Vegetal material

The experiment was conducted in two periods: october 2009 to may 2010 and september 2012 to april 2013, the Scientific Research Center of Yucatán A.C. (CICY) in Merida,Yucatan, Mexico. The average annual temperature of the experimental site is 26.1 °C and precipitation 1.012 mm (Conagua). From the cross between creole papaya line 7 (hermaphrodite, pulp yellow-orange) and papaya Maradol 22 (hermaphrodite, orange-red pulp), the 42 seedlings F1 were developed and self-pollinations of H90B, H77B and H70B (genotypes F1) were performed for the development of 162 seedlings F2 of which 83% of the population was ruled out for evaluation by the attack of a viral illness. This research was conducted opencast in a nursery with perimeter fence. The plants were irrigated handled with proper fertilization and pest and weed control following the technology package described in (^{Cituk, 2006}). The parents are homogeneous and stable materials.

Molecular characterization

The leaf material F1 and F2 seedlings was used and a method of DNA extraction based was used on CTAB suggested by ^{Doyle and Doyle(1990)}, with some modifications. They were amplified by PCR molecular markers CPM1815YC2 (^{Chen et al., 2007}) and CPFC1 (^{Blas et al., 2010}) associated with the character of sex and color of pulp, respectively; QTLs associated markers addition to fruit morphology such as P3K1700CC, ctg-43C0, CPM1556C0 and P3K4311bK0 for weight P3K6372CC and P6K969CC for length, CPM1550C0 for diameter and P3K2426aK3 to form (^{Blas, 2008}). The fragments were visualized by agarose electrophoresis 5%. The expected fragment size was calculated in silico with prfectBLAST software, which indicated the approximate migration of the amplified for molecular markers in parental (L7 and M22) and progeny F1 and F2 product.

Morphological characterization

Throughout the F1 progeny and only 17% of population F2 three fruits per plant were harvested at physiological maturity, this maturity was determined according to ^{Santamaria et al. (2009)}. The fruits were transported to the laboratory of Molecular Physiology CICY and weight were evaluated, the length, diameter and shape and color of the fruit-based descriptors reported by the ^{UPOV (2010)}.

Statistical analysis

The principal component analysis of the correlation matrix between evaluated and standardized morphological characters was performed using the numerical taxonomic system (NTSYS 2.1).

Molecular analysis

The molecular markers linked to QTL characters of commercial interest (quantitative trait loci for its acronym in English) are a tool to increase the efficiency of traditional breeding programs; as they serve to identify, select and maintain genotypes containing the combination of desired alleles and to discard those that do not occur; allowing a decrease in demand for time and resources in studies aimed at the selection of elite genotypes (^{Valadez and Gunter, 2000}).

Molecular marker CPM1815YC2: sex identification Carica papaya L.

The size of the expected fragment molecular marker CPM1815YC2 was predicted bioinformatic program by prfect^{BLAST (Santiago and Ramirez, 2012}) was 167 bp. This molecular marker linked to the sexual nature of Carica papaya L. amplified a double band or single-band, depending on the type of plant analyzed. The amplicon size genotypes dual band corresponds to 175 and 195 bp respectively, while in genotypes with a single band of 190 bp (Figure 1a). The creole parental Line 7 and Maradol 22 had double amplified band, as both are hermaphroditic plants. Similarly, double band appeared in 28 genotypes F1, while the remaining 14 individuals F1 plants corresponding to one female (Figure 2a) band was observed. In the F2 progeny of self-pollination generated H90B, H77B and H70B showed 35, 40 and 39 genotypes dual-band, and 19, 14 and 15 genotypes with a single amplified band, respectively (Figure 3a).

Figure 1 Electrophoresis molecular marker sex CPM1815YC2 (a) and colored pulp CPFC1 (b) amplified parental Creole line 7, Maradol 22 and a female wild plant and light yellow pulp Carica papaya L., and eight QTLs associated length (c), weight (d), diameter (e) and form (f) of the fruit, agarose gel high resolution 5% stained with ethidium bromide. Molecular weight marker 10 bp and 50 bp. 

Figure 2 Electrophoresis molecular marker sex CPM1815YC2 (a) and colored pulp CPFC1 (b) parental Criolla amplified L7, Maradol 22 and F1 progeny derived from the cross of Carica papaya L. L7xM22 L7: population Creole line 7, M22: Maradol 22, CFFM: wild female, H: hermaphroditic plants agarose gel 5% ethidium bromide stained. MM: 1 Kb. The photography is included flower and fruit for each F1 progeny obtained at 6 and 9 months after taking the DNA sample, respectively. 

