Assessment of genetic diversity and cross-compatibility among pitaya (Selenicereus spp.) genotypesAbstract
New pitaya (Selenicereus spp.) cultivars can be obtained through the selection of genotypes and the development of hybrids from intra- and interspecific crosses. This research was carried out to evaluate the compatibility of intra- and interspecific crosses between S. undatus and S. monacanthus through controlled and natural pollination, and to assess the genetic diversity of the plants using SSR molecular markers. Intraspecific and interspecific diversity was assessed using 29 SSR molecular markers. Interspecific crosses between S. undatus and S. monacanthus showed compatibility, while intraspecific crosses involving the studied genotype of S. monacanthus were not viable, and those with S. undatus resulted in low fruit set rate. Controlled interspecific crosses between S. undatus and S. monacanthus ensured a higher fruit set rate and the production of fruits with larger size and a higher number of seeds. Interspecific cross-pollination of S. undatus with S. monacanthus pollen improved the quality of its fruits. The genetic diversity of the orchard favored the production of better-quality fruits. The population of S. undatus and S. monacanthus in the studied orchard was probably formed by a single clone.
Key words
Genetic breeding; hybridization; Selenicereus; molecular markers; compatibility
INTRODUCTION
The pitaya, known as “dragon fruits” belongs to the Cactaceae family, which comprises approximately 100 genera (Barthlott & Hunt 1993). Among these, the Selenicereus genus stands out for including economically important species such as S. undatus (2n = 2x = 22), S. monacanthus (Lem.) D.R. Hunt = Hylocereus polyrhizus (F.A.C. Weber) Britton & Rose (2n = 2x = 22), and S. megalanthus (2n = 4x = 44) (Tel-Zur et al. 2011).
It has become a commercially important fruit species worldwide and presents high levels of genetic diversity (Tel-Zur 2022). Pitaya species can be self-compatible, partially self-compatible, or self-incompatible. Furthermore, in many species, the floral structure exhibits spatial separation between the anthers and the stigma. Herkogamy reduces the probability of autogamy, limiting self-pollination even in self-compatible genotypes (Cho & Ding 2021, Del Ángel-Pérez et al. 2022, Santos et al. 2025).
Pitaya breeding strategies have focused on creating cultivars that exhibit not only improved fruit quality but also other desirable traits, such as resistance to thermal stresses (e.g., temperature and radiation) and the selection of self-compatible genotypes (Oliveira et al. 2020, Tel-Zur et al. 2012, Tel-Zur 2022, Faleiro & Junqueira 2022). However, one of the main challenges is confirming the genetic diversity of the genotypes belonging to each species.
The lack of information about the origin of the cultivated genetic materials, combined with phenotypic similarity among species clones, has hindered the selection of promising genotypes (Faleiro & Junqueira 2022). This is because morphological characteristics can be influenced by the environment (Abirami et al. 2021), which highlights the importance of genetic diversity studies in these species.
In this context, molecular markers have been used to deepen genetic diversity studies, supporting conservation activities and the use of germplasm in the genetic improvement of different pitaya species (Junqueira et al. 2010, Faleiro & Junqueira 2022).
Among the markers that have been used for genetic diversity studies in pitaya, microsatellite markers or Simple Sequence Repeats (SSRs) stand out, due to their high levels of polymorphism compared to dominant molecular markers (Turchetto et al. 2017). For this reason, they are considered more efficient for estimating the levels of genetic variability in pitaya (Pan et al. 2017). Moreover, SSR markers are characterized by their reproducibility and simplicity, requiring only a small amount of DNA and allowing allele differentiation at each locus (Pan et al. 2017, Turchetto et al. 2017, Nashima et al. 2021).
Considering the limited information regarding the origin of the genotypes of the species S. undatus and S. monacanthus in the orchard under study, along with the attractive physicochemical characteristics of the fruits for inclusion in genetic improvement programs, it is essential to evaluate the genetic diversity and the compatibility of crosses.
Given the above, this study was carried out with the aim of evaluating the compatibility of intra- and interspecific crosses between S. undatus and S. monacanthus through controlled and natural pollination, as well as assessing the genetic diversity of the plants using SSR molecular markers.
MATERIALS AND METHODS
The work was conducted in three steps. The first stage evaluated the compatibility of crosses in field-grown plants from fruit set rate. The second to assess the physicochemical characteristics and seed germination of fruits resulting from intra- and interspecific crosses. The third stage analyzed the genetic diversity of the genotypes used in the crosses, using SSR molecular markers.
