1. Introduction

Genetic erosion is one of the main problems in breeding programs (Dar et al., 2019). Genetic erosion leads to the loss of genetic variability and without genetic variability, it is impossible to conduct any plant breeding process. Modern agriculture focuses primarily on the conservation and utilization of valuable genetic resources (Dar et al., 2019). Therefore, knowledge of genetic variation is crucial for classification, evolutionary studies and taxonomy, particularly in relation to specific direct crosses. These factors are essential in a plant breeding program. However, to achieve this, it is necessary to carry out collections, morphological characterizations, and, most importantly, molecular characterizations. Molecular techniques allow more precise determination of intra- and interspecific genetic diversity.

Molecular markers are the most commonly used technique to analyze genetic diversity, and the choice of markers depends on the purpose. Although more sophisticated sequencing techniques exist, these are not always accessible in all laboratory environments. Therefore, with molecular markers and cytogenetic analyses, we can obtain the answers we seek, with a lower cost and greater accessibility. Random amplified polymorphic DNA (RAPD) is widely used because it is the simplest, most cost-effective technique, requires no prior knowledge of the genome, and can be used for various purposes, such as estimating genetic diversity, germplasm management, genetic mapping, and more (Babu et al., 2021). Similarly, the cytogenetic CMA/DAPI Chromomycin A3 and 4′,6-diamidino-2-phenylindole technique can differentiate species based on the number of chromosomes present in each cell.

Portulaca is a genus of plant species being P. grandiflora Hook and P. umbraticola Kunth are grown as ornamentals, and Portulaca oleracea L. can be used in human nutrition (Mitich, 1997; Alam et al., 2014). Additionally, P. oleracea is considered one of the most widely distributed and harmful weeds in the world (Singh and Singh, 1967; Caton et al., 2004). Portulaca umbraticola is widely used for the ornamentation of public roads in Brazil, often found in pots on windowsills or in flowerbeds along sidewalks. These plants produce beautiful flowers in a variety of colors, including hot pink, yellow, red, white, and orange. Their flowers typically have five or more petals, and some cultivars lack reproductive verticillium. P. umbraticola is also considered an unconventional food plant (PANC), containing several nutritious compounds that are safe for human consumption (Alam et al., 2014).

The Portulacaceae family includes about 10 genera and 258 species (The Plant List, 2020). These individuals are mainly distributed in Western North America, South America, and Africa, with a few representatives in Europe and Asia (Coelho and Giulietti, 2010). The genus, Portulaca L. is one of the members of the Portulacaceae family, which includes succulent herbs, annual to perennial, with trichomes in the leaf axils, conspicuous to abundant, opposite to alternate leaves, solitary flowers or forming capituliform inflorescences surrounded by leaves and encapsulated fruit (Nyffeler and Eggli 2010; Ocampo and Columbus, 2012). According to Matthews et al. (1991) the genus Portulaca has a basic chromosome number of (x) 4 or 9, with n = 9 being the most common haploid number, n = 4, P. suffrutescens Engelm. a n=27 in polyploid subspecies of P. oleracea L. (Danin et al., 1978). The P. umbraticola species has between 18 and 54 chromosomes. The highest chromosomal numbers were found in P. centrali-africana R. E. Fr, P. kermesina N. E. Br., P. quadrifida L., all with 2n=4x=108. There are reports that only two suggestive hybrids occur naturally (Kim and Carr, 1990), while artificial hybrids have already been found between P. centrali africana x P. kermesina (Bouharmont, 1965), P. oleracea x P. grandiflora (Kim and Carr, 1990), P. lutea x P. oleracea, P. lutea x P. molokiniensis, P. molokiniensis x P. oleracea, P. sclerocarpa x P. villosa and P. villosa x P. olowalu (Jia et al., 2017).

In the genus Portulaca, some works studied the genetic diversity using molecular markers, like EST-SSR (Alam et al., 2015), ISSR (Alam et al., 2015), AFLP (Feliciano et al., 2022; Ren et al., 2011). On the other hand, works using P. umbraticola is scarce, maybe due to a mistake some authors make among the classifications of species, as seen above, but there’s one using the marker SRAP to investigate the relationship between P. umbraticola and P. grandiflora present in a collection of ornamental purslanes cultivated in China (Jia et al., 2017). Recently research has showed that the importance of P. umbraticola goes beyond the ornamental use, your system of flower opening can be a model to understand the flower behavior in another species under heat stress, this thanks to a first sequencing of transcriptome by RNA-Seq of P. umbraticola (Maguvu et al., 2021). The specie also has a potential to be used in high-throughput phenotyping due its colorful flowers. Researchers developed an imagery protocol to predict the pixel-wise intensity of flower’s color upon the fractality of the architecture of P. umbraticola, thus providing new knowledge about the specie (Souza et al., 2022).

