Ploidy and DNA content of cape gooseberry populations grown in southern Brazil

ABSTRACT

Studies detected chromosomal variability in genotypes of Physalis peruviana L. cultivated in Andean countries, with the existence of diploid and tetraploid genotypes and other variations. Knowledge about the ploidy level in cape gooseberry genotypes grown in southern Brazil is essential to define efficient breeding strategies, for example, in the knowledge of the effects caused by inbreeding and heterosis in obtaining commercial hybrids. The objective of this study was to determine the chromosome number and quantify nuclear DNA by flow cytometry in cape gooseberry populations grown in southern Brazil and in Andean populations. To this end, four cape gooseberry populations of different origins (from Lages and Caçador in Brazil, and Colombia and Peru) were subjected to classical cytogenetic analysis (chromosome counting) and flow cytometry. The chromosome number of the four populations was found to be 2n = 4x = 48, classifying them as polyploid with tetraploid cells. Uniformity was also detected in the amount of DNA, ranging from 12.87 to 13.98 pg, with low coefficients of variation (1.9 to 4.2%). The Tukey’s test confirmed the uniformity between populations as to the amount of DNA. Therefore, tetraploid cape gooseberry populations cultivated in southern Brazil have a 2C DNA mean of 13.23 pg. The chromosomal uniformity reveals that cultivation in Brazil was initially based on the sampling of a small number of plants purchased from Colombia, which may already have been subjected to selection for polyploidy.

KEYWORDS:

• Physalis peruviana L.
• plant breeding
• chromosome number
• flow cytometry
• small fruit

Introduction

Physalis peruviana L. is a fruit species with excellent market expectations and high added value. This is due to the exotic aspect, sensory quality, nutritional and medicinal properties, with particularly high contents of vitamin A and B complex (Fischer et al. 2014; Olivares-Tenorio et al. 2016). Cape gooseberry cultivation is quite recent in regions of Brazil and it is typically produced by small farmers, so that the country is not self-sufficient in the production of fruits. Breeding programs are being carried out for the morphological characterization and selection of superior populations based on fruit yield and quality traits (Trevisani et al. 2016). The success in developing a superior genotype depends on the ability of the breeder to associate information expressed in the plant phenotype with information obtained at the chromosome level (Simmonds 1980).

Studies reveal the existence of variation between and within populations of cape gooseberry regarding chromosome number. For example, studies carried out in Colombia by Rodríguez and Bueno (2006) and Lagos et al. (2005) show the existence of populations with variations between 2n = 2x = 24 and 2n = 4x = 48. Therefore, with x = 12 being the basic number (Quiros 1984), there are diploid and tetraploid genotypes in Physalis peruviana. More recently, Liberato et al. (2014) found variation according to the origin of the accessions, which were classified in landraces (2n = 2x = 24), Colombia (2n = 32) and Kenya (2n = 4x = 48), with a predominance of tetraploids among cultivated accesses. According to Lagos et al. (2007), the variation found in Physalis peruviana is attributed to the current evolutionary process of the species (natural and artificial selection), emphasizing the relatively recent domestication.

The cultivation of cape gooseberry in southern Brazil occurs mainly in the municipalities of Lages, Fraiburgo, Caçador and Urupema in the State of Santa Catarina and Vacaria in Rio Grande do Sul. In most cases the cultivated populations are of unknown origin, which does not guarantee the genetic quality of the seeds, and there are no varieties of cape gooseberry registered in Brazil according to the Ministry of Agriculture Livestock and Supply (Mapa, 2014). Thus, it is possible that there is variability in the number of chromosomes of populations cultivated in the southern region, corroborated by the unknown origin and the cytogenetic diversity within the species found in the scientific community.

Knowledge about the chromosome number is a tool of prime importance in plant breeding, essential for the definition of efficient breeding strategies. It is indispensable in the design of hybridization strategies for the breeding of cultivars or hybrids, in the understanding of gene segregation, and consequently of the resulting inbreeding and heterosis effects (Birchler 2013; Washburn and Birchler 2014) in the establishment of the relation between cultivated plants and their ancestors (Soltis et al. 2016), among others.

Aside from the chromosome number, studies also reported differences in the amount of nuclear DNA between accessions and within the same ploidy level, varying between 5.77 and 8.12 pg (Liberato et al. 2014). Flow cytometry has been widely used as a tool for counting the chromosome number (Ochatt 2008). The great advantage is the possibility of rapidly determining the ploidy level of a large number of plants in an early development stage. In cape gooseberry, the amount of nuclear DNA estimated by means of flow cytometry can serve as a reference in future analyses and thus determine possible changes in the genome of the populations studied. Some factors may alter the amount of DNA in plants: (i) presence of spontaneous or induced mutations; and (ii) duplication of chromosome numbers naturally as well as by artificial techniques (e.g. use of antimitotics).

