Morphological, Sensorial and Chemical Characterization of Chilli Peppers ( Capsicum spp.) from the CATIE Genebank

: In order to assess the potential of 192 accessions of Capsicum L., from 21 countries, a morphological and agronomic characterization was carried out by applying 57 qualitative and quantitative descriptors. Multivariate analyses identified two large groups: the first including C. annuum (G3, G5, G7 and G8) and the second C. frutescens , C. baccatum , C. chinense and C. pubescens (G1, G2, G4, G6 and G9). The discriminant qualitative descriptors were the colour of the corolla, the colour of the anthers and position of the flower. The quantitative discriminant characteristics were length, weight and width of the fruit. The participatory selection identified 15 materials by colour, aroma, texture, flavour, size and thickness of fruits. Chemical analyses determined the highest concentration of flavonoids in the accessions 10,757 (16.64 mg/g) and 15,661 (15.77 mg/g). Accessions 17,750 (11.68 mg/g) and 10,757 (11.41 mg/g) presented the highest polyphenol contents. The highest capsaicin concentration was recorded in accessions 16,209 (55.90 mg/g) and 10,757 (48.80 mg/g). The highest antioxidant value was recorded in accessions 17,750 (90.85 mg/g) and 15,661 (87.03 mg/g). All these characteristics are important with a view to increasing industrial use and genetic improvement processes. These results show the existence of significant genetic variability within the genus Capsicum .


Introduction
Chilli pepper (Capsicum L.) is one of the most important spice and vegetable crops in the world in agricultural, cultural and economic terms [1][2][3]. The genus Capsicum, native to Tropical America, comprises 27 species [4,5]. The five most widely used species worldwide are: C. annuum L., C. chinense Jacq., C. frutescens L., C. baccatum L. and C. pubescens Ruíz & Pav. [6]. Capsicum annuum and C. frutescens-which according to The Plant List [7] are considered conspecific, but which are here maintained as separate taxa as they are in The Tropical Agricultural Research and Higher Education Center (CATIE) genebank-are known to have been domesticated in Mesoamerica [5], however the original description of C. frutescens by Linneaus was based on a cultivated specimen from India. In the Andean region, chilli peppers have been consumed for more than 5000 years; C. baccatum, C.

Morphological Characterization
The morphological characterization was set up in a field experiment at CATIE location, in Turrialba, Costa Rica (9° 54′ North Latitude, 83° 40′ West Longitude, altitude 625 m a.s.l.). The average annual precipitation is 2700 mm, and the annual average temperature is 22 °C according to CATIE [Centro Agronómico de Investigación y Enseñanza Tropical, CR] [26]. For morphological characterization, we worked with 192 accessions of chilli (Capsicum spp.). These accessions come from various collections carried out in Central America and Mexico between 1976 and 2006 and germplasm exchanges carried out between institutions from five continents corresponding to 21 countries (Table  1). Table 1. Origin and number of accessions of each of the species of Capsicum spp.

Capsicum Species Origin
C. annuum C. frutescens C. chinense C. baccatum C. pubescens Not Identified Twenty plants per accession were installed in the field and the middle 10 evaluated (to eliminate border effect). The descriptors jointly defined by IPGRI [International for Plant Genetic Resources Institute, now BIOVERSITY, AVRDC and CATIE [27] were used, which comprise 57 qualitative and quantitative descriptors (Tables 2 and 3). The data obtained from the morphological characterization were analysed using the InfoStat/Professional program version 2011 [28]. Qualitative and quantitative variables were used in a hierarchical cluster by using Ward's method and Gower distance, Also, contingency tables were used, using Chi-square, and multiple correspondence analyses were made.

Participatory Sensory Evaluation
For this stage the 192 morphologically characterized accessions were evaluated by 50 invited untrained participants representing producers, industrialists, chefs, scientists and ordinary consumers from Costa Rica, Mexico, Colombia, Ecuador and Peru. The evaluation was carried out in four phases. In the first phase, farmers/producers were invited to the field trial in CATIE to select accessions based on criteria such as colour, shape, size and production. In the second phase, with previously selected accessions, participants made a second evaluation related to shape, colour and size of the fruit. In the third phase, a sensory evaluation was carried out using the 9-point hedonic test proposed by Lawless [29]. However, this scale caused confusion for the attendees, so we decided to change to a friendlier scale, in which six criteria were recorded colour, smell, texture, flavour, size and thickness of the pulp. Each criterion was evaluated based on a 5-point scale: Excellent 5; Very Good 4; Good 3; Regular 2; Bad 1, proposed by Hernández [30]. In the fourth phase, the samples were tasted to determine the degree of fruit spiciness as reported by Hernández [30], the following scale was used: Very spicy 3, Medium spicy 2 and Regular 1. In each scale the assistants marked with an X the criterion they considered adequate based on their preferences. For the analysis of the results, frequency tables were prepared.

