The papaya, Carica papaya L., is a native of Central and South America. It is a member of the family Caricaceae. This family consists of 55 species (Dallwitz 1980). Badillo (1971) placed these species into four genera, namely: Carica, Cyclimorpha, Jacaratia and Jarilla. Carica is the largest genus, with 22 species (Purseglove 1968). Of these 22 species, Carica papaya is the most economically valuable, and is widely cultivated for its edible fruit. The family is characterized by trees or shrubs that are erect, small, soft-wooded and fast-growing. Often dioecious or monoecious, they grow to a height of 3-6 meters. The trunk is usually unbranched, and crowned with large spirally arranged leaves which give the tree a palm-like appearance. The stem and the leaves produce a milky sap when wounded, a feature found in all members of this family (Purseglove 1968).
In general, there are three sex types in papaya: male, female and hermaphrodite. However, they cannot be distinguished at the seedling and vegetative stages of growth. In an open-pollinated species such as papaya, the selection of the appropriate sex type of the progeny for commercial planting would be beneficial, since only the female and hermaphrodite plants are grown for fruit. The identification of sex types prior to propagation, especially in polygamous plant species with a long juvenile cycle such as papaya, would result in higher fruit production and increased profitability.
In sub-tropical areas, dioecious varieties such as Honey Gold (Africa) and Richter Gold (Australia) are preferred, because of their high yields (Allan 1981). Conversely, in the Philippines, hermaphrodite lines are preferred for commercial production because of their high yield potential, inbreeding ability and crop uniformity over several generations. In both cases, being unable to identify the sex type of the seedling prior to planting is a limiting factor.
The sex type of a papaya plant can only be identified 3-6 months after transplanting, when the flowers develop. Several morphological characteristics such as seed coat color and root morphology have been associated with the sex type of papaya. Females have been described as having a seed coat which is lighter in color and branched root morphology, while males are believed to have darker seed coats and straight root morphology. However, these claims have not been proven scientifically. A loose linkage between flower morphology and sex type has been identified (Storey 1953), but sex determination based on flower morphology is not possible until four months after planting.
Cytological studies have also been conducted to determine sex types in papaya, in the hope of identifying the possible existence of a heteromorphic pair, an unpaired chromosome or any chromatin body that may indicate a sex difference (Datta 1971). However, none have been recognized.
A colorometric test for total phenol content could distinguish females (86%) from males (77%), but could not detect the hermaphrodites (Jindal and Singh 1976). Paper chromatography also indicated that transcinamic acid is dominantly expressed in the leaves of hermaphroditic seedlings, but females and males could not be differentiated (Paller 1988). In addition, isozymes (gene products that are phenotypically multiple forms of an enzyme) have been exploited to identify markers that could co-inherit with sex type in papaya. Using the banding pattern of cationic peroxidase, males could be differentiated from females, but females could not be distinguished from hermaphrodites (Sriprasertsak et al. 1988).
The failure of morphological tags, cytological evidences and isozyme markers to determine the sex type of papaya at the seedling stage has led to the analysis of the DNA to determine sex differences. Markers for sex type in papaya that could be generated through DNA analysis using PCR technology is seen as a reliable strategy. This is because direct analysis of the genomic DNA allows an accurate determination of genetic variations and assessment of similarities between individuals and populations, as well as between species and cultivars, thereby eliminating the components of phenotypic variation and interaction between different alleles of a locus or different loci (epistasis). For example, through PCR, markers were generated by DNA amplification fingerprinting (DAF) for male, female and hermaphrodite progenies of a cross between Khaeg Dum (Thai cultivar) and Richter Gold (Australian cultivar) (Somsri et al. 1997). In addition, randomly amplified polymorphic DNA (RAPD) markers were also developed for interspecific Carica hybrids (Magdalita et al. 1998). However, these techniques are more complicated than ordinary PCR, so that their use is very limited. Therefore, the development of simple PCR markers linked with the sex determining locus, and those that co-segregate with sex, is needed as a reliable genetic marker for predicting the sex of papaya at the early seedling stage.