The purpose of this study was to determine the level of ploidy and the amount of nuclear DNA of populations of cape gooseberry cultivated in the southern region of Brazil in relation to populations of Andean origin, as a way of providing subsidies for the genetic improvement of cape gooseberry.

Material and methods

Cape gooseberry populations (treatments)

The treatments involved four cape gooseberry populations (genetic constitutions). The populations come from different locations, representative of the genetic variability of cape gooseberry currently under cultivation. The places of origin were the municipalities of Lages and Caçador in Brazil, and seeds obtained commercially from Colombia and Peru. The municipalities of Lages and Caçador were chosen to represent a cape gooseberry-producing region in the south of the country. The populations were obtained from commercial plantations. The cape gooseberry populations from Colombia and Peru were purchased on the retail market (of imported fruits), being thus considered of Colombian and Peruvian origin.

Determination of the chromosome number

The cape gooseberry populations of Lages and Caçador were sampled to obtain seeds by selecting 10 healthy plants, from which approximately five fruits per plant were picked. The number of sampled plants of the Colombian and Peruvian populations, whose fruits were purchased commercially, is unknown. The seeds of all four populations were removed from the fruits manually, washed in running water, air-dried for seven days, and then stored until analysis.

To count the chromosomes, a 50-seed sample of the total stored seeds per population was used. The seeds were germinated on moist filter paper, in a germination chamber at 25–30°C. After germination, 0.5–1.0 cm radicles were pretreated with 8-hydroxyquinoline (2 mM) for 3 h at room temperature. Thereafter, they were fixed in Carnoy (3 ethanol:1 acetic acid) for 24 h, washed in distilled water and subjected to enzymatic maceration in pectinase: cellulase (2 U:20 U) for 3 h at 36°C. They were then washed again in distilled water and fixed for approximately 24 h in ethanol:acetic acid (3:1) to mount the slides. The slides were mounted by the cell dissociation technique (Carvalho and Saraiva 1993) and stained in 5% Giemsa solution for 6 min. For each treatment, 10 slides were evaluated and at least one metaphase phase cell per slide was recorded. Cell images were captured with an Olympus BX-60 epifluorescence microscope (Curitibanos, Santa Catarina, Brazil) coupled with an Olympus DP70 camera to count the chromosome number.

Determination of DNA content by flow cytometry

For the cytometric analysis we used young leaves of the four studied populations. Five seeds per population (Lages, Caçador, Peru, Colombia) were placed to germinate in polystyrene trays filled with substrate. After the seedlings reached approximately 15 cm in height, they were transplanted to the final location, for vegetative growth of the plants and leaf sampling. For the analysis, two plants per treatment (population) were selected and three samples with approximately five young leaves per plant were collected.

The leaves of Physalis peruviana and Pisum sativum (internal standard) (9.09 pg/2C) (Cavallini and Natali 1990) were cut with a scalpel in 50 μl of LB01 extraction buffer and filtered through a 50 μm nylon mesh. The buffer-dye containing 25μl propidium iodide (1mg ml^–1) and 5 μl RNase was added. The resulting cell suspensions were left to stand at room temperature for 60–120 min in the dark until analysis and then analyzed using a FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer. The histograms were obtained and analyzed with software Cell Quest (Becton Dickinson) and WinMDI 2.8 (Windows Multiple Document Interface for Flow Cytometry, Juiz de Fora, Minas Gerais, Brazil); the DNA content was estimated from the following formula: Sample (2C) = (value in the peak channel of the standard) × 9.09 pg. The genome size in Mbp was estimated according to Bennett et al. (2000), where 1 pg DNA = 978 Mbp.

Results and discussion

Chromosome analysis revealed metaphases with excellent morphology, which allowed the determination of the chromosomal number equal to 2n = 4x = 48 for the four analyzed populations (Colombia, Lages, Caçador and Peru) (Figure 1(a)). Considering that the basic chromosome number of the genus Physalis is x = 12 (Quiros 1984), chromosomal uniformity between the populations cultivated in Andean countries and those grown in the southern region of Brazil was observed, which were therefore classified as tetraploid.

