Introduction

Understanding the genetic inheritance of traits is essential for selecting effective breeding strategies that harness the genetic potential of crops (Navhale, Dalvi, Wakode, Bhave, & Burondkar, 2017; Rêgo, Rêgo, & Finger, 2015). Trait inheritance information aids decision-making in breeding programs (Cazassa et al., 2019) and streamlines the process, optimizing resources, and guides breeders to select optimal parents for initial generations (Zorzetto, Motta, Morais, Kiihl, & Silva, 2008).

Pepper plants (Capsicum spp.) serve various purposes in industries and local businesses, including food production, pharmaceutical and cosmetic applications, personal and collective defense weaponry, and recently, ornamental cultivation (Gomes et al., 2019). Ornamental pepper plants (Capsicum annuum L.) are chosen for their unique aesthetic attributes, such as variegated foliage, compact stature, and multi-colored fruits on the same plant (Rêgo & Rêgo, 2016). Their ease of cultivation, durability in pots, and resistance to pests and diseases make them ideal for ornamental purposes (Neitzke, Fischer, Vasconcelos, Barbieri, & Treptow, 2016).

Few breeding programs for ornamental peppers exist both in Brazil and globally. Consequently, there is a limited number of distinct cultivars available in the market. This situation contrasts with the substantial genetic variability present within the species, which remains largely untapped at the genetic breeding level. An initial step in establishing a successful breeding program, following the identification of parent plants with ornamental potential, involves conducting studies on the genetic inheritance of key contrasting traits present in these parents.

Pessoa, Rêgo, Santos, Carvalho, and Rêgo (2019) assessed the genetic inheritance in ornamental pepper plants and identified potential genetic improvements in traits such as plant height and leaf length. Similarly, Zixiang, Xiaowei, Jianguo, Zhujun, and Biao (2021) conducted a comparable study and revealed that the fruiting pattern is a recessive genetic trait controlled by a single gene.

Qualitative traits are typically governed by simple inheritance patterns and controlled by one or just a few genes, with minimal influence from the environment. These traits are often categorized into distinct classes. In the case of ornamental pepper plants, descriptors such as flower color, anther color, leaf characteristics, and fruit color tend to exhibit this straightforward pattern of inheritance. In contrast, quantitative traits are more complex in nature and are significantly influenced by environmental factors (Falconer, 1981).

The breeding program for Capsicum spp. at Universidade Estadual de Montes Claros (Unimontes) in Janaúba, Minas Gerais State, Brazil, identified two promising parents, UNI01 and UNI05, with the potential to be used as ornamental pepper varieties (Pimenta et al., 2020). This program was initiated based on the attributes of these parent plants, with a primary focus on developing new ornamental cultivars. Understanding the genetic inheritance of traits that contribute to a desirable ornamental ideotype is a crucial strategy for the successful execution of a breeding program. The selection of specific descriptors for the current study was guided by the contrasts observed between these parents (Pimenta et al., 2020).

In this context, our primary goal was to elucidate the genetic inheritance of traits related to ornamental characteristics in segregating populations of pepper plants (C. annuum L.) derived from these contrasting parents, while also characterizing them based on their flowering and fruiting cycles.

Material and methods

Experiment location

The experiment took place in a greenhouse, which was covered with a 50% shade screen. This greenhouse was located in the experimental area of Universidade Estadual de Montes Claros (UNIMONTES), Janaúba, Minas Gerais State, Brazil (latitude 15°48'09" S, longitude 43°18'32" W, at an altitude of 533 meters). The climate in this region is classified as AW, a tropical rainy savanna with a dry winter, according to the Köppen classification (Ometto, 1981).

Generation acquisition and experiment conduct

The study involved the analysis of seven generations: P₁ (parent UNI01), P₂ (parent UNI05), F₁, F₂, RC₁, RC₂, and F₃. The F₁ generation was obtained through biparental crossbreeding between the accessions UNI01 (P₁, Capsicum annuum var. annuum L.) and UNI05 (P₂, C. annuum var. glabriusculum), both sourced from the active germplasm bank of the pepper plant breeding program (Capsicum spp.) at Unimontes, Janaúba, Minas Gerais State, Brazil.

UNI01 displays flowers with a white corolla without any spots, along with blue anthers and white filaments. Its leaves have a medium width, strong roughness, and a moderately intense green color. The fruits are pungent and small, initially white-greenish before ripening and turning red (Pimenta et al., 2020).