Importance of Sex Prediction in Papaya
Propagation of papaya by seed is still the most practical method of raising the crop, because it is efficient and economical. Vegetative methods of propagation, such as the use of cuttings, grafting and tissue-cultured materials, are available but they are laborious and expensive. Commercial papaya planters and most backyard growers depend solely on seed as planting materials. There are several reasons why a desirable sex type of papaya plant be known to the grower before planting.
The papaya usually flowers 3-6 months after transplanting, and produces ripe fruits at the age of 9-14 months. The waiting time from planting to harvesting is long, so growers need to be sure that a seedling is a female or a productive hermaphroditic plant, either of which will give a good harvest. The determination of the sex type of papaya seedlings prior to the flowering stage would avoid the need for removing undesired sex types (e.g. males) from the field, thus saving labor, time and other resources.
Under Philippine conditions, since papaya ringspot virus (PRSV) is often present, the grower should be assured that the seedling he is planting will bear fruit a year after planting. Papaya is presently being grown as an annual crop because of the virus problem. The hermaphroditic papaya exhibits sex reversibility depending on prevailing environmental conditions. Selection of the productive hermaphroditic type (ie. elongata) with desirable fruit form is necessary to assure the grower of a productive crop.
A knowledge of the sex type of papaya is important in selecting parents for use in hybridization work. Crosses between females and hermaphrodites will give all fruit-bearing progenies. In addition, for micropropagation, the early detection or identification of the sex type of a particular papaya seedling would be advantageous, since the desired sex type can be selected prior to micropropagation. This will ensure that the resulting micropropagated plants are 100% either females or hermaphrodites.
There are three types of flowers in papaya: male, female and hermaphrodite or bisexual ( Fig. 1(0)). The inflorescence is a cyme (Storey 1969). Male inflorescences are long, pendulous and freely branching. They are in panicles, 25-100 cm long, pendent, sessile with a small five-toothed cup-shaped calyx, trumpet-shaped corolla with light yellow lobes and 10 stamens in two whorls alternating with the petal lobes. The female flowers are solitary or in few-flowered cymes, 3-5.5 cm long with a yellowish green cup-shaped calyx. The corolla has five petals that are twisted, whereas the ovary has numerous ovules and a fan-shaped stigma. There are two types of hermaphrodite flowers: the elongata type with flowers with short peduncled petals, 10 stamens in two series and an elongated ovary, and the pentandria type, with flowers similar to female flowers but with five stamens. Intermediate forms occur in which some or all of the stamens become carpelloid and produce ridged or irregularly shaped or cat-faced fruits. The proportion and type of hermaphrodite flowers produced may vary, depending on the variety, the age of the tree, the season, the soil moisture, the nitrogen content of the soil, the temperature, daylength, defoliation, defloration, etc. (Awada 1958).
Papaya flowers are pollinated by natural agents. They may be wind-pollinated, since the pollen is light and abundant. A few insects, including thrips and moths, can assist in pollinating the sweet-smelling flowers (Purseglove 1968). The stigma is receptive prior to anthesis, and remains receptive for several days or until the stigmatic lobes turn brown. Self-pollination of hermaphrodite flowers can easily be achieved by bagging each flower in a glassine bag before anthesis, and then keeping it covered for at least five days until the fruit begins to develop. Cross-pollination is done by dusting the pollen onto the stigma of a female flower that is at the balloon stage, and then covering the flower with a glassine bag immediately to prevent contamination. Fresh pollen is preferred for hybridization. However, pollen stored for about six months at 10oC and 10% relative humidity can be also used (Cohen et al. 1989). The pollinated fruit is ready for harvest 4-5 months after pollination. Fruits that develop from hermaphrodite flowers are generally elongated, cylindrical, obovoid or pyriform, depending on the variety, while those from female flowers are rounded or spherical to oval in shape ( Fig. 2(0)). In Australian varieties, fruits that develop from sexually ambivalent males (SAM) are elongated in shape (Aquilizan 1986).