Therefore, in view of the great importance of knowing the species to be improved and the fact that some accessions have characteristics not described within the species, such as hundreds of petals and flowers with no reproductive whorls. The objective of this work was to analyse the genetic diversity among 18 accessions of P. umbraticola using molecular markers, besides determine the chromosome number of 20 accessions of P. umbraticola and one of P. oleracea, comparing them intraspecifically and interspecifically.

2. Materials and Methods

2.1. Botanical collection and documentation

Two species were analyzed: one accession of Portulaca oleracea and 20 accessions of Portulaca umbraticola. The accessions originated from collections carried out in the field in the municipalities of Areia and Santa Rita in Paraíba state, Brazil, and kept in cultivation at the experimental garden of the Laboratory of Plant Biotechnology, Department of Biological Sciences of the Centro the Agrarian Sciences, Universidade Federal da Paraíba (UFPB). Subsequently, exsiccated of all the studied material were provided, which are deposited in the collection of the Herbarium Prof. Jayme Coelho de Morais. For the identification of the genotypes, pertinent literature and consultation with specialists were used, in addition to comparisons with previously identified genotypes.

2.2. Plant material and extraction and quantification of DNA

Eighteen accessions of P. umbraticola were used to analyze the genetic diversity. The plants were kept in glass bottle with water duly identified to obtain clean roots, were placed 3 branches of each individual to emit roots, after 10 days, the root were sufficient to continuous the experiment, the issued roots were collected and kept in aluminum foil envelopes and conditioned in freezer until the extraction of DNA.

To extract the DNA from roots, they were submitted to disinfestation in 1% sodium hypochlorite for 5 minutes, to eliminate possible microorganisms, after this, the material was washed in distilled water for three times and dried in a towel paper. The CTAB procedure was used to isolate genomic DNA (Doyle and Doyle, 1987). By running on 0.8% agarose gel electrophoresis, the quality of extracted DNA was analyzed, and photographed under UV light, in a Gel logic 112 photodocumenter.

2.3. PCR reaction and RAPD analysis

To amplify the DNA in the polymerase chain reaction, a total of 14 primers (Table 1) were used, containing 25μl volume (2.5μl of loading buffer 1X; 1.5μl MgCl₂ [3mM]; 0.5μl of each DNTP [200mM]; 2.5μl primer [1mM]; 0.2μl of Taq DNA polymerase; 15.8μl ultrapure water, and finally 2μl DNA template diluted with ultrapure water). PCR amplification was carried out in a thermocycler following this cycles and conditions: three minutes initial denaturation step was carried out at 94ºC, after these forty cycles of 15 seconds at 94ºC, thirty seconds at 40ºC and 60 seconds at 72ºC, in the end of these forty cycles, the final extension step was performed for seven minutes at 72ºC.

To confirm the amplifications, the final product was electrophoresed on 1.5% agarose gel in 1x TAE buffer, and were run in 80V, at the end the amplifications were visualized and photographed under UV light, in a Gel logic 112 photodocumenter. The DNA band was confirmed according to the knowledge pattern of the Ladder 1Kb (Invitrogen™ 1 Kb Plus DNA).

2.4. Genetic diversity analysis

To estimate the genetic diversity of the accessions was made a binary matrix based on the electrophoretic profile of the gels of each primer. The comparison was made with the pattern of the Ladder and scored visually as “1” for the presence or “0” for the band absence. A polymorphic locus was defined as one with the frequency of the most common allele equal to or less than 0.95.

The genetic distance (dissimilarity) was estimated by using the Genes program (Cruz, 2006) based on the Sokal binary distance (Sokal and Rohlf, 1962). From the dissimilarity matrix was performed a dendrogram clustering based on ward.D2 method (Ward Junior, 1963). To a cutting on dendrogram was used the rule proposed by Mojena (Mojena, 1977), which is based on the relative size of the merger levels (distances) in the dendrogram. Finally, the accessions were clustered with an optimized method by Tocher from of dissimilarity matrix (Rao, 1952).

2.5. Cytogenetic analysis

Root tips obtained directly from the cultivated material were pre-treated with 0.002 M 8-hydroxyquinoline (8HQ) for 24 h in a refrigerator and subsequently fixed in Carnoy 3:1 (absolute ethanol:glacial acetic acid, v/v) for 2–24 h at room temperature and stored at −20 °C. (Guerra and Souza, 2002). For fluorochrome staining (CMA/DAPI), roots were digested in an enzymatic solution containing 2% cellulase and 20% pectinase at 37 °C for 1 h. Subsequently, they were squashed in a drop of 45% acetic acid. Coverslips were removed in liquid nitrogen.

