Morphological diversity among Brazilian Capsicum peppers

ABSTRACT: This research aimed to morphologically characterize and estimate the genetic diversity of 21 Capsicum accessions belonging to the Capsicum Germplasm Active Bank at the Universidade Federal do Piauí (BAGC-UFPI) using uni- and multivariate analysis. The experiment was carried out in a greenhouse, by completely randomized experimental design with four repetitions, with one plant per plot. Analysis of variance (ANOVA) and the comparison of means for seven quantitative variables were performed, followed by clustering the averages by the Scott-Knott test (P < 0.05). The analysis of the seven quantitative and thirteen qualitative descriptors was estimated based on the Gower distance. Later, it was performed the principal component analysis and the UPGMA hierarchical cluster method. Results characterized and identified a wide intra- and interspecific genetic variability related to the fruit size, colors, and shapes among the Brazilian Capsicum genotypes belonging to the BAGC-UFPI. The descriptors used in this research were effective in the discrimination of the pepper accessions, especially the closely related C. frutescens and C. chinense species.

Understanding the genetic variability of the plant species based on the morphological, geographical, cytogenetics and molecular traits is essential for the agronomic, genetic and evolutionary studies, especially for that poorly researched and economically under-exploited Capsicum species (ASSIS et al., 2020;BIANCHI et al., 2020). Moreover, the agronomic and genetic characterization is important for the conservation and the maintenance of the germplasm banks and for supplying additional knowledge for the genetic breeding programs Ciência Rural, v.53, n.1, 2023.
Brazil is considered a secondary center of Capsicum species. Aiming to preserve the genetic diversity of peppers in the country, the Universidade Federal do Piauí created a Capsicum Germplasm Active Bank (BAGC-UFPI). Until November 2021, the BAGC-UFPI has more than 250 peppers accessions belonging to four out of the five different Brazilian regions (Northeast, Southeast, Midwest, and South). To date, genetics, cytogenetics, agronomical and morphological studies have been performed in the pepper accessions of the BAGC-UFPI (MARTINS et al., 2018;COSTA et al., 2019).
The morphological characterization remains a classical and efficient approach to estimating the genetic diversity within and between species and/or accessions belonging to the germplasm banks (MELO et al., 2014). Pessoa et al. (2018) and Soares et al. (2019), for instance, used morphological traits to quantify and identify the genetic dissimilarity of heterotic groups of pepper accessions at BAGC-UFPI. The authors identified genotypes with potential usage in the breeding programs for ornamental peppers and with desirable agricultural characteristics for registration as cultivars, respectively.
Uni-and multivariate analysis has become routine as a research approach for genetic breeding programs, as in the divergent progenitor's studies, and methods for selection of segregation population (CRUZ et al., 2012;LEITE et al., 2018). This research aimed to morphologically characterize and estimate the genetic diversity of 21 Capsicum accessions belonging to the BAGC-UFPI by using uni-and multivariate analysis.

MATERIALS AND METHODS
The experiment was carried out in the greenhouse using Sombrite ® 40% in the Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal do Piauí (UFPI), Teresina, PI, Brazil, from May 2019 to January 2020. The 21 pepper accessions belonging to BAGC-UFPI used in research are represented in table 1. Seeds of each accession were germinated in polystyrene seedling trays (128 cells each) with the commercial substrate Plantmax ® . After two weeks, the plants were transferred to polyethylene vessels (14 L) containing substrate with vegetable soil and humus. The fertilization was performed using a mixture of 2 g of ammonium sulfate, 17 g of single superphosphate and 2.5 g of potassium chloride, as recommended by Alcântara and Ribeiro (2008). The phytosanitary treatment for pest control and irrigation was performed during all the plant cycles, by using micro-sprinkle. The low germination rate during the experiment resulted in different numbers of evaluated accessions within species.
The Four plants were evaluated per accession, being 10 mature fruits per plant. The measurement of the plant height was performed by measuring tape, while the fruit pedicel length, fruit length and weight were obtained by digital caliper. The fruit weight was calculated using a digital scale.

