolution of the results is better.

The results of the comparison between matrices of genetic distances without resampling and the average matrix of 1 000 resampling, both for ISSR and RAMP were corroborated with the ^{Mantel test (1967)}, which identified high, positive and significant correlation for both molecular techniques (r= 0.999), with 99.9% congruence between genetic distances and clusters of genotypes, about ^{Beyene et al. (2005)}, mention that significant correlations indicate that the data sets reflect the same pattern of genetic diversity and validate the use of these data to perform statistical analyzes of different types, such as clusters on different populations.