Meiotic analysis of interspecific hybrids between Capsicum frutescens and Capsicum chinense

The aim of this study was to observe the genetic relationship between C. frutescens (UENF 1636) and C. chinense (UENF 1785) based on the meiotic behavior, on the meiotic index, and on pollen viability of their F1 hybrids. For meiotic analysis and meiotic index, flower buds were collected and fixed for 24 hours in ethanol: acetic acid (3:1), and then transferred to 70% ethanol, and stored in a freezer. Slides preparation for meiosis, meiotic index and pollen viability was carried out according to the laboratory’s protocol. Hybrid meiosis was regular, and 12 pairs of chromosomes were observed in diakinesis, as well as a few anomalies. The MI of 88.16%, as well as the pollen viability of 72.5%, were satisfactory. Given the results, it was confirmed the genetic proximity of these species, which are classified in the Capsicum annuum gene complex.


INTRODUCTION
The genus Capsicum belongs to the Solanaceae family, and is represented by pepper and bell peppers (Eshbaugh 1993).Most of the Capsicum species are self-compatible, except for C. cardenasii, whose incompatibility is gametophytic, also found in other Solanaceae genera (Pickersgill 1991).According to Barbosa et al. (2011), among the 38 species, which have already been identified, five of them are grown and used by humans: C. annuum var. annuum; C. baccatum var. baccatum; C. chinense; C. frutescens, and C. pubescens (Pickersgill 1991, Moscone et al. 2007).Within this genus, species can be distinguished in two groups, according to the basic number of chromosomes: one with n = x = 12 chromosomes, and another with n = x = 13 chromosomes (Moscone et al. 2007).
Wild species are considered as gene repositories, and several varieties of agronomic importance have been improved through interspecific hybridization (Hajjar and Hodgkin 2007).Interspecific crosses may allow the improvement of varieties by the introduction of important alleles, such as those which provide resistance to pathogens (Pickersgill 1997).However, for the success of gene transfer between species, it is necessary that the species are genetically close, i.e., they must present chromosome homology, reducing incongruity problems, and thus making the hybrid viable (Prestes and Goulart 1995).In addition, the knowledge of the direction of the cross is essential, since, in some species, interspecific cross is effective and unique to a given direction (Prestes and Goulart 1995).

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In Capsicum, species are grouped in three complexes in order to obtain fertile hybrids.The complex Capsicum annuum gathers the species C. annuum, C. chinense and C. frutescens and their botanical forms; the complex Capsicum baccatum gathers the species C. baccatum var.baccatum and C.baccatum var.pendulum; and the complex Capsicum pubescens gathers wild species and only one domesticated species, C. pubescens (Pickersgill 1991).
Interspecific hybrids can be obtained between the domesticated species of the Capsicum genus; however, these hybrids present different degrees of fertility, which may be due to chromosome pairing problems in the hybrid, or due to the presence of different chromosome structural arrangements (Kumar et al.1987, Lanteri andPickersgill 1993).According to Shifriss (1997), the degree of male sterility of interspecific hybrids of Capsicum depends on the accessions used in the crosses.Monteiro et al. (2011) observed that the degree of viable interspecific hybrids of Capsicum ranged from high, in combinations involving C. chinense x C. frutescens, to complete male sterility, or hybrid unviability.The authors also observed that pollen viability of the interspecific hybrid depended on the accessions involved in the combination, and this was a genotype-specific viability.
The knowledge of the fertility of the species involved in an interspecific hybridization program is important in order to obtain successful crosses.The fertility of a species, usually measured by the pollen viability, is the result of normal and balanced gametes formation.According to Defani-Scoarize et al. (1996), the success of both intra and interspecific hybridization, in genetic terms, is also determined by a regular meiosis, resulting in the formation of viable gametes.
Considering the importance of evaluating the fertility of interspecific hybrids obtained between C. frutescens (UENF 1636) and C. chinense (UENF 1785), and of following up with further researches, this study aimed to analyze the meiosis of an interspecific hybrid, and to estimate pollen viability and the meiotic index of this hybrid.