Figure 3 Electrophoresis of molecular marker sex CPM1815YC2 (a) and colored pulp CPFC1 (b) parental Creole amplified L7, Maradol 22 F2 progeny from selfing of H90B, H77B and H70B in Carica papaya L. L7: population Creole line 7, M22: Maradol 22, CFFM: wild female, H: hermaphroditic plants agarose gel 5% ethidium bromide stained. MM: 1Kb. The photography flower and fruit for each progeny F2 obtained at 9 months after taking the DNA sample is included. 

These results agree with those reported by ^{Chen et al. (2007)} who tested this molecular marker in Hawaiian papaya materials and identified as female and hermaphrodite genotype AU9 and Sunup, respectively with a double band respectively. Our data show that 42 plants in the F1 progeny are hermaphrodites 66.6% and 33.3% are female (ratio 2: 1; hermaphrodites: female). This relationship is expected in an allelic segregation of a selfed hermaphrodite plant or both parents herms as reported by ^{Ming et al. (2007)}; ^{Niroshini, et al. (2008)}.

While in the F2 progeny derived from self-pollination of H70B, H77B and H90B presented on the one hand 72.2%, 74.4% and 64.8% of allelic condition hermaphrodite, and 27.8%, 25.9% and 35.2% female, respectively allelic condition. These results are consistent proportionately with those reported by ^{Esquivel et al. (2008)} for HGxMR cultivars (65.5 and 34.5%, hermaphrodite and female, respectively), HGxMA (57.8 and 42.2%, hermaphrodite and female, respectively) and Red Maradol (68.9 and 31.04%).

Sexual Carica papaya L. type can only be displayed until started flowering (from three months after transplant approximately) as reported ^{Ming et al. (2007)}; ^{Niroshini, et al. (2008)}; ^{Reddy et al. (2012)}. So determining the sex of papaya seedlings at the molecular level before transplantation would be an advantage in saving time, space and money for the breeder, being a complement to the traditional selection of suitable plants for fruit production agreement with market demands or to incorporate them into the program selection and breeding of the species (^{Sánchez-Betancourt and Nuñez, 2008}).

In addition, papaya fruits from hermaphrodite flowers are generally elongated, with sufficient strength to withstand the mechanical post-harvest and higher commercial demand for occupying less space per unit volume at the time of packaging damage, representing savings freight; mainly for export markets (^{Muñozcano and Martinez, 2009}). The validation of the marker CPM1815YC2 to discriminate female plants hermaphroditic in 42 genotypes F1 (from L7xM22) and 162 genotypes F2 derived from self-pollination of H70B, H77B and H90B is reliable because it was achieved at the molecular level to identify and classify 100% both sex types in papaya plants which shows great potential for use in marker-assisted selection (SAM) in other genotypes wild, creoles and commercial.

Molecular marker CPFC1: color identification Carica papaya L.

The coloring papaya pulp, besides the size and shape of the fruit is an aspect that determines its acceptance by the consumer. From a nutritional standpoint, identifying and obtaining materials aimed at obtaining Carica papaya L. with orange-red pulp favors the presence of a higher content of lycopene, which gives it the orange-red color to the fruit pulp. The intensity of the color depends on the concentration of this pigment, which acts as antioxidants to benefit human health by reducing the risk of lung, stomach and prostate (^{Giovannucci, 1999}) cancer. While in genotypes yellow fruit pulp, the content of β-carotene is higher than the content of lycopene. With the color marker pulp (CPFC1) three allelic forms were observed in the progeny evaluated.

The parental M22 whose phenotypic characteristic was having reddish-orange pulp and 13 genotypes F1 showed a single band with size of 600 bp, which is homozygous recessive condition allelic. While the parental L7, which has yellow flesh and 15 individuals F1 dual band presented a 600-bp and 550 bp another characteristic allelic heterozygous genotypes. In addition, a wild material (FCam) was analyzed with color from light yellow flesh and a band of 550 bp was observed; that is, its condition is homozygous dominant allele (Figure 1b and 2b), these data were close to 568 bp calculated by prfectBLAST.

At the molecular level, the ratio F1 population was approximately 53.57% for genotypes with yellow pulp, and 46.43% for genotypes with reddish-orange pulp (ratio 1:1 approximately) (Figure 2b). Within the F2 progeny tested was observed that the recessive allele of orange-red color introgression 100% for F2 progeny from genotype H90B, in 98.1% of the genotypes from H70B and 92.6% of the genotypes from H77B (Figure 3b). Significantly, in the F2 population from H70B, heterozygous genotype H70-57 presented segregating parental condition L7, moreover, in the F2 population from H77B, the genotypes H77-12, H77-52, and H77- 54, they presented segregating parental heterozygous condition L7 and only genotype homozygous dominant H77-43 presented condition similar to that presented the FCam.