The fluorochrome staining protocol was done as described by Carvalho et al. (2005). The slides were stained with 10μL of CMA (0.5 mg / ml), covered with a coverslip and stored in a darkroom for one hour. Subsequently, the coverslip was removed with a jet of distilled water, and then dried at room temperature. Subsequently, 10μL of DAPI (2 μg / ml) was added in Glycerol / Mcllvaine buffer, the best slides were aged in a darkroom for three days to stabilize the fluorochrome. The best metaphases were photographed in an AxioCam MRm Zeiss epifluorescence photomicroscope equipped with a video camera, using an image capture program. The images were processed using Photoshop CS6.

2.6. Self-fertilization and crossings

A diallel cross was performed with parents without reciprocal combinations. Five flowers from each access that have reproductive whorls were emasculated, pollinated, and covered with an aluminum foil. At least five crosses between each accession were performed. Aborted intercrosses or self-fertilizations were repeated.

3. Results and Discussion

3.1. Genetic diversity

The PCR reactions with the 14 RAPD markers amplified a total of 169 bands, with 44 polymorphic loci. No monomorphism was observed for any of the loci, indicating the potential for significant genetic diversity (Table 2).

In this study, the RAPD molecular markers was utilized to identify genetic divergences among genotypes, and it proved to be effective for this purpose. It amplified numerous distinct bands among the 18 genotypes, and multiple polymorphic loci were detected, varying according to the primer used. Alam et al. (2015), when assessing the genetic diversity of Portulaca oleracea using ISSR markers, identified a high degree of polymorphism among the studied individuals and successful band amplification. This suggests that both RAPD and ISSR markers are effective for evaluating the genetic diversity of the Portulaca genus.

One of the events that introduces significant genetic variation in nature is gene flow, which occurs naturally as new individuals migrate between populations, resulting in different gene combinations. Sexual reproduction is responsible for allele recombination, and this variation in populations or species is referred to as genetic diversity (Chung et al., 2023).

Assessing the genetic diversity of plants is of paramount importance for plant breeding, whether for improving the characteristics of a particular species or for its conservation in germplasm banks (Souza et al., 2024a). Given that climate change is occurring globally, it is necessary to conserve biodiversity to prevent genetic erosion of species. Molecular markers are widely used for genetic diversity studies due to their extensive genomic coverage, high reproducibility, easy automation and freedom from environmental fluctuations (Bhandari et al., 2017).

When analyzing the dissimilarity matrix among the 18 genotypes of P. umbraticola (Table 3), an index of 0.7671 was found between individuals 12 and 16, indicating that they are the most dissimilar. This suggests the need for conventional breeding methods to cross these individuals. However, individuals 5 and 15 were found to be genetically identical, as their dissimilarity was zero, presuming no genetic distance between them. Nevertheless, this might be due to the use of the random RAPD marker, and it is necessary to employ other specific markers to confirm this identity.

Some genotypes, such as PU-04, PU-06, PU-09, PU-10, PU-11, PU-14, and PU-15, lack well-established reproductive structures (androecium and gynoecium), making conventional crossing impossible. However, among those with functional reproductive structures, individuals PU-16 x PU-05 and PU-08 x PU-17 showed higher dissimilarity compared to the others, suggesting that crossings should be performed to obtain new accessions with ornamental potential. Additionally, collecting seeds from these genetically distinct genotypes for storage in germplasm banks is recommended to preserve the species' biodiversity (Table 3).

Among the 18 genotypes of P. umbraticola studied (Figure 1), most of them exhibit significant morphological differences, especially in the colors of their flowers. However, some genotypes show high similarity, such as PU-1, PU-5, PU-16, PU-17, and PU-18; PU-02 and PU-08; and PU-09 and PU-10. To understand the similarity between these genotypes, genetic distance is studied. Beaumont et al. (1998) define distance as a quantitative measure of genetic difference that can be calculated between individuals or populations. In other words, the more similar genes genotypes share, the smaller the genetic distance between them. Elucidating the similarity or dissimilarity between individuals contributes to better planning genetic improvement programs.

In the dissimilarity matrix, the 18 genotypes exhibited significant dissimilarity, with values fluctuating. The highest dissimilarity was observed between individuals PU-12 and PU-16 (0.7661 – Table 3). The genetic diversity found in P. umbraticola genotypes is mainly attributed to cross-fertilization performed by various insects and native bees, attracted by its delicate, small, and colorful flowers (Aizen and Harder, 2009). Considering that P. umbraticola is an outcrossing species, it undergoes cross-fertilization.