Compatibility of the intraspecific and interspecific crosses
The crosses were carried out in an experimental orchard of the Federal University of Jequitinhonha and Mucuri Valleys (UFVJM), located in Minas Gerais State, Brazil, at 18° 04’ 15” S latitude and 43° 28’ 15” W longitude at an altitude of 726 meters, using controlled and natural pollination. The region has an Aw climate type, classified as high-altitude tropical, characterized by a well-defined dry and rainy season.
Five-year-old genotypes of S. undatus (Haw.) D.R. Hunt (= Hylocereus undatus (Haw.) Britton & Rose) and S. monacanthus (Haw.) D.R. Hunt (= H. polyrhizus (F.A.C. Weber) Britton & Rose), obtained through asexual propagation (cuttings), was studied. The plants were alternately arranged between planting lines, with a spacing of 2 m between plants and 3 m between rows.
A total of 24 plants from each species were identified, and their floral buds were emasculated and protected during the pre-anthesis phase for intra- and interspecific cross-pollination (Figure 1). Plants of the species S. undatus and S. monacanthus were left close together as pollen donors to ensure natural open pollination. For controlled interspecific cross-pollination, pollen grains were collected and transferred at the time of anthesis.
Controlled intraspecific and interspecific crosses between S. undatus and S. monacanthus; protection of closed flower buds during the pre-anthesis phase (A); emasculation of flower buds during the pre-anthesis phase (B); collection of pollen (C); and controlled pollination during anthesis (D).
Six crosses were carried out through natural open pollination (S. undatus ♀ × open ♂ and S. monacanthus ♀ × open ♂) and controlled pollination (S. undatus ♀ × S. monacanthus ♂, S. monacanthus ♀ × S. undatus ♂, S. undatus ♀ × S. undatus ♂, and S. monacanthus ♀ × S. monacanthus ♂). The experiment followed a completely randomized design with four replications and 40 flower buds per type of cross.
Fruit set was evaluated based on the number of developed fruits. Counting was performed 15 days after pollination. The fruit set rate was calculated as the ratio between the number of fruits from each pollination and the number of pollinated flowers, multiplied by 100. Fruits were bagged (in a non-woven fabric bag) to protect them from pest attacks until harvest, ensuring the evaluation of physicochemical characteristics after harvest.
Physicochemical characterization of the fruits
All harvested fruits were evaluated to characterize size, flavor, and seed formation. Fruit weight was determined using an electronic analytical balance (0.001 g). The longitudinal (length) and transverse diameters, as well as skin thickness, were measured with a digital caliper (CaliperWithin® 300 mm), with an accuracy of 0.01 mm. Pulp yield was determined by fractioning the fruits and calculated as the ratio between the pulp weight (seeds and pulp) and the whole fruit weight, multiplied by 100.
The physicochemical characterization of the fruits was carried out by sampling 12 fruits from each pollination. The pH was determined using a Marconi pH meter (PA200) from a solution prepared with 1 g of pulp diluted in 20 mL of distilled water (AOAC 2007). Soluble solids (SS) content was measured with a digital refractometer, model PR-100 Pallet (Atago Co., LTD, Japan) (AOAC 2007). Titratable acidity (TA) was determined by titrimetry, based on the neutralization of fruit acids with a standardized alkali solution (Zenebon et al. 2008). The SS/TA ratio was then calculated.
Number and germination rate of seeds
The total number of seeds per fruit was determined by counting all seeds extracted from the fruits of each cross. Fruits were cut crosswise, and the seeds were separated using a sieve and by washing the pulp under running water. The seeds were then shade-dried for approximately 72 hours.
The weight of 1,000 seeds was estimated from eight subsamples of 100 seeds. For each treatment, seeds were randomly selected, counted, and weighed on an analytical balance with 0.0001 g precision, following the methodology described by Brazil (2009).
The germination test was carried out using four replications of 50 seeds per pollination, arranged in a completely randomized design. Seeds were placed in plastic boxes of the “gerbox” type to germinate on germitest paper moistened with distilled water in a proportion of 2.5 times the weight of the substrate (Brazil 2009).
The seeds were then incubated in a Biochemical Oxygen Demand (BOD) germination chamber set at a constant temperature of 25 °C (Alves et al. 2011). Evaluations of the number of germinated seeds were carried out on the fifth and tenth days. Only seeds producing normal seedlings were recorded, following the criteria established in the Rules for Seed Analysis (Brazil 2009).