Data analysis
The univariate analysis was performed by the analysis of variance (ANOVA) (P < 0.05) and the hierarchical Scott-Knott (P < 0.05) algorithm executed by GENES program (CRUZ, 2013).
For the multivariate analysis, principal component analysis (PCA) and the biplot graphic were performed by FactoMineR (HUSSON et al., 2008) and factoextra (KASSAMBARA; MUNDT, 2019) libraries. The construction of the UPGMA dendrogram was performed by adopting the Gower coefficient as dissimilarity measure (GOWER, 1971). The dendrogram validation was obtained by the cophenetic correlation by Biotools (SILVA et al., 2017) and Cluster (MAECHLER et al., 2019) packages executed in the computational and statistical environment R (R CORE TEAM, 2019).

RESULTS AND DISCUSSION
The results showed significant differences (P < 0.05) observed in the 21 pepper accessions for the quantitative descriptors, indicating genetic variability among them. The coefficient of variation (CV%) results for the quantitative descriptors (Table 2) ranged from 2.77% Number of days to flowering (NDF) to 23.26% Fruit weight (FWe).
The quantitative trait analysis based on the Scott-Knott test identified 10 clusters regarding the Number of days to flowering (NDF) ( Table 3). The Table 2 -Mean squares (MS) and coefficients of experimental variation (CV) obtained by the variance analysis of seven quantitative descriptors of peppers evaluated among the 21 accessions belonging to the BAGC-UFPI.  width among the accessions, ranging from 0.77 to 5.63 cm in C. baccatum and C. frutescens, respectively. For the Fruit weight (FWe) descriptor, seven clusters were observed, with higher values for BAGC 130 and 131 (28.48 g and 23.88 g, respectively). The accessions BAGC 110,117,122,156,157,160,178,208,209,222 and 250 belong to the group with lighter fruits, with an average less than 4.38 g. The accession BAGC 130, the red bell pepper (C. annuum), showed a higher weight of 28.48 g. Similarly, Sudré et al. (2005) reported the weight variation in sweet pepper occurred from 25.54 g to 48.24 g. The C. annuum species include sweet pepper, which has larger fruits and greater weight.
The mature red bell pepper fruits can be dehydrated by heating and then ground for paprika production (food colorant). The paprika extract is one of the most consumed flavorings in worldwide cuisine (BAENAS et al., 2019). The Capsicum fruits owe their intense red color to different carotenoid pigments, especially capsanthin and capsorubin (KONISHI et al., 2019). Carotenoids, not naturally synthesized by humans, have a range of functions and properties in human health, such as antioxidant effects and cancer cell proliferation inhibition (KIM et al., 2016). We suggested the selection of the accession BAGC 130 for the paprika market or to be consumed in natura.
Our principal component analysis results indicated the variance percentage for each component, being that, 77.37% of the variance in the third component (CP3) was accumulated (Table 4). Our results corroborate with BELAY et al. (2019) research. In a study analyzing 64 hot pepper genotypes by using 19 morphoagronomical traits, the authors indicated that 69.25% of the variance in the data set was related to only three principal components.
Our Biplot results (Figure 1) showed the first and the second principal components, with BAGC 130 genotype the most divergent among the clusters. As we highlighted, this accession (C. annuum) exhibited a higher fruit weight. Moreover, the accession BAGC 130 showed prostrate plant growth habit, with no firmness nor sustentation (needed tutoring), intermediate leaf density, corolla with no spot, pallid blue anthers, no calyx annular constriction, pendants fruits with dark brown color in their intermediate maturation stage and rectangular fruits with apex sunken. Our graphic also identified the accessions BAGC 91, 131, and 251 are closely related and have a correlation with the weight and width of their fruits.
The accessions BAGC 114 and 242 created a cluster of fruit length (FL). The genotypes BAGC 122, 160, 208, 222, and 250 created a cluster for plant height (PH) ( Table 3). The later accession, commonly known as "pimenta-de-cheiro" (C. chinense), showed the higher average, with176 cm. Similar results were obtained by Araújo et al. (2018) that analyzed 20 genotypes of peppers belonging to Peruvian, Colombian, and Brazilian Amazon Forest, and observed C. chinense with a greater high of 100.12 cm. The accessions BAGC 110, 177, and 209 were clustered for NDF and NDM, presenting later reproductive cycle and later days to fruiting (Table 3).
