Acta Agronómica

Introduction

The genus Capsicum belongs to the Solanaceae family, which is represented by approximately 3000 species, distributed in 150 genera (Barth & Duarte, 2008). Capsicum comprises a large group of hot and sweet peppers with different morphological varieties and degree of fruits pungency. In fact, due to their active ingredients are widely used and marketed worldwide (Büttow, Barbieri, Neitzke, Heiden & Carvalho, 2010; Neitzke, Barbieri, Vasconcelos, Fischer, Vilella & Castro, 2014).

The taxonomy of this genus is still confused, there is controversy as to the number of species, ranging from 25 (Büttow et al., 2010), 30 (Moscone, Scaldaferro, Grabiele, Cecchini, Garcia, Jarret, Daviña, Ducasse, Barboza & Ehrendorfer, 2007) and 31 (Martins, Pereira, Souza & Costa, 2010) described species, being classified as domesticated (five species), semi-domesticated and wild (Souza, Martins & Pereira, 2011).

The origin center of this genus is the Tropical America (Pickersgill, 1997). Spanish and Portuguese people were the first to discover this kind in the Americas, one of the products most commonly used in cooking and traditional medicine for these people (Ribeiro, Lopes, Carvalho, Henz & Reifschneíder, 2008). In cooking, as constituents of salads and condiments; in traditional medicine, in the fight and prevention of diseases (Signorini, Renesto, Machado, Bespalhok & Monteiro, 2013), some countries like Peru and Mexico use peppers in large-scale, because of the color characteristics and assets of the fruit principles (Teodoro, Garcia & Corona, 2007).

According to Rufino & Penteado (2006), Asia is the continent that more grows peppers, with approximately 89% of the total cultivated. India, Korea, Thailand, China, Vietnam, Sri Lanka and Indonesia are the most prominent production countries. Production of the United States and Mexico is around 7%, followed by countries in Europe, Africa and Middle East, with 4% of the total cultivated peppers. In Brazil, the production of peppers is growing, being an important diversity center, and harboring domesticated, semi-domesticated and wild species, with emphasis on the cultivation of domesticated species C. annuum, with greater economic importance in this country (Rufino & Penteado, 2006; Neitzke et al., 2014).

Among the widely consumed species and commercially cultivated are C. annuum (pepper, sweet pepper); C. baccatum (finger-to-girl in-hat-friar); C. chinense (pepper-of-smell, pepper-goat, murici); C. frutescens (chilli pepper) and C. pubescens (hairy pepper, rocoto), which is a no cultivated species into Brazil (Carvalho, Maciel, Beckman & Poltronieri, 2014).

The basic chromosome number usually found in domesticated species, C. annuum, C. frutescens, C. chinense and C. baccatum is x=12 (2n=24) (Moscone et al., 2007), but in some wild species as C. buforum, C. campylopodium and C. cornutum, a number of x=13 (2n=26) was found (Moscone et al., 2007; Pozzobon & Wittmann, 2006; Teodoro, Garcia & Corona, 2007).

Given these concerns, this study aimed to analyze cytogenetically C. annuum, C. frutescens and C. chinense genotypes from Valle del Cauca, Colombia. In addition, chromosome number, meiotic behavior, meiotic index and pollen viability were studied, in order to detect chromosome abnormalities related to the ploidy level and pollen fertility. Since, to amplify cytogenetic information for this group of plants, which have allowed a basis for plant breeding programs.

Materials and methods

We collected flower buds, flowers and fruits of plants of three species of Capsicum: C. annuum (Cayenne), C. frutescens (Tabasco) and C. chinense (Habanero) grown at the Experimental Center at the Universidad Nacional de Colombia, campus Palmira - CEUNP, in the municipality of Candelaria, Valle del Cauca, Colombia.