Analysis of genetic diversity using SSR molecular markers
Cladode samples were collected from individuals of both S. undatus (n = 48) and S. monacanthus (n = 47) populations at an intermediate stage of development, before the complete transition to the intense green hue. Genomic DNA was extracted using the CTAB method, with modifications (Doyle & Doyle 1990, Faleiro et al. 2003).
The quality of genomic DNA was assessed for each sample by electrophoresis on a 1% agarose gel (MetaPhor®) in 1X Tris-borate-EDTA solution, current 90 V, stained with ethidium bromide (0.25 μg mL⁻¹; ThermoFisher®). DNA concentration was then estimated by spectrophotometry, measuring absorbance at 260 nm and 280 nm using a UV-Vis spectrophotometer (UV5100). Afterwards, DNA was diluted for subsequent analyses.
Twenty-nine SSR primers were selected based on their high polymorphic information content (Pan et al. 2017, Nashima et al. 2021) and used to assess diversity in S. undatus and S. monacanthus populations. The primers are listed in Table I. The amplification of the target regions was performed by polymerase chain reaction (PCR) in 25 μL reaction volumes, with the final concentrations of each component listed in Table II.
Amplifications were performed in a thermocycler (Bio-Rad: T-100) using Touchdown PCR (Don et al. 1991). The reaction cycle consisted of an initial denaturation step of 5 minutes at 95 °C, followed by two amplification steps and a final extension. The first amplification step comprised 15 cycles at 95 °C for 30 seconds, 65 - 52 °C for 30 seconds (decreasing by 1 °C per cycle), and 72 °C for 30 seconds. The second step consisted of 20 cycles at 95 °C for 30 seconds, 52 °C for 30 seconds, and 72 °C for 30 seconds, followed by a final extension at 72 °C for 5 minutes.
Amplification products were separated by electrophoresis was performed at 120 V on a 3% agarose gel (MetaPhor®) in 1X Tris-borate-EDTA solution. The gel was stained with ethidium bromide at a concentration of 0.25 μg mL⁻¹ (ThermoFisher®), and the amplified fragments were visualized using a photodocumenter under UV light (Loccus®).
Data analysis
The data related to fruit set, physicochemical characteristics of fruits, and germination rate were subjected to a normality test and analysis of variance. When a significant difference was observed, means were compared using the Tukey test at a 5% probability of error.
Intraspecific diversity was assessed through a descriptive analysis of the data, including the count of monomorphic, polymorphic, and non-amplified SSR markers. PolyGene software version 1.5 (Huang et al. 2020) was utilized to analyze interspecific genetic diversity.
The total number of alleles (N), expected heterozygosity (He), observed heterozygosity (Ho), and polymorphic information content (PIC) were analyzed. The genetic distance of Nei (1972) and the Unweighted Pair-Group Method with Arithmetic Averages (UPGMA) clustering were determined.
RESULTS
Fruit set, physicochemical characteristics of the fruits
Differences between the compatibility of intraspecific and interspecific crosses were observed. Interspecific crosses (S. undatus ♀ × S. monacanthus ♂ and S. monacanthus ♀ × S. undatus ♂) exhibited notably high fruit set rates, ranging from 96% to 100% in both self-compatible and self-incompatible genotypes. However, intraspecific crosses resulted in lower fruit set rates. S. undatus intraspecific crosses showed a maximum of 28.5%, while no fruit set was observed in S. monacanthus intraspecific crosses (Figure 2).
Intra- and interspecific fruit set rate between the species S. monacanthus and S. undatus from open and controlled natural pollinations. Vertical bars indicate the standard deviation of mean of each cross.
The fruits resulting from intra- and interspecific crosses presented distinct differences in weight, longitudinal and transverse diameters, pulp yield, and skin thickness, which were influenced by pollen origin (Table III). Controlled interspecific crosses generally produced fruits with greater longitudinal and transverse diameters and higher pulp yield compared to open-pollinated crosses. Specifically, fruits from the cross S. monacanthus (♀) × S. undatus (♂) exhibited the highest averages for weight (545.8 g), transverse diameter (100.5 mm), and pulp yield (84.4%), along with thinner skin (2.1 mm), when compared to fruits from the S. undatus (♀) × S. monacanthus (♂) cross.