MATERIAL AND METHODS
In this study, it was used an interspecific hybrid plant obtained between the accessions UENF 1785 (C.chinense) and UENF 1636 (C.frutescens) (Martins et al. 2015).Hybrid seeds were initially germinated in B.O.D. chamber at 27.5 ºC, with photoperiod of 8 hours of light and 16 hours of darkness, and then planted in polystyrene trays with the vegetable substrate Vivatto®, in a greenhouse at the Unit for Research Support (UAP) in the campus of the State University of Northern Rio de Janeiro (UENF).When seedlings presented four to six true leaves, they were transplanted from the tray to 500 mL plastic cups with vegetable substrate Vivatto®, in a greenhouse at the UAP of UENF.About two months after germination, plants were transferred to 5 L plastic pots, using the same type of vegetable substrate, with 15 hybrid plants.Management was employed according to the recommendations for the culture (Filgueira 2000).
During the flowering period, flower buds of different sizes and at different development stages were collected at random in bulk, in fifteen plants of the interspecific hybrid, and fixed in ethanol: acetic acid solution at a ratio of 3:1, for 24 hours.Afterwards, they were transferred to 70% ethanol solution, and kept in the refrigerator until being used.For slides preparation, three anthers of flower buds were macerated (squash) on the slide, in 1% acetic carmine solution, according to the laboratory's protocol.Slides were then observed under optical microscope, in brightfield, and the different stages of meiosis were analyzed.Possible meiotic abnormalities were also recorded, such as laggard chromosomes, early segregation of chromosomes, and problems in the spindle fibers.
To estimate the meiotic index (MI), according to Love (1951), flower buds at anthesis were collected in 70% ethanol solution and kept in the refrigerator.At the time of slides preparation (5 slides), three anthers were macerated in 1% acetic carmine solution and observed under a microscope.For the estimate of the meiotic index, the numbers of the post-meiotic products in the five slides analyzed (500 pollen grains, totaling 1500 post-meiotic products) were counted.The meiotic index is estimated by the ratio of the total normal tetrads by the total post-meiotic products.Tetrad with four nuclei of the same size was considered normal, and any deviation (monad, dyad, triad and polyad) was considered abnormal.

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Pollen viability of the accessions was estimated by the Alexander's triple solution (Alexander 1969).To this end, flower buds at anthesis were collected in 70% ethanol solution and stored at 4 °C.In the slides preparation, the anthers were macerated in a drop of the Alexander's triple solution, in which viable pollen grains were stained red, and unviable pollen grains were stained green, simultaneously.In order to obtain a random sample of the stained pollen grains, the scanning method was used in the slide (8 slides), and 250 pollen grains/slide were counted.All the slides were observed under optical microscope (Olympus BX60), and the images were captured with the Image-Pro Plus Software (version 5.1, Media Cybenertics).

RESULTS AND DISCUSSION
The meiotic analysis of the hybrids allowed observing 12 chromosomes in bivalent association in diakinesis (Figure 1a), corroborating the literature for diploid parental species, with 2n=2x=24 chromosomes (Pozzobon et al. 2006, Moscone et al. 2007).This result is expected, since the parental species, C. frutescens and C. chinense, differ only by the presence of an annular constriction in the calyx, typical of the species C. chinense (IPGRI 1995).For some authors, both species are in fact a single species, depending on the results of morphological analysis (Pickersgill et al. 1979), enzyme analysis (Jensen et al. 1979), and cytogenetic (Egawa and Tanaka 1986).However, Baral and Bosland (2004), when investigating the morphology, crossability, and polymorphism of the DNA of the accessions of these species, concluded that Capsicum frutescens and C. chinense represent two distinct species.Meiotic abnormalities were observed in the form of early chromosome segregation (Figura 1b) at the second meiotic division; of laggard chromosomes (Figures 1c and 1d); and of the lack of synchrony.Usually, laggard chromosomes are lost during cell division, causing aneuploid daughter cells (Weinert 1998).
The abnormalities found may result in unbalanced gametes at the end of cell division.According to Pagliarini (2000), the most common meiotic abnormalities observed in several species is the irregular chromosome segregation, characterized by early migration or laggard chromosomes at metaphase I and anaphase I. Considering that the meiotic behavior of a plant is directly related to their degree of fertility, the changes observed during cell division may be reflected in the viability of pollen grains (Defani-Scoarize et al. 1996).
In the hybrid analyzed, it was observed that cytokinesis is not symmetrical, with triad post-meiotic products (Figure 1f).However, there was predominance of normal tetrads (Figure 1E), which is expected at the end of a normal meiotic division.Triads can originate from errors that occurred at the time of the cytoplasm division during meiosis I or II, and are