The plant materials collected were stored in plastic bags and transferred to the cytogenetics laboratory of the Universidad Nacional de Colombia, campus Palmira, for the meiosis analysis in flower buds, and mitosis in root meristems. Then, floral buds were fixed in Carnoy I solution (3:1 ethanol:glacial acetic acid), for a maximum time of 3 to 4 hours, transferred to fresh solution and then preserved under refrigeration until the moment of preparation of the slides. To prepare the slides, technique of squash were employed; the anthers were macerated into a drop of Carnoy I solution and stained by acetic carmine 2%.

Chiasmata frequency were analyzed at 10 cells in diakinesis of each species. Spindle patterns were evaluated at meiosis II (metaphase, anaphase and telophase, respectively). The meiotic index (MI), analyzing a minimum of 100 tetrads of microspores. Pollen viability was counted in 400 grains for each genotype.

For the root meristem analysis, the seeds were germinated in Petri dishes, with moistened filter paper, at 25°C. The onset of germination varied from 5 to 10 days to C. annuum and C. frutescens, and 10 to 20 days to C. chinense. Roots were subjected to a pretreatment by using 8-hydroxyquinoline (8-HQ) solution followed by cooling for 2 hours at 14°C. Thereafter, the roots were washed with distilled water three times for 5 minutes to eliminate 8-HQ. Then, this material was fixed in 3:1 Carnoy I solution and stored under refrigeration for slide mounting. For slides preparation, the rootlets were washed in distilled water and hydrolyzed 1N HCl for 20 minutes at room temperature to the degradation of the cell wall. After hydrolysis, were washed again in distilled water. The rootlets were cut vertically, macerated, stained with orceine 2% and viewed under an optical microscope with objective at 10, 40 and 100x.

To determine the morphological floral structure patterns of the Capsicum 10 flowers were preliminarily evaluated, registering the variables as floral radius, length of peduncle, number and color of the petals, and number of stamens, respectively.

The quantitative and morphological traits were studied according to the standard procedure of recording data in chili crop and were analyzed by using the GLM procedure from Statistical Analysis Systems-SAS (r). To identify significant difference among treatments and statistical significance for all comparisons was made at p<0.05. Tukey's multiple range test was used to compare the mean values of treatments.

Results

Both mitosis and meiosis analysis for C. annuum, C. frutescens and C. chinense, confirmed chromosome number 2n=2x=24 (Figures 1 a, b, c), In the genotypes of C. annuum and C. frutescens, there was a numerical variation of 2n=26 chromosomes (Figure 1d), suggesting that resulting from a process of aneuploidy or an upward disploidy, causing an increasing in chromosome number.

Figure 1: Mitotic cells in prometaphase (a) and metaphase (b) in C. annuum, with 2n=24; meiosis (diakinesis), C. frutescens with 2n=2x=24 (c) and 2n=2x=26 (d).

Some chromosomal abnormalities as additional nucleoli (Figure 2a-b), micronuclei (Figure 2c) and nuclear reconstitution at telophase II (Figure 2d) possibly leading to triad; irregular segregation in both metaphase and anaphase II (Figures 2e-f), and asyncronic chromosome division (Figure 2i) during meiosis II were observed in C. annuum and C. frutescens.

Figure 2: Spindles patterns and chromosomal abnormalities during meiosis II in C. annuum (a-e) and C. frutescens (f-i). a) parallel spindles in telophase II (TII), with additional nucleoli; b) perpendicular spindles in TII, with additional nucleoli; c) TII with micronuclei; d) nuclear restitution in TII, which possibly giving rise to an unreduced gamete; e) early ascension of chromosomes at metaphase II (MII); f) perpendicular spindle at anaphase II, with lagging chromosome; g) convergent spindle at MII; h) parallel spindle at MII; i) asynchronous division (prophase-metaphase) in parallel spindle.

C. frutescens presented polyploid cells with 2n=4x=48 to 2n=6x=72, which could be the result of defective cell wall formation during pre-meiotic mitosis or processes of endomitosis (Figures 3a, 3b).

Figure 3: Poliploidy in C. frutescens; a. 2n=6x=72; b. 2n=4x=48

The meiotic index (MI) of the three evaluated species of Capsicum can be seen in Table 1 and 2. C. annuum, showed 98.7% and C. frutescens 100%, respectively.