The diversity in chromosome number observed in the scientific literature gave rise to the hypothesis that populations from different locations could manifest chromosomal differences. For example, in Colombia, Lagos (2005) observed chromosome numbers of 2n = 24, 36 and 48, classifying them as diploids, triploids and tetraploids respectively. The origin of triploid genotypes possibly results from the natural crossing of diploid and tetraploid plants, resulting in triploid progenies. Lagos (2005) also recorded cases of myxoploidy in Physalis peruviana. Yamamoto and Sakai (1932) found 2n = 2x = 24, characterizing the species as diploid. According to Rodríguez and Bueno (2006), in the so-called Colombian accession, the chromosome number is 2n = 32. However, Betancourt (2014) characterized the same accession (“Colombia”) and reported a ploidy level of 2n = 4x = 48, classifying gooseberry cape as tetraploid. The same was found for the four populations under study, also in agreement with the findings of Vilmorin and Simonet (1928), Menzel (1951), Liberato et al. (2014). These results indicate that the chromosomal conformation of Physalis peruviana has not yet been established and that this is possibly due to the recent process of domestication of the species when compared, for example, to the domestication of Solanum tuberosum, a member of the Solanaceae family.

The uniformity in chromosome number between populations cultivated in southern Brazil (Lages and Caçador) and the populations of Andean origin suggests a possible common origin and, therefore, the existence of only one genic pool in cultivated cape gooseberry. This may be due to cultivation in a large area of the Andean region, for example, of the so-called “Colombia” accession, which was introduced in Brazil through fruit obtained commercially. This assumption is based on the following facts. (i) Colombia is the largest cape gooseberry producer and exporter, and obtaining seeds and seedling production are easy, which favors the wide distribution of Colombian genotypes in different regions of the world, particularly in Southern Brazil. Possible selection/breeding processes in the country of origin and the sampling of small numbers of plants (improved genotypes) to harvest seeds resulted in the genetic uniformity detected in this study. (ii) Populations are also morphologically similar. In comparisons of morphological traits of the Colombian accession in morphological studies of Trillos et al. (2008) and Herrera et al. (2012), uniformity was found for the traits described in the four populations under study (Trevisani et al. 2016), i.e. fruit diameters between 1.25 and 2.50 cm, a mean fruit weight between 3 and 6 g and mean sugar content of 14 ºBrix (total soluble solids). As shown by Rodríguez and Bueno (2006), the chromosomal variations observed in different accessions are manifested in phenotypic traits, explaining the relation between ploidy level and magnitude or number of fruit and plant traits.

According to Criollo et al. (2014), more than 80 cape gooseberry accessions were described and are being maintained in germplasm banks in Colombia. These accessions are differentiated by plant size, shape of the capsule surrounding the fruit, and fruit size, color and flavor. The accession known as Colombia is characterized by bright-colored fruits, a high sugar content and higher weight, resulting in its widespread cultivation. Possibly, events of natural polyploidization (from possible diploid ancestors) resulted in increased cell volume (Dhooghe et al. 2011) and consequently in improved morphological traits of agronomic importance. In species marketed in the small fruit group, traits related to fruit quality, among them fruit size, color and flavor, are crucial at the moment of commercialization. In this way, the selection processes favored the maintenance of polyploid plants, precisely because of their superior agronomic traits.

A major purpose of this study was to understand the ploidy of cape gooseberry populations cultivated in the southern region of Brazil, as a guideline for future breeding efforts of the crop, both for the development of cultivars or the establishment of commercial hybrids. Moreover, the objective was a comparison of the ploidy of populations of the southern region with those cultivated in Andean regions, to draw conclusions about the possible origin. Therefore, the results are extremely relevant for the continuity of the cape gooseberry breeding program in the southern region and the development of gooseberry cape genotypes with promising commercial traits. This leads to important issues such as knowledge about the inbreeding effects and heterosis, which may be related to the ploidy degree of the species. These aspects also condition the success in outlining hybridization strategies among populations with the same chromosome complement.

The controversies in the scientific literature regarding the reproduction mode of Physalis peruviana are worth mentioning, although there are strong indications for the predominance of cross-fertilization (Santana and Angarita 1999; Lagos et al. 2008). No studies have addressed the performance of cape gooseberry populations over several selfing generations (inbreeding) and few studies describe the importance of hybridization as a way to increase productivity, capitalizing on heterosis (Leiva – Brondo et al. 2001; Lagos et al. 2007). Due to the relationship between the ploidy level and inbreeding effects on the development of inbred lines and the heterosis for the development of commercial hybrids, quantification is of paramount importance. In diploid species, if the two alleles of a locus are contrasting for a trait, a reduction in heterozygosity of 50% occurs in each selfing generation. In tetraploid species, the reduction of heterozygosis is practically 10 times smaller, i.e. 5.6% in each generation (Husband and Schemske 1997). Therefore, in tetraploid species, the inbreeding depression is less marked during selfing generations. The complexity is greater for quantitative traits, for which the four alleles of a locus must reach homozygosis.