Fruit weight (FW), transverse diameter (TD), longitudinal diameter (LD), pulp yield (PY), peel thickness (PT), soluble solids (SS), titratable acidity (TA), SS/TA ratio, and pH of fruits from intra- and interspecific crosses between S. monacanthus and S. undatus, through open and controlled pollination.
Regarding intraspecific crosses, as previously mentioned, no fruit production occurred in S. monacanthus (Figure 2). However, fruits from the S. undatus (♀) × S. undatus (♂) had reduced size, with an average weight of 309.0 g and lower pulp yield (33%) compared to fruits from natural pollination (64.7%) and controlled interspecific crosses (79.4%) in this species, possibly due to thicker peel, observing negative correlation (Table IV).
Pearson’s linear correlation coefficient between the variables fruit weight (FW), transverse diameter (TD), longitudinal diameter (LD), pulp yield (PY), peel thickness (PT), seed number (SN), thousand-seed weight (TSW), soluble solids (SS), titratable acidity (TA), and pH of fruits from intra- and interspecific crosses between S. monacanthus and S. undatus, through open and controlled pollination.
Number and germination rate of seeds
The fruits with greater weight and diameters were those with the highest number of seeds, presenting positive correlations (Table IV). Fruits from interspecific crosses with controlled pollination had seed number averages ranging from 3,760 (S. undatus ♀ × S. monacanthus ♂) to 5,143 (S. monacanthus ♀ × S. undatus ♂), while in open pollinations, seed number averages of 1,552.7 in S. monacanthus and 461 in S. undatus were observed (Table V). Furthermore, in addition to variations in the number of seeds per fruit, differences were observed in the weight of a thousand seeds and the germination rate. Fruits with the highest number of seeds showed lower average seed weights (1.8 g) and lower germination rate (88%).
Seed number (SN), thousand-seeds weight (TSW), and seed germination rate (SG) of fruits seed from intra- and interspecific crosses between the S. monacanthus and S. undatus through open and controlled pollination.
In the seeds from S. undatus fruits, regardless of the pollen source, the highest germination rates were observed (Table V). However, seeds from crosses in S. monacanthus (♀) showed lower germination rates, ranging from 79.5% to 88.5%. In this species, smaller seeds were observed, resulting in lower thousand-seed weights.
Genetic Diversity
The intraspecific genetic characterization involving 48 individuals from the S. undatus population and 47 individuals from the S. monacanthus population showed that, out of the 29 SSR primers evaluated, 16 primers (HLG02, HLG04, HLG06, HLG07, HLG09, HLG013, HLG014, HLG018, HLG021, TsuHu35, TsuHu40, TsuHu44, TsuHu45, TsuHu48, TsuHu300, and TsuHu301) were identified as monomorphic for both species (Table I). Additionally, the primers HLG05, HLG08, TsuHu31, TsuHu50, TsuHu304, and TsuHu312 did not amplify.
In S. undatus, the primers HLG016, HLG020, TsuHu38, and TsuHu47 (Table I) were monomorphic, revealing one to two alleles per locus, with band sizes ranging from 110 to 200 bp. However, HLG03, TsuHu46, and TsuHu296 did not amplify in this species.
In S. monacanthus, the primers HLG03, TsuHu46, and TsuHu296 (Table I) were monomorphic, showing one to two alleles per locus and band sizes ranging from 140 to 260 bp. For this species, the primers TsuHu38 and TsuHu47 did not amplify, while only HLG016 and HLG020 amplified polymorphic fragments within the population.
Despite the prevalence of monomorphic primers in the intraspecific evaluations, differences in amplified alleles between the two species revealed interspecific polymorphism. Five specific primers (HLG013, HLG016, HLG020, TsuHu35, and TsuHu301) displayed polymorphism when comparing S. undatus and S. monacanthus. The primers HLG013, HLG016, HLG020, and TsuHu35 presented two alleles, while TsuHu301 showed three alleles (Figure 3), enabling the estimation of genetic relationships between the studied species (Table VI).
Estimation of interspecific genetic diversity using five SSR microsatellite markers in populations of S. monacanthus and S. undatus.
SSR gel profile using the TsuHu301 primer to detect genetic differentiation between S. undatus and S. monacanthus. On the left is the molecular weight standard (100 bp ladder); A represents S. undatus (genotypes 1 at 25), and B represents S. monacanthus (genotypes 26 at 40).