Table 1: Spindle patterns at meiosis II, tetrads of microspores and meiotic index in C. annuum.

Table 2: Spindle patterns at meiosis II, tetrads of microspores and meiotic index in C. frutescens.

It was found a pollen fertility equal to 94.6% in C. annuum, while C. frutescens showed 19.8% and C. chinense, 97.0% (Table 3, Figure 4). The genotypes of C. annum and C. chinense, because they have a high pollen fertility rate, are recommended as parental in breeding programs. C. frutescens genotype can be considered as a male sterile.

Table 3: Pollen fertility of C. annuum, C. frutescens and C. chinense.

Figure 4: Pollen grains in C. annuum; a. no viable; b. viable

The genotype of C. frutescens showed a high degree of no viable pollen, as to be regarded a male-sterile, since pollen is the final product of male meiosis not recommended as a parental. This large number of no viable grains shows a meiotic instability of this species, or a post-meiotic event which leads to partial or complete sterility.

The number of floral structures of the species of C. annuum and C. frutescens varied between 5 and 7, while in C. chinense this variation was higher, 4 to 7 (Tables 4-6; Figure 5).

Table 4: Floral structure in C. annuum.

Table 5: Floral structure in C. frutescens.

Table 6: Floral structure in C. chinense.

Figure 5: Floral structures in Capsicum. a. C. annuum , numbers ranging from 5 to 7; b. C. frutescens, numbers between 5 and 7; c. C. chinense with a greater variation, between 4 and 7.

Discussion

Both mitosis and meiosis analysis for C. annuum, C. frutescens and C. chinense, confirmed chromosome number 2n=2x=24 (Figures 1 a, b, c), similarly to reported by Shopova (1966); Souza et al. (2011); Pozzobon et al. (2015).

In addition, Moscone et al. (2007), reported the same chromosome number for the three species. Pozzobon & Wittmann (2006), and Teodoro, Garcia & Corona (2007), reported the same chromosome number for some wild species, among them C. buforum, C. campylopodium, C. cornutum, C. villosum var. villosum, C. schottianum and C. pereirae.

Following Moscone et al. (2007), the chromosome basic number of Capsicum would be x=12, where x=13 would have arisen with the evolution of the genus. According to Pozzobon & Wittmann (2006), the genus has two lines of separate evolution, one of the wild species with x=13 chromosomes, and the ancestral number of domesticated, x=12, emerged after the loss of a pair of chromosomes.

These abnormalities were also reported by Caetano (2003), in maize and Caetano, Sandoval, Posada, Caetano-Nunes & Lagos (2008), in Vasconcellea. By other hand, Martins, Pereira, Souza & Costa (2010), reported an early chromosome ascension and laggards, which may be lost during cell division, resulting in aneuploid cells.

Pickersgill (1977), reported a chromosome number of 2n=4x=48 for C. annuum, indicating the occurrence of polyploidy, or a natural tetraploid.

This ploidy level phenomenon, could facilitate the emergence of self-fertilization of these plants the self-incompatibility as a result of the same process, affecting pollen fertility and reducing the likelihood of genetic variability (Bateman, 1952).

According to Love (1951), meiotic index larger than 90% indicate elite materials to be used as parental in plant breeding programs. Martins et al. (2010), and Pozzobon, Bianchetti, Santos, Carvalho, Reifschneider & Ribeiro (2015), observed pollen viability higher than 90% for the species C. chinense and C. annuum, C. frutescens. Conversely, Pozzobon, Souza, Carvalho & Reifschneider (2011), found a large number of unviable pollen grains, suggesting that due to the high temperature and physical factors, can influence the low viability of this species.

Moscone et al. (2007), suggest that changes may occur in chromosome number and morphology of the genus Capsicum, making necessary cytogenetic studies for chromosomal determination of this group, which has distinct genetic variation.

Conclusion

The occurrence of differences in ploidy level, including polyploidy of Capsicum annuum, C. chinense and C. frutescens, is believed to be an outcome of morphological traits changes seen in the flowers, with the number of floral structures.