Aside from the classical cytogenetic approach of counting the chromosome number, flow cytometry was performed to analyze the amount of DNA of the populations and also as a basis of comparison for future studies, to establish a standard of comparison of the amount of DNA of tetraploid plants. The analysis revealed that plants of the four analyzed populations had a mean 2C nuclear DNA content ranging from 12.97 to 13.98 pg, with a mean of 13.23 pg (Table 1). According to the Tukey test results (Pr < 0.05), the four populations did not differ statistically as to the amount of DNA. The coefficients of variation were low (between 1.9 and 4.2%), indicating reliability of the result interpretation. Figure 1(b) expresses the quantification of nuclear DNA in the population of Caçador (of 13.26 pg), and a similar performance in all four populations evaluated.

Table 1. Mean 2C nuclear DNA content in pg of four cape gooseberry populations (Lages, Caçador, Peru, and Colombia), considering three samples (01, 02, 03) taken from a same plant in each population. Lages, UDESC-IMEGEM, 2016.

	DNA content (pg)
Physalis population	Sample 01	Sample 02	Sample 03	Means*
Colombia	13.36	13.18	13.35	13.29a
Lages	12.97	13.10	13.98	13.35a
Caçador	13.31	13.26	13.28	13.28a
Peru	12.87	13.05	13.09	13.00a
Overall average				13.23

* Means followed by the same letter do not differ by Tukey test at 5% of error probability.

Figure 1. Mitotic metaphase (2n = 4x = 48) with chromosomes of a plant of a studied cape gooseberry population (a) and a representative histogram of the mean 2C nuclear DNA content of the population, using Pisum sativum as internal standard. Bar = 10μm (b). The population represented in this diagram is that of Caçador. Lages, UDESC-IMEGEM, 2016.

The nuclear DNA of the cape gooseberry accessions analyzed by Liberato et al. (2014) varied between 2.33 and 8.12 pg; the lowest amount was measured for the only diploid accession (2.33 pg), and a variation from 5.77 to 8.12 among tetraploid accessions. From this, a relationship between the ploidy level of the species and the respective DNA amount can be established. In tetraploid gooseberry cape genotypes, Betancourt (2014) found a medium amount of DNA (2n = 4x = 48) of 10.23 pg. The DNA contents of the studied cape gooseberry populations exceeded those reported in the literature.

The differences in the DNA content may be associated with a greater or lesser degree of repetitive DNA (Martel et al. 1997), making cape gooseberry a species in evolution and domestication. This situation may be due to: (i) adaptation to constant changes in environmental conditions (Cavallini and Natali 1990), related to the origin of the plant material, specific climates and environments of each growing region; and (ii) phenomena of hybridization, in case of predominance of cross-fertilization, by the loss or increase of repetitive DNA sequences. Information about the DNA content of a species can be used to estimate the genome size, allowing conclusions on relationships between genotypes and predictions of the most effective crosses (Özkan et al. 2010).

The applications of flow cytometry in plant breeding may also be related to monitor the stability of the ploidy level, e.g. of in vitro-propagated plants (Ochatt et al. 2011), to identify haploid plants for subsequent breeding of dihaploid lines, i.e. pure fertile lines (Gemes-Juhasz et al. 2002), in the establishment and detection of new ploidy levels, e.g. of tetraploid plants, which may present new traits of economic interest, such as higher fruit yield and in the development and detection of inter-specific hybrids.

In view of the above, the results detected a uniform chromosome number, suggesting a common origin of the cape gooseberry populations contemplated in this study. Uniformity was also observed in the nuclear DNA content. However, a large amount of scientific research reports variation in the chromosome number and DNA content within the species, indicating plasticity of the genome, which may be related to the adaptation of the species to new cultivation environments. Currently, the predominance of tetraploid populations indicates that the species is in a possible process of natural and directed (diploid to tetraploid) evolution. The natural selection for the maintenance of polyploidy possibly occurred due to a better adaptation to environmental conditions and also to anthropogenic evolution, by selecting plants with agronomically desirable traits, as pointed out by Schifino-Wittmann (2012). With regard to the cape gooseberry breeding program in the south of the country, the information obtained so far will be extremely important for the adoption of effective breeding methods, focusing on the increase of fruit yield, by, for example, exploitation of hybrid vigor.

Conclusion

The cape gooseberry populations of Andean origin (Colombia and Peru) sold in Brazil, as well as the populations cultivated in the southern region of the country (Lages and Caçador), are tetraploid (2n = 4x = 48).

The tetraploid cape gooseberry populations cultivated and sold commercially in the south of Brazil have a mean 2C nuclear DNA content of 13.2 pg.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The State University of Santa Catarina (UDESC), the Federal University of Santa Catarina (UFSC), the Federal University of Juiz de Fora (UFJF), the National Council for Scientific and Technological Development (CNPq), the Personnel Development Coordination Higher Level (CAPES) and Foundation of Support to Scientific and Technological Research of the State of Santa Catarina (FAPESC) provided scholarship of an author and financial support for the development of this research.