Chemical Characterization
The chemical analysis of the samples was carried out on the best 15 accessions selected by the morphological characterization and the participatory process. One kilogram of fruits, at the same maturity state, was harvested per accession from different parts of the plants to avoid differences in biochemical properties due to plant position according to Zewdie et al. [31]; Kirschbaum-Titze et al. [32]; Mueller-Seitz [33]. These samples were dried out in oven at 60 °C for 30 h, after which 100 g per accession were sent for analysis. The determination of flavonoid content was conducted using the method proposed by Miean and Mohamed [34]. The determination of antioxidant activity was completed using the method of measurement of the absorption capacity of oxygen radicals proposed by Álvarez-Parrilla et al. [35]. The determination of the content of total phenolic compounds was done according to the method proposed by Blainski et al. [36]. The determination of the concentration of capsaicin and dihydrocapsaicin was completed according to the method proposed by Juangsamoot et al. [37]. Finally, the analysis of the results was carried out through multivariate descriptive statistics.

Descriptive Analysis of Morphological Characters
Evaluation of the characteristics of stem, leaves, flowers and fruits from 192 accessions are summarized follow.

Stem
The purple hypocotyl character was present in 70 accessions, purple stem in 5 accessions, purple knot anthocyanin in 78 accessions, light purple anthocyanin in 46 accessions and dark purple in one accession. Having accessions with purple characteristics determines the presence of anthocyanins, which are classified as nutraceuticals and appetizing agents. Bhattacharya et al. [38] indicate that anthocyanins minimize the proliferation of cancer cells, prevent lipid damage in food and protect against diseases of the heart. Likewise, Rodríguez and Kimura [39] mention that antioxidants can neutralize or reduce the activity of free radicals, associated with cardiovascular diseases.

Leaves
The variable leaf shape of the individual cotyledon was recorded according to the lanceolate, elongated-deltoid and oval categories, where the oval category was the most dominant since it was found in 134 accessions, corresponding to 69.79% of the materials evaluated. Likewise, the colour of the mature leaf was mostly green in 159 accessions corresponding to 82.81%. The majority of our accessions (170) had reduced leaf pubescence corresponding to 88.54%. This is in agreement with the results reported by Smith and Heiser [40] who mention that for C. frutescens leaf pubescence tends to be scarce.

Flowers
The colour of the corolla, the colour of the anther, position of the flower, the length of the placenta, and the pubescence of the stem had a marked influence on the discrimination of species. To the extent that the C. annuum were characterized by presenting white flowers and a single flower per leaf axil and C. frutescens presented a greenish-yellow flower without calyx constriction. It is worth noting that in our study the length of the ovary influenced the grouping of the samples, which differs from IPGRI [27], which do not consider it as highly discriminating for species differentiation. According to Sreelathakumary and Rajamony [41], the length of the ovary is correlated with the size and weight of the fruits and the most extended shelf-life at the market. This descriptor is more significant in C. frutescens than in C. annuum, and capsaicin is stored in it, also, both weight and size influence good filling of fruit cavities and seed production.
In the same way, this study agrees with the results reported by Hernández et al. [42] who mention that the characteristics of the flowers per leaf axil are an important variable to establish differences between the C. annuum and C. frutescens species. Note that the two taxa here are treated as separate taxa, though taxonomists in general consider them as being conspecific, as already mentioned; however, for practical reasons they are here considered as representing separate cultivar groups. Capsicum annuum accessions were characterized by having solitary flowers and C. frutescens for presenting more than one flower per leaf axil. The white colour of the corolla appeared in 47.40% of the accessions evaluated mainly in the G3 and G7, represented by the C. annuum species. The corolla's greenish-yellow colour appeared in 36.46% of the accessions, specifically in the G4, G9 and G6 constituted by the species C. frutescens. In comparison, the colour of the corolla was light yellow at 7.29%, and in G1 mainly made up of the C. baccatum species (Figure 1). The above agrees with Pickersgill [43], who mentions that in Capsicum, two groups of flowers are defined: white and purple. In the group of white flowers, there are two subgroups, the one made up of C. baccatum and a second that groups C. annuum, C. chinense, C. frutescens. The group of purple flowers are the species C. eximium Hunz., C. cardenasii Heiser & P.G.Sm., and C. pubescens. On the other hand, Smith and Heiser [35] reported that in C. frutescens, the flowers are greenish-yellow, and for C. annuum, they are white.