UNI05 exhibits flowers with a purple corolla without any spots, along with purple anthers and filaments. Its leaves are narrow, dark green, and slightly rough. The fruits are pungent and short, initially dark purple before maturation and eventually turning red (Pimenta et al., 2020).

The F₁ generation was produced through manual hybridization between UNI01 and UNI05 (Cycle 01), followed by the generation of the F₂ generation through the self-pollination of F₁ individuals. Segregating populations (F₂, RC₁, RC₂, and F₃) were obtained by sowing the F₁ population alongside the parents and conducting targeted crosses. The F₂ generation resulted from the self-pollination of F₁ plants. Backcrosses were achieved as follows: RC₁ was produced by crossing UNI01 (female parent) with F₁ (male parent), and RC₂ was produced by crossing UNI05 (female parent) with F₁ (male parent). Additionally, seeds for F₁, P₁, and P₂ were renewed.

All crosses were manually performed using flower buds at the pre-anthesis stage. In the morning, the plants were emasculated, and pollen from another plant's anther was immediately transferred to the stigma of the recipient flower. Subsequently, the flowers were labeled and covered with aluminum foil to prevent contamination. Seeds for sowing the segregating population were collected from mature fruits.

The segregating population was evaluated through a greenhouse experiment. Seeds were sown in 128-cell plastic trays, with each cell containing a commercial substrate (Bioplant®) and a single seed. The number of seeds sown for each category was as follows: 10 seeds for parent P₁, 10 seeds for parent P₂, 15 seeds for the F₁ generation, 200 seeds for the F₂ generation, 200 seeds for the F₃ population, 60 seeds for RC₁, and 60 seeds for RC₂. The trays were kept in the greenhouse and received daily irrigation.

Seedlings with four to six definitive leaves were transplanted into 1-liter pots filled with a mixture of clayey soil, coarse sand, and bovine manure in a 1:1:1 ratio. These pots were arranged in rows and labeled for subsequent evaluations. Throughout the experiment, cultural practices were applied following conventional cultivation recommendations for the crop (Filgueira, 2012), with necessary adaptations for protected and potted cultivation.

Evaluations

Qualitative descriptors

The plants underwent morphological evaluation based on four qualitative descriptors: corolla color, the predominant shape of the transversal fruit section, the predominant shape of the longitudinal fruit section, and fruit color at the immature stage. These descriptors followed the recommendations of the National Service for Plant Variety Protection (Serviço Nacional de Proteção de Cultivares [SNPC], 2015), an agency affiliated with the Brazilian Ministry of Agriculture, Livestock and Supply - MAPA (Brasil, 2015).

For corolla color, pepper plants were categorized as having one of the following colors: white, white with a violet base, white with a violet margin, white-greenish, white greenish with a violet margin, light yellow, yellow, greenish-yellow, violet with a white base, or violet (Brasil, 2015).

For the predominant shape of the longitudinal fruit section, plants were classified as having one of the following shapes: flattened, rounded, heart-shaped, square, rectangular, trapezoidal, triangular, narrow-triangular, horn-shaped, oval, or elliptical (Brasil, 2015).

Regarding the predominant shape of the transversal fruit section, plants were classified as having one of the following shapes: elliptical, angular, or rounded (Brasil, 2015).

Fruit color before ripening was assessed based on the colors whitish-green, yellowish, green, or purple (Brasil, 2015). This evaluation considered immature fruits located at the second or third node of the plant.

These qualitative descriptors were evaluated through side-by-side comparisons, with distinctiveness determined by direct observation of genotypes in greenhouse tests. The evaluations were visual, and scores were assigned to each descriptor and genotype involved in the experiment, without using measurements.

Quantitative descriptors

Two quantitative descriptors were assessed: flowering and fruiting cycles, measured in days. The flowering cycle was determined by counting the days from sowing until 50% of the plants had at least one open flower. The fruiting cycle was measured by counting the days from sowing until the first fruit on each plant reached full maturation.

Statistical analyses

Qualitative descriptors

Parents and all generations resulting from the crosses were evaluated to determine genetic control, based on phenotypic segregations. For traits controlled by a single gene, segregation ratios of 3:1 and 1:2:1 were tested in the F₂ generation. In the F₃ generation, ratios of 5:3 and 3:2:3 were tested, while ratios of 1:1 and 1:0 were tested in the backcrosses (RC₁ and RC₂).

For traits controlled by two genes, segregation ratios tested included 9:7, 13:3, 15:1, 9:6:1, 12:3:1, and 9:3:4. For traits controlled by three genes, ratios of 27:37 and 63:1 were examined. Additionally, epistatic segregations, involving one or two genes, were tested.