Allelic condition obtained with the color marker CPFC1 in F1 and F2 progeny of this research coincided with the reporting ^{Blas et al. (2010)}, who indicate that the material SunUp color red pulp amplifies a single band and has homozygous recessive condition, while Richter and Rainbow hybrids show yellow flesh and are heterozygous condition; i.e. contain two alleles showing a double amplification bands. They also mentioned that the Kapoho material has yellow flesh color but has a dominant homozygous condition. In this regard, ^{Rodriguez (2008)} reported that the characteristic yellow-fleshed (dominant allele) predominates over the orange-red flesh, so in wild populations rarely papaya appear orange-red fruits, because this color is given by recessive genes.

The molecular marker flesh color (CPFC1) the F1 and F2 genotypes derived from the cross L7xM22 facilitated early detection of genotypes with reddish-orange pulp (phenotypically could be obtained 7-9 months after transplantation) and achieved a correlation molecular much as morphological in relation to the expected segregation ratio. So this specific marker can also have an important potential use in marker-assisted selection (SAM) for the character color of pulp, used in early in nursery plants without having to wait up to nine months to display color fruit of the genotypes used in breeding programs papaya.

QTLs associated markers fruit morphology of Carica papaya L.

The sizes of fragments obtained in silico with values between 84 bp to 174 bp (data not shown) it was as expected and approximate the QTLs microsatellite (SSR) amplified by PCR in parental L7, M22 and progeny F1 and F2 (Figure 4) also consistent with those reported by ^{Mittal and Dubey (2009)} who mentioned that the amplified fragments of SSR are variant size, usually several tens to several hundred base pairs (bp), codominant, polymorphic and Mendelian segregation, attributes that could be seen in this investigation.

Figure 4 Electrophoresis is an of molecular markers associated with QTLs fruit morphology amplified in line 7 and Maradol 22 Criolla Carica papaya L. parental and progeny F1 (a) and F2 (b). The photography fruit includes corresponding each progeny F1 and F2, obtained 9 months after taking the DNA sample. MM: 50bp. 

The eight QTLs associated characters morphology fruit parental L7 and M22, are shown in Figure (1 cf) 2 associated length (1c), 4 associated with weight (1d), one associated diameter (e) and 1 to form (f) of the fruit. None of the eight QTLs or parental and F1 and F2 progeny showed molecular and phenotypic correlation (Figure 4, Table 1); i.e. not provide useful for use in marker-assisted selection for papaya fruit characteristics such as length, weight, diameter and shape of fruit information. It is possible that the lack of correlation is because these characters exhibit a wide variation along a continuous gradient attributed to the interaction of two or more genes (polygenic) (^{Obando et al., 2008}). It is not excluded that there pleiotropic effects on QTLs associated with papaya fruit morphology, which are closely related to the genes ovate, sun and fw.2.2 present in the genome of tomato and orthologous regions in the genome of papaya (^{Paull et al., 2008}; ^{Blas et al., 2012}.

Table 1 Mean values associated morphological characters the size and shape of fruit progeny F1 and F2 intraspecific crosses from Carica papaya L. L7xM22. 

In other species such as tomato, it has been suggested that factors such as fertilization, temperature and humidity will directly influence the expression of the sun gene regulating early elongation pericarp ovary, ovate gene involved in elongation pericarp ovary after fertilization and fw2.2 gene that regulates cell division and fruit development. It is assumed that the effect may be similar papaya, so that the QTL that are influenced by these genes will be affected, such is the case of some genotypes. Or, these QTLs initially evaluated in Hawaiian papaya, are not associated with morphological characteristics of the fruit such as length, weight, diameter and shape of the fruit in hybrids Maradol x creole material Carica papaya L.

Morphological analysis

The molecular identification of sexual coincided with phenotypic data recorded and evaluated three months after transplantation, both parental and F1 and F2 of Carica papaya L. (Figures 2a and 3a) progeny. The F1 population obtained from intraspecific crosses L7xM2 and F2 from self-pollinations of H90B, H77B and H70B genotypes developed only hermaphrodite and female plants. For F1 values were 28 hermaphrodites and 14 female; it is representing 67% of hermaphroditism and 33% of feminism. Within the F2 percentages were 64%, 72% and 67% of hermaphroditism and 36%, 28% and 33% of feminism, for H90B, H77B and H70B, respectively, who remained close to the ratio of 2:1 expected.

Similarly the molecular identification of fruit color was compared with the morphological characterization of the color of the fruits made between 7 and 9 months later, since the plants in both parental and progeny F1 and F2 corresponding issued fruits (Figures 2b and 3b). Similarly 7 and 9 months after taking samples for molecular analysis of QTLs associated with fruit shape, variability in the length, diameter, weight and shape of the corresponding fruits it was characterized and shown in Figure 4 and in Table 1.