Fruits
According to Andrews [43], the accessions belonging to the C. annuum species are characterized by having small, ovoid fruits with two locules, the fruit-a bloated berry-with different colours, e.g., light green, green, purple, yellow, orange and deep red. The C. frutescens species is characterized by presenting elongated fruits ending in a blunt point, with two locules per fruit, which agrees with the results found in this work where most of the accessions belong to C. frutescens and C. annuum.
In the characterized accessions, the following high variation coefficients were registered for the fruit characteristics: • anthocyanin spots or streaks on the fruit, These characters indicate the importance of the descriptor to discriminate variability within a collection. The results corroborate those reported by Smith and Heiser [40,44] who maintain that in each species of chilli pepper there are various fruit shapes and colour of immature fruits. In the accessions evaluated, the shape of the fruit was mostly triangular and elongated; however, smallfruited species tended to be round and conical, especially sweet pepper species. The variability of the genus is mainly due to the characteristics of the fruit, followed by the architecture of the plant, flower structure and the number of flowers per leaf axil [16].
Pickersgill [45] mentions that in Capsicum, the annular constriction of the calyx is characteristic of C. chinense and is absent in the other four species. For the colour of the immature fruits, it is typical for the fruits among the Capsicum spp. to start with a green colour before reaching the final colour at full maturity; however, the fruits in a mature state have mostly red tones and an elongated shape, while C. chinense matures with fruit shades of yellow and orange. Yellow colours of fruits in the intermediate maturity stage, i.e., apparent from the results obtained, were recorded in some accessions of both C. chinense (7300, 12,154, 5489), and C. frutescens (10,946,10,793,165,654). Furthermore, the shape of the fruit apex was mostly pointed in the accessions studied and the epidermis of the fruit was smooth, i.e., characteristics that correspond to the C. frutescens and C. annuum species.

Grouping of Accessions Based on Morphological Variables
The hierarchical cluster, using the qualitative and quantitative variables identified significant differences among nine accession groups of Capsicum spp. Significance (p < 0.0001) was obtained employing a multivariate analysis and the differences between mean vectors were obtained using the Hotelling comparison test corrected by Bonferroni [46,47].

Combined Analysis of Qualitative and Quantitative Variables
The result of the grouping of the accessions-obtained with the Ward method and the Gower distance-allowed us to identify the taxonomic structure of the collection, where the relationship between the groupings could be seen, i.e., C. annuum (groups 7, 8, 5 and 3), C. frutescens (groups 9, 6, 4 and 2). However, within these groups there are also accessions of C. chinense and C. pubescens and group 1 formed by the C. baccatum species. Group 3 contains the highest number of accessions (36); while Group 1 is made up of nine accessions. The groups with the highest similarity for the qualitative and quantitative variables are Group 1 and Group 2 (Table 4, Figure 1).