GENETICS OF SEX EXPRESSION
Sex expression in papaya is controlled by a single gene, with three alleles which have a pleiotropic effect (Hofmeyr 1941, Storey 1953). The sex homologues were designated as: M for male, MH for hermaphrodite and m for female. All combinations of dominant alleles, such as MM, MHMH and MHH, are lethal to the zygote. This makes all males and hermaphrodite into enforced sex heteroxygotes. Twenty-five percent of the seeds in their fruits are non-viable. The genotypes for sex are Mm for female, MHm for hermaphrodite and mm for female. Using these sex genotypes, there are eight possible cross combinations that could be made with various segregation ratios, as indicated in Table 1(0).
Self-pollination in males, cross-pollination between males, and cross-pollination between males and hermaphrodites, can all be done using the sexually ambivalent males (SAMs) that produce perfect flowers during certain periods of the year. Male and hermaphrodite trees undergo various degrees of sex reversal, depending on seasonal changes and climate (Awada 1958). The female tree is the most stable form.
Sex Prediction Studies in Papaya Using PCR
Our understanding of the complex genomes of a eukaryotic organism has been enhanced by the discovery of the in vitro synthesis of a specific stretch in the DNA. This technique of gene amplification was originally conceived by Kary B. Mullis in 1985, while he was pondering a proposed experiment on DNA sequencing (Mullis 1990). The technique is now popularly known as polymerase chain reaction (PCR). It is an in vitro and sterile method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize into opposite strands and flank the region of interest in the target DNA. It replicates a specified nucleic acid sequence in a complex DNA template, and consequently produces millions and even billions of copies of DNA fragments with defined length and sequence. This in vitro synthesis requires deoxynucleotide triphosphates (dNTPs), two oligonucleotide primers, a relatively pure DNA template and a DNA polymerase that remains stable and active at high temperatures.
The cycles involved in PCR are a reflection of in vivo DNA synthesis. PCR is made up of three basic cycles: 1) denaturation of the heteroduplex DNA template, 2) annealing of the primers at the homologous region sequences of the template and, 3) binding of the polymerase enzyme and subsequent extension of the primer to synthesize the specified DNA segment. After the first cycle, two double-stranded DNA fragments are produced which serve as templates for the following reaction. The repetition of the cycles to about 40 therefore leads to an exponential amplification of the defined DNA segment.
In predicting the sex type of papaya, the four 20-Mcr oligonucleotide primers shown in Table 2(0) are used in the PCR amplification. Primers T1-F and T1-R (primer sequence provided by the University of Hawaii) produced a 1.3 kb PCR product in both females and hermaphrodites, while W11-F and W11-R produced a 0.8 kb PCR product in hermaphrodites only. Thus, the hermaphrodites are distinguished by having two distinct bands (1.3 and 0.8 kb). Females have a single band (0.8 kb), while males have no band.
The standardized reaction mixture being used for PCR has a volume of 25 ?L. The components of the reaction are indicated in Table 3(0). The genomic DNA should be loaded individually into each reaction tube. The other components are mixed together in one tube and suspended over ice. This mixture is called the master mix. From the master mix, 21 ?L is transferred by aliquot into each reaction tube. The reaction tubes are placed into the PCR block, which has already been brought to 95oC. In addition to this initial denaturation at 95oC, the denaturation, annealing and polymerization steps are run for 25 cycles. After these are completed, the reaction remains in the reaction block at 4oC until ready for electrophoresis. Conditions used for PCR using a MJ PCR machine are shown in Table 4(0).
To test the reliability of the PCR technique in predicting the three sex types in papaya, three genotypes producing progenies with known segregation ratios were used. These genotypes included the following.
Cariflora. This is a dioecious inbred line producing male and female progenies only. It is used as a female parent of the Sinta F1 hybrid. It was released originally by Conover, Litz and Malo (1986) in Florida as a variety tolerant to papaya ringspot virus.