The clustering analysis based on the Ward.d2 method using the Sokal binary genetic distance (Figure 2) resulted in the formation of 3 groups for the 18 individuals of P. umbraticola, with a cutoff point at 1.164. Group I included 13 out of the 18 genotypes (72.22%), while Group II and III included S3 and 2 genotypes, respectively. Based on the dendrogram, it is possible to suggest genotypes 14 and 15 as potential parents for crosses between genetically dissimilar genotypes to produce segregating populations.

In Table 4, it can be observed that clustering using the Tocher optimization method (1952), based on the dissimilarity matrix, resulted in the formation of 6 distinct groups. This method groups individuals based on the criterion that distances within the group are smaller than those between groups (Cruz et al., 2012). Therefore, this clustering provides valuable information for selecting distant parents for the development of new cultivars. Group I includes the largest number of genotypes, encompassing 12 out of the 18 genotypes (1, 2, 4, 5, 8, 9, 12, 13, 14, 15, and 18), while Group II consists of only two genotypes (16 and 17). Groups III, IV, V, and VI consist of a single genotype, specifically 3, 10, 11, and 6, respectively.

The Tocher clustering method groups different accessions based on the evaluated descriptors in a way that maximizes homogeneity within the group and heterogeneity between groups (Cruz et al., 2012). Therefore, Group I, which accommodated the most accessions, can be considered internally homogeneous and distinct from the other groups. Genotypes within this group are genetically similar and, consequently, less suitable for cross breeding (Gerrano et al., 2015). On the other hand, individuals in the other groups can be used among themselves because they are distinguished between groups, indicating a higher potential for hybrid generation (Gonçalves et al., 2017).

The coefficient of cophenetic correlation (CCC) obtained from the dissimilarity matrix and the clustering matrix serves to analyze the fit between them. Matta et al. (2015) suggest that satisfactory correlations have a distortion of more than 20%. In other words, a CCC greater than 80% is considered ideal. In this study, a CCC of 86% was obtained (Table 5), indicating that the formed clusters are consistent and intact, with no distortion in the representation of the groupings and similarities formed.

Genetic variability among individuals of the same species is crucial for conservation and breeding programs (Souza et al., 2024b). Without genetic diversity and interaction with the environment, it becomes more challenging to obtain diversified genotypes through genetic improvement (Glick et al., 2014).

To obtain diverse materials, especially when it comes to ornamental plants, where the market constantly demands novelty, it is essential to understand the existing diversity for better planning. Several ornamental plants have demonstrated a broad genetic variation, such as Ilex crenata (Geukens, 2023), Chrysanthemum (Mekapogu et al., 2023), Caladium (Zhou et al., 2023), Canna edulis (Le et al., 2023), among others.

Species lacking sufficient genetic diversity may not be able to respond to environmental changes, compete with other plants, or successfully defend against pests and predators, which can lead to a decrease in their population and eventual extinction (Geukens et al., 2023).

3.2. Cytogenetics

The chromosomal number observed in P. oleracea was 2n = 52 (Figure 3A, B, C), being the second record for the species. The species P. oleracea has a basic chromosome number x = 9, with the occurrence of polyploids and ranging from 2n = 18, 36, 45 to 54 (Glick et al., 2014).

The chromosomal number observed for the 20 accessions of P. umbraticola was 2n = 18 (Figure 3D, E) in all the individuals analyzed, corroborate with previous records in the literature (Table 6). Although the individuals of P. umbraticola analyzed in this work have flowers of different color, number of different petals and reproductive system present or absent, there were no differences in the chromosomal number and in the composition of heterochromatin. Indicating that all accessions belong to the same species (P. umbraticula). In this species, two CMA + interstitial bands (Figure 3F) were observed, which may be related to satellite DNA.

The species P. oleracea has a basic chromosome numbser of x = 9, with the occurrence of polyploids and ranging from 2n = 18, 36, 45 to 54 (Glick et al., 2014). This variation may be related to the different geographic areas in which they are found, corroborating with Ocampo and Columbus (2012) who also found chromosomal number 2n = 52 in eight taxa from the P. oleracea complex including transient forms, whereas the expected would be 54. About 21% of the species of the Portulacaceae family have a number chromosome recorded in the literature, ranging from 2n = 8 in P. suffrutescens to 2n = 108 in P. centrali-africana, P. kermesina and P. quadrifida (Table 2). The variation in the chromosome number can provide a better understanding of the evolutionary processes (Mayrose and Lysak, 2021). In Portulaca, the evolution of the chromosome number was investigated with the aid of probability models from the chromosome number.