Hypotheses concerning expected ratios were assessed using the chi-square test (χ²) to analyze phenotypic segregation in parents and all generations resulting from the crosses, following Equation 1:

χ² = ∑ (Observed frequency-Expected frequency)²/(Expected frequency) 1

The hypothesis of specific segregation (H0) for each locus was tested at a 1% significance level. These analyses were performed using the GENES software (Cruz, 2016).

Quantitative descriptors

Descriptive statistics, including frequency distribution, were employed to categorize values into proposed classes for each descriptor: the flowering cycle included early (<99 days), medium (100 to 114 days), and late (>114 days), while the fruiting cycle included early (<146 days), medium (147 to 158 days), and late (>158 days).

Results and discussion

The segregation ratio for flower corolla color was not significant, indicating agreement with the expected segregation pattern (Table 1). The evaluated generations conformed to the 1:2:1 ratio, suggesting that this trait is controlled by codominant inheritance and governed by a single gene with two alleles, resulting in three distinct phenotypes. Notably, one of these phenotypes displayed a combination of colors (white corolla with purple borders) (Figure 1).

The contrast in corolla color between the parental lines UNI05 (C. annuum var. glabriusculum) and UNI01 (C. annuum var. annuum) resulted in the F₁ generation exhibiting flowers with an intermediate color, white with a violet margin. This intermediate phenotype alone is insufficient to determine the species of the F₁ genotypes, necessitating consideration of other descriptors. However, our results confirm that the type of interaction observed is codominance, as evidenced by observations in the F₂, RC₁, and RC₂ generations.

The purple corolla color is associated with the production of anthocyanin by the genotype. Chaim, Borovsky, Jong, and Paran (2003) mapped the A gene on chromosome 10, responsible for anthocyanin synthesis, in an F₂ population resulting from a cross between C. annuum L and C. chinense Jacq. Anthocyanin synthesis is partially controlled by the A gene, displaying partial dominance due to variations in pigment distribution in tissues. This variation includes both qualitative differences in the presence of purple color and quantitative differences in the intensity of purple color among genotypes.

The existence of distinct flower colors within this species can be harnessed for ornamental purposes. Combining these colors with other descriptors can lead to specific combinations, resulting in diverse products that cater to various consumer preferences. This descriptor is particularly important for species identification and phenotypic selection, as taxonomists often focus on flower characteristics (Carvalho & Bianchetti, 2008).

Similar to corolla color, the results for immature fruit color indicated a non-significant difference from the expected segregation pattern (Table 1). A segregation ratio of 3:1 (purple: white) was observed, suggesting that immature fruit color is governed by a gene with two alleles, and the allelic interaction is of the complete dominance type.

In Capsicum species, the presence or absence of green color in immature fruits is considered a qualitative trait (Brand et al., 2012), and in C. chinense, it has been molecularly linked to a single gene locus (Shu et al., 2023), detailed as an allelic series by Wang and Bosland (2006). It is worth noting that in Capsicum species, various fruit color stages are commonly observed during ripening (Song et al., 2022; Wu et al., 2022). However, this study focused solely on the coloration of immature fruits at the initial stage. A more comprehensive investigation considering the various possible colorations in segregating populations, as conducted by Votava, Balok, Coon, and Bosland (2000), would provide a better understanding of the genetic relationships governing distinct and expected phenotypic expressions during ripening.

Fruit color is a crucial trait for the appearance of peppers (C. annuum L.), as consumers primarily use it as a reference when selecting and purchasing peppers (Jang et al., 2020). Lightbourn et al. (2008) investigated two C. annuum L. genotypes with black and violet fruits and correlated fruit color with concentrations of carotenoid and anthocyanin pigments in plant tissues. They attributed the difference between violet and black colors in immature fruits to varying concentrations of chlorophylls and carotenoids.

In the case of fruit shape, genetic control could not be identified based on the tested hypotheses. The parents exhibited contrasting longitudinal fruit shapes, with P₁ showing a triangular shape and P₂ exhibiting an oval shape (Figure 2). However, the filial generation predominantly had a triangular shape (Figure 2).

Although allelic interaction and dominance may play a role in the observed results for P₁, P₂, and F₁ generations, the F₂, F₃, RC₁, and RC₂ generations displayed significant variability and the emergence of an additional phenotype not observed in the parents and the F₁ generation: a narrow-triangular longitudinal fruit shape (Figure 2). These results highlight greater phenotypic variability in the F₂ populations, as this generation experienced increased segregation due to the self-pollination of the F₁ generation.