Cavite. This is a gynodioecious inbred line producing hermaphrodite and female progenies. It is the male parent of the Sinta F1 hybrid. The original tree, which has moderate tolerance to papaya ringspot virus, was selected in Silang, Cavite in 1983. Inbreeding, selection and screening for PRSV were conducted on the progeny until a tolerant line was derived.
Sinta hybrid. This is a gynodioecious hybrid from a cross between Cariflora x Cavite (Villegas 1995). It produces hermaphrodite and female progeny only. This hybrid is moderately tolerant to papaya ringspot virus. The sex types and ratios of the progeny of these genotypes are shown in Table 5(0).
Forty-five Sinta hybrids, and 25 Cariflora and 25 Cavite Special plants, were used. All plants were at the vegetative stage (before flowering commenced). The genomic DNA of these samples was extracted using the Doyle and Doyle (1987) method, and used for PCR. After the gel was stained with ethidium bromide and visualized under UV light, it was found that there were 23 and 13 hermaphrodite plants of the Sinta hybrid and Cavite Special, respectively. Three hermaphrodite plants had two distinct bands: a 1.3 and 0.8 kb fragment. On the other hand, there were 22 female plants of the Sinta hybrid, while there were 12 Cavite Special and 14 Cariflora hermaphrodite plants. The female plants were found to have only one distinct band, a 1.3 kb fragment. In addition, it was found that there were 11 male plants of Cariflora, none of which had a band. In the male plants, there is no available binding site for any of the primers used, hence the failure to flank any stretch in the DNA template, resulting in the absence of PCR products. In this way, the male genotype can be differentiated from the female and the hermaphrodite. The negative control did not show any PCR product.
A limiting factor in the use of this marker is the possible occurrence of false-negative results. Thus, during the PCR run, the male should be run under an optimal genotype and the negative control should always be present. In addition, the PCR conditions and the PCR reaction mixture should be accurately prepared, to ensure the accuracy and consistency of PCR amplification reactions.
The 45 Sinta hybrid plants and the 25 plants each of Cavite Speical and Cariflora were inspected during the flowering stage to determine their sex type, in order to verify the results of the prediction obtained using the PCR technique. The results of the prediction by PCR was consistent with the observed sex type of the plants tested, as shown in Table 6(0). This result shows a 100% accuracy in the prediction of the three sex types in Sinta hybrid, Cavite Special and Cariflora varieties. For instance, in the Sinta hybrid, 22 females and 23 hermaphrodites were identified using PCR. This prediction corresponded with the number of females and hermaphrodites actually observed during the flowering stage in the field ( Table 6(0)). A sample of the gel representing the female and hermaphrodite genotypes of the Sinta hybrid is shown in Fig. 3(0) and Fig. 4(0).
For the Cavite Special, 12 females and 13 hermaphrodites were identified using the PCR technique. The same number of females and hermaphrodites were observed at the flowering stage in the field ( Table 6(0)). The gel representing the females and hermaphrodites of the Cavite Special variety is shown in Fig. 5(0). For the Cariflora variety, 14 females and 11 males were predicted by PCR. A sample of the gel representing the females and males is shown in Fig. 6(0). The same number of females and males were actually observed in this variety when the same plants grew to the flowering stage in the field ( Table 6(0)).
The observed frequency of females, hermaphrodites and males, identified as such both by PCR and field observation, in the three varieties (Sinta hybrid, Cavite Special and Cariflora) were tested for goodness of fit (using Chi-square) with the expected sex segregation ratios given by Storey (1953). The derivation of the female, hermaphrodite and male progenies of Sinta hybrid, Cavite Special and Cariflora, including the result obtained by Chi-square tests, are as follows. Female and hermaphrodite progenies of Sinta hybrid were obtained from a cross between female Cariflora and hermaphrodite Cavite Special. The expected sex types are female and hermaphrodite at a ratio of 1:1. Chi-square goodness of fit at 1% level of significance indicates that the observed frequencies match the predicted segregation with a X2 value of 0.36 ( Table 7(0)). The sibbed (cross of female x male) female Cariflora that is expected to segregate at 1 female: 1 male also matched with the expected sex segregation ratio, with a X2 value of 0.36.