Demi-polyploidy transitions are good when there are only haploid even numbers, so demi-polyploidy from n=9 can lead to n=13 or n=14, according to Glick et al., (2014). However, according to Glick et al., (2014), neither of the possibilities can represent a tripling event, with a subsequent doubling event, a tripling 9! 27 would be next, involving an additional gain in chromosome number (9!13!26!27) or loss through (9!14!28!27), artificially increasing dysploidy rates. That indicates the group's basic number x = 9 and inferring that dysploidy and polyploidy are the main mechanisms involved in its evolution (Ocampo and Columbus, 2012).

Portulaca is polyphyletic, and its complex is divided into two clades, one of American origin and the other of the old world, clade OL with P. quadrifida and clade AL with P. oleracea and P. pilosa, P. umbraticola and P. grandiflora (Ocampo and Columbus, 2012). Multiple long-distance events are frequent within the AL clade, with representatives distributed throughout the delimited area under study (Ocampo and Columbus, 2012).

As for the heterochromatic bands, P. oleracea has six terminal bands CMA + / DAPI- (Figure 1C), which are regions of the chromosome rich in GC. Walter et al., (2015) also observed terminal bands CMA + in the pentaploid individual of P. oleracea, which was proportional to its level of ploidy compared to the diploid species, where it was possible to suggest the occurrence of a neopoliploid.

3.3. Self-fertilization and crossings

It was found that the self-fertilizations carried out in this species, P. umbraticola, in the different accessions evaluated, did not bear fruit. Demonstrating that these plants are self-incompatible. For the values of the crossings between different accesses, except for the crossings PU2 x PU8, PU1 x PU8, PU15 x PU5, all the other 52 crossings formed fruits and consequently seeds (Figure 4).

All crosses had a maximum of 5 flowers crossed in the same environmental conditions and performed together with the self-fertilizations that did not obtain fruits and seeds. Self-fertilizations had a higher number of crossings compared to cross-fertilizations (Figure 4). In these crossings, the accessions share similar characteristics as the colors of their flowers. This may indicate that these accessions are genetically equal, requiring a molecular analysis to prove it, but since self-fertilization does not occur, this hypothesis is very likely to be correct and these accessions that did not cross over to be clones.

It is known that for the conservation of species to be effective, it is necessary to know the genetic diversity of populations, and there must be a monitoring of the patterns of genetic variation over time (Chung et al., 2023). However, according to Kageyama et al. (2001) population fragmentation and reduction is related to genetic drift, increased inbreeding, and decreased gene flow. In this way, gene flow or gene escape is defined as the exchange of genetic material between individuals, that is, the transfer of alleles from one variety or species to another. Something that occurs among P. umbraticola from the point of view that they do not self-fertilize.

Levin and Kerster (1974) define the condition necessary for gene flow to occur in field conditions: the existence of sexually compatible individuals. In addition to individuals' temporal and spatial coincidence, cross-pollination, long pollen longevity, viable hybrids, gene transmission in subsequent generations, gene recombination between genomes and non-exclusion of the receptor genome gene (Chung et al., 2023). However, some environmental factors can interfere with the viability of the gene flow, such as wind speed and direction, relative temperature, and humidity, which can affect the distance, duration and the amount of pollen released (Okubo and Levin, 1989).

Thus, accessions PU-04, PU-06, PU-09, PU-10, PU-11, PU-14 and PU-15 (Figure 1) do not have the capacity exchange of genetic material, because they do not have reproductive whorls. Thus, their gene flow is affected, however, as they are plants with easy vegetative propagation, these accessions are not at risk of extinction, because they are maintained through exchange between neighbors, where the human is its main disperser, taking materials from one region to another.

4. Conclusion

This study revealed that the RAPD molecular marker was efficient in differentiating 20 different accessions of Portulaca umbracitola, a non-conventional edible plant with ornamental potential. Genetic diversity elucidates how divergent these species are, thus, we can suggest some genotypes for crosses, such as PU-16 x PU-05, PU-08 x PU-17 or PU-12 x PU-16. Through cytogenetic studies, it was possible to verify the difference between the species P. umbraticola and P. oleracea (2n = 52 and 2n = 18, respectively), determining that all accessions in the bank used in this work are P. umbraticola. The crosses allowed us to verify that the species are allogamous and self-incompatible. Thus, these results help in directing the most divergent crosses and in obtaining seeds to contribute to the genetic improvement of this species.

Acknowledgements

Laboratório de Botânica UFPB/CCA, Prof. Dr. Leonardo Pessoa Felix, Capes, CNPq, Unesp.

References

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