Concerning the shape of the transversal fruit section, the parents presented elliptical and rounded shapes (Figure 3). In contrast, the offspring generation predominantly expressed an elliptical shape, similar to parent P₁. The fruits of the segregating generations F₂, F₃, and RC₂ predominantly exhibited a rounded transverse shape (Figure 3).

These variables related to fruit shape are influenced by multiple genes, indicating polygenic action. Other factors, such as the number of locules, can also impact pepper fruit shape. The number of locules has various effects on fruit shape and size in Solanaceae species (Munos et al., 2011). Ma, Qiao, Li, Yu, and Gong (2022) suggest that CaBRX plays a significant role in locule development in peppers and is associated with 17 other QTLs influencing fruit shape. Van der Knaap and Tanksley (2003) observed that previously identified fruit morphology loci in tomato, eggplant, and pepper may result from pleiotropic effects of the same orthologous loci in these species. According to Pimenta et al. (2020), the parents UNI01 and UNI05 have the same number of loci, which is two. Yet, despite this, the variation observed in the segregating generations (F₂, RC₁, and RC₂) confirms the influence of genetic multi-loci.

Species of the Capsicum genus are renowned for their extensive variation in fruit color and shape, offering a rich resource for ornamental purposes. The fruit shape in Capsicum species is under the control of multiple genes. Wang and Bosland (2006) have identified six genes that influence fruit shape in these species.

Mapping studies of quantitative trait loci (QTL) have played a crucial role in pinpointing various genetic loci responsible for controlling fruit shape in Capsicum spp. (Zygier et al., 2005; Barchi, Lefebvre, Sage-Palloix, Lanteri, & Palloix, 2009). Chaim et al. (2001) reported the identification of three QTLs associated with fruit shape in peppers, with one major QTL on chromosome 3 explaining over 60% of the phenotypic variation in fruit shape. Chunthawodtiporn, Hill, Stoffel, and Van Deynze (2018) suggested three candidate genes responsible for regulating fruit size and shape in bell peppers. In the case of C. annuum, fs3.1 and fs10.1 have been identified as controlling fruit shape, particularly the length-to-width ratio of the fruit (Han et al., 2016).

In the context of the frequency distribution analysis, the flowering cycle was categorized into three classes: early (<99 days), medium (100 to 114 days), and late (>114 days). Both parental plants and the F₁ generation displayed an early flowering cycle (Figure 4). Over 54% of individuals in the other generations also exhibited an early cycle, with only a small proportion of plants demonstrating a late flowering cycle (Figure 4).

The fruiting cycle was categorized into three groups: early (<146 days), medium (147 to 158 days), and late (>158 days). Both the parents and the F₁ generation exhibited an early fruiting cycle. Furthermore, more than 51% of individuals in the other generations also displayed an early fruiting cycle (Figure 5).

Growers have a preference for plants with early flowering cycles because it shortens the time it takes for the product to become commercially available (Silva, Rodrigues, Bento, & Pimenta, 2017). However, the classification used in this study can be a subject of debate and is inclined to complexity due to its association with a quantitative trait. According to Falconer (1987), such traits are significantly influenced by environmental factors, which can diminish the genetic contribution to the observed phenotypic expression.

The duration of the flowering cycle in pepper plants is directly linked to the timing of the fruiting cycle. In the context of ornamental pepper plants, the commercial stage is reached when the plants bear ripe fruits, as these are the primary attractors for consumers. Consequently, these cycles must be relatively short, as this reduces production costs related to irrigation, management, and inputs. Additionally, shorter cycles minimize the exposure of crops to potential risks such as pests and diseases.

The insights gained from studying the segregation patterns of the traits assessed in this research will prove invaluable for researchers involved in this breeding program when selecting the most suitable breeding strategy. These findings are expected to enhance the precision of the selection process, making it more effective. Furthermore, the characterization of flowering and fruiting cycles can aid in the planning of various activities within the breeding program, whether about management practices or the choice of breeding methods to be employed.

Conclusion

The key findings comprised that codominance is the genetic control mechanism for flower corolla color in ornamental pepper plants (Capsicum annuum L.) and dominant genetic inheritance plays a role in determining immature fruit color. However, genetic inheritance patterns for transversal and longitudinal fruit shapes cannot be elucidated based on the tested segregations. The parental and segregating generations under evaluation exhibit similar flowering and fruiting cycles. Both parents and the F₁ generation displayed a 100% incidence of early flowering and fruiting cycles, while the segregating generations had more than 50% of individuals with early cycles.

Acknowledgements

Thanks to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), for the infrastructure provided and financial support

References

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