The five polymorphic primer pairs revealed a total of 11 alleles in the interspecific evaluation, resulting in an average number of alleles (K) per locus of 2.2. Observed heterozygosity (Ho) ranged from 0 to 1, with an average of 0.439. Expected heterozygosity (He) values ranged from 0.423 to 0.625 across loci, with an average of 0.509. For the polymorphic information content (PIC), values ranged from 0.334 to 0.555, with an average of 0.403 (Table VI). These results indicate that the evaluated population is predominantly homozygous.
When evaluating the primers separately, HLG013 and TsuHu35 exhibited the lowest Ho values (both at zero) compared to an He value of 0.50, suggesting a prevalence of homozygous individuals for these loci. However, the TsuHu301 displayed the highest Ho value (1.00) and an He of 0.625, indicating a higher number of heterozygous individuals at this specific locus. HLG016 presented Ho and He values trending towards equilibrium (Table VI).
DISCUSSION
Fruit set, physicochemical characteristics of the fruits
The high fruit set rates observed in the interspecific crosses in this study, ranging from 96% to 100% (for both self-compatible and self-incompatible parental genotypes), clearly demonstrate the reproductive compatibility between S. undatus and S. monacanthus. Crucially, no fruit abortion was observed during the initial developmental stages, with fruits consistently reaching full maturity on the maternal plants. These results are in agreement with other studies on interspecific Selenicereus crosses (S. monacanthus × S. megalanthus; S. monacanthus × S. undatus; and S. undatus × S. costaricensis), which also report the absence of crossbreeding barriers between these species (Oliveira et al. 2020, Tel-Zur 2022, Faleiro & Junqueira 2022).
The observed fruit set from controlled pollinations (96% to 100% between S. undatus ♀ × S. monacanthus ♂ and S. monacanthus ♀ × S. undatus ♂ ) ( Figure 2) is consistent with previously reported results (72.6% to 100%) for S. monacanthus and S. undatus genotypes when pollinated with pollen from different genotypes, compatible genotypes (Tran et al. 2018, Moreira et al. 2022).
A critical finding regarding the intraspecific crosses was the complete absence of fruit production in S. monacanthus (Figure 3), highlighting the necessity of pollen from a distinct genotype for fruit formation in these individuals. This result strongly suggests that the S. monacanthus plants within the experimental orchard are clones of the same genotype, belonging to the group known to be self-incompatible in this species. Self-incompatibility is a common characteristic reported in various pitaya genotypes (Mizrahi 2014).
Self-incompatibility in S. monacanthus is attributed to gametophytic self-incompatibility, a mechanism controlled by a single S allele that prevents the growth of pollen tubes from grains produced by the same or closely related individuals. Recent research has identified specific molecular components of this system, such as HuS-RNase2 as the gynogenetic S gene, involved in ribosomal, ubiquitination-mediated, and phytosignaling pathways (Wang et al. 2023). This mechanism relies on the interaction between pistil S-RNase, a ribonuclease, and an SFB-S haplotype-specific F-box protein from the pollen, leading to RNA degradation in genetically related pollen and thus inhibiting pollen tube growth (McClure et al. 2011).
The fruit set rates observed from open pollination in S. undatus (87.9%) and S. monacanthus (87.5%) in this study did not differ significantly from rates reported by other authors, which range from 61% to 92% (Tran & Yen 2014). However, it is well established that environmental variations can significantly contribute to flower bud abortion (15% to 20%), primarily by interfering with pollen supply. This interference may manifest as reduced pollen grain viability or as limited activity of floral visitors crucial for pollination (Menezes et al. 2015b, Rech et al. 2018, Guimarães et al. 2022).
The challenge posed by environmental factors and pollen supply (in terms of fertility and availability) is further intensified in S. undatus and S. monacanthus due to their floral morphology. Herkogamy reduces the likelihood of autogamy, particularly in S. monacanthus, where the spatial positioning of the stigma above the anthers is more pronounced than in S. undatus (Santos et al. 2025). This morphology feature limits self-pollination, even in genotypes considered self-compatible (Cho & Ding 2021, Del Ángel-Pérez et al. 2022, Santos et al. 2025).
The observed differences in the physicochemical characteristics of fruits from controlled and open crosses may be influenced by several factors, such as fertilization (Rabelo et al. 2020, Alves et al. 2021, 2024), the number of fruits per cladode (Santos et al. 2023), the quantity of pollen deposited on the stigma and pollen grain viability (Lone et al. 2017, Chu & Chang 2022, Guimarães et al. 2022), and the influence of the pollen source on maternal tissues, a phenomenon known as metaxenia (Mizrahi et al. 2004).