The primers used in this prediction study are highly specific, because of the large number of bases (20-mer nucleotides). Hence, the chance that the primers would find a homologous sequence in any DNA other than the target site is low. The technique of sex prediction by PCR is much more precise than the colorometric test (Jindal and Singh 1976), chromatographic analysis (Paller 1988) or isozyme markers (Sriprasertsak et al. 1988).
The PCR-based markers for sex prediction in papaya have the advantage that they are not affected by environmental conditions in which the plants are grown or by epistasis (gene interaction). Thus, sex prediction can be done at any developmental stage of plant growth. However, the PCR technique for sex prediction in papaya at this point in time is costly. It also requires technical expertise and sophisticated equipment (PCR machine, electrophoresis). At present it would seem that the technique is useful only for experimental purposes, particularly in developing countries. However, if this technique could be converted into a dip-stick type method (eg. color reaction type) that is fast, reliable, practical and cheap, it would become affordable and of great value to plant biologists and papaya growers.
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Index of Images
Figure 1 Different Sex Types of Papaya Flowers (a) Female (Pistillate) (B) Hermaphrodite (Bisexual/Perfect); and (C) Male (Staminate)
Figure 2 The Shapes of Papaya Fruit Are Related to the Sex of the Flower Types of Papaya Plants. Hermaphrodites Produce Elongated Fruits (a), While Females Produce Rounded Fruits (B).
Figure 3 PCR Banding Pattern of 'Sinta' Papaya Plants Grown in the Field Generated by Primers T1-F, T1-R, W11-F and W11-R. Lanes 1 and 13: Molecular Size Markers (???? X174 Dna, Digested with Hae III, Lane 2: Negative Control (N, without Dna Template), and Lane 3-12: 'Sinta' Papaya Hybrids
Figure 4 PCR Profile of 'Sinta' Papaya Hybrids Cultured in Vitro. Lanes 1 and 17: Molecular Size Marker (?, ??X 174 Dna Digested with Hae III), Lane 2: Negative Control (N, without Dna Template), and Lane 3-4: 'Cariflora' Female, Lane 5-6: 'Cavite Special' Hermaphrodite, and Lane 7-16: Their Progenies ('Sinta' Papaya Hybrids)
Figure 5 PCR Profile of the 'Cavite Special' Generated by Primers T1-F, T1-R, W11-F and W11-R. Lane 1 and 13: Molecular Weight Markers (???? X 174 Dna Digested with Hae III), Lane 2: Negative Control (N, without Dna Template), and Lanes 3-12: Predicted Sex Types (Females and Hermaphrodites) of the Representative Plants of 'Cavite Special'.
Figure 6 PCR Profile of the 'Cariflora' Papaya Generated by Primers T1-F, T1-R, W11-F and W11-R Lanes L and 13: Molecular Weight Markers (???? X174 Dna Digested with Hae III), Lane 2: Negative Control (N, without Dna Template, and Lane 3-12: Predicted Sex Types (Males and Female) of 'Cariflora'.
Table 1 Cross Combinations Involving the Three Sex Genotypes of Papaya, and the Resulting Sex Segregation Ratios of the Progenies
Table 2 Primers Used for PCR and Their Nucleotide Sequences. the Primer Sequence Was Provided by the University of Hawaii.
Table 3 Components of the PCR Mixture and the Volume Needed for One Standard Reaction Used for Sex Determination in Papaya
Table 4 The PCR Cycling Conditions Used for Sex Determination in Papaya
Table 5 Genotypes, and the Ratios of the Sex Types of the Progeny
Table 6 Comparison of the Predicted Sex Type Shown by PCR, and the Actual Sex Type of the Plants As Observed in the Field at the Flowering Stage for the Three Papaya Genotypes
Table 7 Segregation Ratio of Various Crosses of the Three Types of Sexes Involving Sinta Hybrid, Cavite Special and Cariflora
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