The dendrogram generated by the UPGMA method ( Figure 2) using Gower distance by the qualitative and quantitative variance showed five clusters, considering a cut-off value representing 40% of dissimilarity among the analyzed accessions. Cluster 1 represents C. baccatum (BAGC 156) and C. annuum species (BAGC 163,114,130,and 131). Despite belonging to different species, the accessions  (BAGC 117), which presented plants with stem and pubescence leaves, besides erect and black fruits in the immature stage ( Figure 2). These peppers, known as "malaguetas", are one of the most consumed peppers in Brazil, especially in Zona da Mata Mineira, which are consumed in natura or as in hot pickled sauce production (CARVALHO et al., 2006). The domesticated C. annuum, C. chinense, C. frutescens and the wild C. galapagoense species belong to Annuum clade, previously known as the "white flower group" (CARRIZO GARCÍA et al., 2016). The closely related C. frutescens and C. chinense are easily confused regarding their morphological features. The presence of the calyx annular constriction with no geniculate pedicel in C. chinense is the main morphological difference between these species (CARVALHO et al., 2014). In our research, the quantitative and qualitative multicategorical descriptors showed to be effective in the discrimination and identification of C. chinense and C. frutescens accessions.
Cluster 3 presented a higher number of accessions: BAGC 176,177,208,157,81,222,178,110 and 122, all belonging to C. baccatum ( Figure  2). These peppers, known as "dedo-de-moça" (BAGC 81,110,122,156 and 157) are commonly used in sauce and jelly production (CARVALHO et al., 2006;LIMA et al., 2017). Within this cluster, we noted phenotype similarity between the accessions BAGC 110 and 122. No differences were observed in the qualitative variance, regardless of the different provenance of the accessions.
Cluster 4 is represented by C. chinense genotypes (BAGC 160, 242, 91 and 251) (Figure 2). We highlighted the accession BAGC 160 (murupi pepper), with elongated and yellow fruits, besides a wrinkled surface. Murupi pepper is widely used in the North of Brazil as seasoning because it exhibits characteristic flavor and chili taste. Its pepper commercialization usually occurs in marketplaces as in natura or as homemade sauces (NASCIMENTO FILHO et al., 2007). The accessions belonging to cluster 4 show a wide range of shapes, sizes, and color of their fruits. Similarly, Moreira et al. (2018) reported wide differences in 65 C. chinense accessions belonging to different Brazilian regions, in terms of color, shape, size, and pungency of their fruits, being widely used in the Brazilian cuisine ( NASCIMENTO FILHO et al., 2007;ARAÚJO et al., 2018). The authors highlighted the importance of understanding the genetic variability of C. chinense for its current conservation and future status in the genetic breeding programs. Cluster 5 is composed by two C. chinense accessions, BAGC 209 and 250, being the later with the higher average plant height, besides the purple fruits in the immature stages ( Figure 2). "Cheiro" pepper fruits (BAGC 91,242,209,and 250) are known because of their strong flavor and sweet pungency. They are mostly commercialized as in natura and used as rice, salads, and fish seasoning (CARVALHO et al., 2006). These accessions have the potential to be used in several breeding programs or be commercialized as fresh vegetables for gastronomic usage.
Our results confirmed the high morphological diversity among Capsicum species and varieties. The continued development of the genetic diversity exploration among peppers, including accessions belonging to germplasm banks, is fundamental for understanding the genetic diversity of this genus. Additionally, morphological data are essential for the germplasm characterization and provide aids for current and future efficient strategies of the genetic breeders for usage and conservation of the genetic resources of this socioeconomic important Capsicum genus.

CONCLUSION
Quantitative and qualitative descriptors showed high intra-and interspecific genetic variability among the genotype collection of Capsicum accessions at BAGC-UFPI. The descriptors used in this research showed to be effective to differentiate the analyzed pepper accessions, especially the closely related C. frutescens and C. chinense species. The evaluated genotypes have economic potential and usage in breeding programs.