The smaller fruit size (both in weight and diameter) observed in fruits resulting from open pollination, compared to those from controlled pollination in this study, is attributed to a lower number of seeds within these fruits (Table IV). The influence of seed number on fruit size has been reported by several authors (Menezes et al. 2015 a,b, Renfiyeni et al. 2018, Muniz et al. 2019, 2020), suggesting that fruit size is influenced by the number fertilized ovules due to reduced pollen quantity and/or low pollen viability.
The correlation between fruit size and number of seed in pitaya species is biologically explained by the development of the fruit pulp from the funicles, which are structures connecting the ovules to the placenta (Dag & Mizrahi 2005). Thus, in regions with a lack of native pollinators, or where their activity is deficient, leading to limited natural pollination, increasing genetic diversity in the orchard, combined with optimized manual pollination, offers an effective and necessary alternative for achieving consistent commercial fruit production (Moreira et al. 2022).
Another factor contributing to limiting the quantity of pollen reaching the stigma is the herkogamy observed in the genotypes of S. undatus and S. monacanthus. This morphological characteristic reduces the probability of self-fertilization (Guimarães et al. 2022, Santos et al. 2025) and thereby increases dependence on external pollinators for successful pollen transfer.
In this study, despite the availability of sufficient pollen in the experimental area, open-pollination crosses may have been affected by a lack of visits from nocturnal pollinators. This is probably due to frequent rainfall coinciding with the flowering periods of these species in spring and summer. Even in self-compatible species, climatic variations, such as precipitation and high temperatures, can interfere with the fertility of stamens and pistils, reducing pollen grain germination and resulting in smaller fruits with fewer seeds (Chu & Chang 2022, Guimarães et al. 2022).
Differences observed in the physicochemical characteristics of fruits may be related to the pollen source (Table III). According to Mizrahi et al. (2004), the pollen origin, specifically the donor species, can significantly influence various fruit traits, including fruit size, pulp yield, number of seeds per fruit, and even the time required for ripening. Fruits of S. undatus (♀) pollinated with pollen from S. monacanthus (♂) are generally larger in size and mass, in addition to exhibiting lower acidity and higher soluble solids content (Silva et al. 2011, Lone et al. 2017, Moreira et al. 2022).
The results observed regarding the improvement of fruit quality (Table III) show that the S. monacanthus genotype has promising characteristics that can be used in genetic improvement programs. Improving fruit quality is a primary objective in the hybridization of pitaya genotypes, aiming to develop new superior cultivars (Lima et al. 2013, Silva et al. 2017). In this context, the S. monacanthus genotypes are particularly noteworthy for characteristics that increase consumer acceptance, such as the purple-fleshed and consistently higher soluble solids and SS/TA ratio content compared to the white-fleshed S. undatus (Rabelo et al. 2020, Alves et al. 2021, Santos et al. 2023). These positive characteristics significantly influenced the quality of fruits resulting from the interspecific cross (S. undatus ♀ × S. monacanthus ♂), as observed in this study.
Number and germination rate of seeds
Regarding the seeds number of, the lower quantity observed in the fruits resulting from open pollination may be attributed to the low number of fertilized ovules (Cho et al. 2013), possibly due to the lower amount of pollen grains deposited on the stigma of the flowers by pollinators.
The germination rates observed in seeds from open and controlled pollination in the S. monacanthus (♀) genotype (Table V), although numerically high, suggest a possible influence of the female parent’s cytoplasmic effect on this trait. This is evidenced by the fact that, even when S. undatus was used as the male parent, germination rates above 90% were not achieved, unlike what was observed in seeds obtained both intra- and interspecifically in S. undatus. This difference may be related to seed size, as smaller and lighter seeds were noted in S. monacanthus, which may have influenced germination. This may occur because lighter seeds have been shown to contain fewer reserves, which can negatively affect their germination capacity (Cremasco et al. 2021).
These results highlight the importance of evaluating both parental lines to ensure the selection of genotypes capable of producing seeds with high germination potential. Crosses between genotypes that promote the production of viable seeds are crucial in genetic improvement, as they ensure the efficiency and continuity of the selection process and the development of new cultivars.
Genetic Diversity
The number of monomorphic primers observed in both S. undatus and S. monacanthus populations in this study is consistent with the results of Li et al. (2018) and Martínez (2021), who also reported a prevalence of monomorphic primers, nonspecific amplification, or amplification failures when using SSR markers in intraspecific evaluations of pitaya. The high number of monomorphic primers in these evaluations may be attributed to the low allelic diversity within the species’ populations. This is likely related to the asexual propagation method, as observed in other species, considering that pitaya is predominantly propagated through cuttings. Furthermore, the inbreeding effect resulting from self-fertilization and crossings between related individuals in S. undatus leads to a loss of heterozygosity in the population, which negatively affects various morphological, physiological, and biochemical processes in the plants (Kodad et al. 2021).
The hypotheses mentioned may be related to the results observed in the intraspecific crosses between S. undatus (♀) × S. undatus (♂), which resulted in low fruit set rates (Figure 2) and smaller fruit size (Table III) in the present study.
The results regarding the variables K, He, Ho, and PIC obtained in this study (Table VI) were lower than those reported by Pan et al. (2017) and Nashima et al. (2021), who analyzed accessions from species of the genera Hylocereus, Selenicereus, Cactus, and Epiphyllum oxypetalum using 18 and 16 pairs of SSR markers, respectively. These authors reported higher values for these indicators, particularly for the PIC index, with mean values ranging from 0.70 to 0.72. This difference can mainly be attributed to the greater genetic diversity of the materials analyzed, as their studies were conducted on germplasm banks composed of accessions from different species and geographic origins.
While our study observed a higher number of monomorphic primers due to lower allele frequency among individuals from the same population, the microsatellite markers successfully facilitated differentiation between the two Selenicereus species. The results indicate that the populations evaluated in the present study exhibit low intraspecific genetic diversity. This limited variability is likely associated with the predominant use of vegetative propagation in cultivation and the historical lack of introduction of new, genetically diverse genotypes into the study area.
The low intraspecific diversity, however, contrasts with the observation that pitaya species generally do not exhibit interspecific reproductive barriers (Tel-Zur et al. 2012). This implies a high potential for obtaining hybrids with unique genetic characteristics through interspecific crosses when diverse parental material is used, even if intraspecific diversity is low in certain cultivated populations.
The UPGMA clustering analysis, based on the five polymorphic primers identified between the species (Figure 4), revealed the formation of only two main groups. This clustering suggests that the S. undatus individuals in the orchard are likely all clones, whereas S. monacanthus showed the presence of other distinct individuals, indicating some degree of genetic variation within that specific population. However, a comprehensive cluster analysis incorporating fruit and seed traits, along with molecular markers, was beyond the scope of this study but would provide a more robust understanding of the phenotypic and genetic relationships within these populations and is recommended for future research.
Dendrogram based on Nei’s (1972) genetic distance using the UPGMA method in the two pitaya populations.
The results regarding the genetic diversity of these pitaya genotypes underscore the importance of such information, not only for guiding genetic improvement programs but also for the strategic establishment and management of commercial orchards. Information on the specific characteristics of each species, such as their production potential, fruit quality attributes, compatibility mechanisms, and susceptibility to diseases, influences the long-term productivity, sustainability, and longevity of the orchard. Future implications of this study include guiding the selection of diverse parental lines for breeding programs aimed at development pitaya cultivars to meet market demands. Specifically, the identified compatibility in interspecific crosses and the promising quality traits of S. monacanthus highlight the potential for creating superior hybrids.
CONCLUSIONS
Interspecific crosses between S. undatus and S. monacanthus showed compatibility, while intraspecific crosses involving the studied genotype of S. monacanthus were not viable, and those with S. undatus resulted in low fruit set rate. Controlled interspecific crosses between S. undatus and S. monacanthus ensured a higher fruit set rate and the production of fruits with larger size and a higher number of seeds. Interspecific cross-pollination of S. undatus with S. monacanthus pollen improved the quality of its fruits. The genetic diversity of the orchard favored the production of better-quality fruits. The population of S. undatus and S. monacanthus in the studied orchard was probably formed by a single clone.
Acknowledgements
We thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and for providing the scholarship Brasil, Finance Code 001, to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - 400904/2019-5, to Federal University of Jequitinhonha and Mucuri Valleys, the Program in Plant Production and Federal University of Viçosa for the infrastructure made available to carry out the research Project.
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Edited by
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Handling editor
Pablo Bolaños-Villegas
The datasets generated and analyzed in this study are available in the author’s doctoral thesis.