Discriminant Values
Regarding the qualitative descriptors, from the 39 characters analysed using the X 2 test, 30 of them were identified with high significance (p < 0.0001) (**), and nine were not significant (ns). These results indicated the presence of a large number of descriptors making an essential contribution to separate the nine genetic groups (Table 5), moreover they presented high association coefficients. Likewise, 15 characters with the highest discriminant value were recorded, which can be used to establish genetic differences between groups. The colour of the corolla and the colour of the anthers were the characters with the highest discriminant value (325.34 and 323.7 respectively) and presented the highest association coefficients. The position of the flower presented an X 2 value of 166.04 and the highest value according to the Cramer test (0.54); therefore, it has a high contribution to discriminate between genetic groups, as has the colour of the corolla that also provides a discriminating value. These results indicate that the G4 and G9 groups are associated with the white character of the corolla.
Regarding the quantitative descriptors, six were identified with the highest discriminant value: leaf length/width ratio, width mature leaf, fruit length, fruit width, fruit wall thickness, plant height; these descriptors allowed to differentiate the nine groups (Table 6). In addition, we determined that accessions within the groups maintain a close relationship, once there is not much variation since they present small values of standard deviation. The discriminant analysis found less distance between the species of C. annuum, C. frutescens and C. chinense. These three taxa are separated from C. baccatum and C. pubescens, because these two species grow in highlands (2800 m a.s.l.), where the climatic conditions differ from the low altitudes (200 m a.s.l.) where C. pubescens is distributed through the middle region of the Andes mountain range (1300 m a.s.l.). Capsicum baccatum is widely distributed throughout the lowlands of South America, as mentioned by Pickersgill [43]. The studies by García [48] corroborate the previous results; García [48] points out that the morphological characterization did not allow the species of C. annuum, C. chinense and C. frutescens to be differentiated. These observations are in agreement with Pardey et al. [18] who concluded that the species C. annuum, C. chinense and C. frutescens make up the same morphological group; like Vallejo et al. [49] who managed to discriminate the C. pubescens and C. baccatum species, but were unable to discriminate C. annuum, C. frutescens, C. chinense.
The Capsicum population presented morphological variation in the qualitative characteristics of the C. annuum, C. chinense and C. frutescens species because of the shared morphological features among the three species, making taxonomic classification difficult. This agrees with the results of García [48] and Palacios [50] who confirm this intraspecific variability; they also mention that as a result of the morphological description, it could be assumed that the three taxa constitute the same group. Similarly, the studies by Vallejo et al. [49], and Palacios Castro and García [51] managed to discriminate the species of C. pubescens and C. baccatum, but not between the species C. annuum, C. frutescens and C. chinense. The results of this study continue to corroborate the hypothesis that these three species are a large group in the process of differentiation, which is consistent with the studies conducted by Pickersgill [52].
The results of the present study are in agreement with those by Chávez-Servia [53] and Chávez-Servia and Castillo [54], who reported that variables such as length, width and shape of the Capsicum fruit showed considerable genetic variation. The purple colour of the anther was observed in the C. chinense species; while C. baccatum anthers presented a yellow colouration. On the other hand, C. annuum had pale blue anthers and C. frutescens blue. The species tended to have no stain on the corolla, except for C. baccatum, which is the typical characteristic of this species. The flowers in C. frutescens are erect; while in C. annuum and C. chinense the position of the flowers varied between intermediate and/or hanging.
According to Martín and González [55] and Fernández [56], chilli peppers with large-sized fruits and a thick epidermis tend to be less pungent. In contrast, in the accessions with smaller fruits where also the epidermis is thinner, the concentration of capsaicinoids increases, which is consistent with the results of the investigation, here accession 16,209 presented higher capsaicin content (5590 ug/g), while accession 16,450 registered low capsaicin content (200 ug/g).

Participatory Sensory Evaluation
The 50 people attending the workshop represented the following categories: producers (30), industry (5), chefs (4), scientists (5) and people who like to consume chilli (6). In the first phase, 134 accessions were selected as 'Very Good', presenting characteristics such as fruits with characteristics such as colour (pale orange, red, dark red and orange), shape (bell-shaped, triangular, elongated, bell-shaped and thick), size (medium and large) and fruit production. In the second phase, participants chose 64 accessions using the shape, colour and size of the fruit as selection criteria. In the third phase, 34 samples were selected with the criteria: taste, odour, texture, pulp thickness and size (Appendix B, Table A1); and, in the fourth phase, the most relevant fruits with orange, pale orange and red epidermis colours were selected. The shapes of the fruit are bell-shaped, and triangular, or bell-shaped and thick; the epidermis are of the fruit is semi-wrinkled and rough and fruits of medium to large size.
At the end of the process, the samples of the accessions were rated as follows: • As excellent-accession 15,661 (dark red fruit colour, bell-shaped and thick shape, large size) • As very good-accession 7818 (dark red fruit colour, triangular shape, medium size) • As good-accessions 16,304 (red fruit colour, flared shape, large size), 10,757 and 22,119 (red fruit colour, flared shape and compact, small size), 9892 (red fruit colour, flared shape, medium size), 9916 and 17,750 (orange fruit colour, bell-shaped, large size), 8994 and 16,209 (red fruit colour, triangular shape, medium size), 17,268 and 9902 (red fruit colour, bell-shaped, medium size).

Chemical Characterization
The nutritional value was determined in 15 accessions, selected as promising in morphological characterization, corresponding to the taxa C. annuum, C. chinense, C. frutescens, C. baccatum and Capsicum spp. Furthermore, these accessions were rated as excellent in the participatory selection. The accessions that had the highest concentration of capsaicin (5.59 mg/g), polyphenol (18.68 mg/g) and flavonoid (16.64 mg/g) were 16,209, 17,750 and 10,757, respectively ( Table 7). The content of total polyphenols, flavonoids and capsaicinoids varied in the accessions evaluated, with a tendency to present an association with the morphological classification described by Morán et al. [57]. Appedino [58] studied 13 cultivars of C. annuum, finding concentration levels of flavonoids (0.028 and 0.551 mg/g) lower than those found in the accessions in the present study. The results in Table 7 coincide with those found by Cázares et al. [59], who report that the populations of Ma'x ik and Sukurre belonging to the C. chinense species presented the highest capsaicin values (2.93 and 4.35 mg/g); while the lowest values reported for C. annuum sweet pepper populations (0.20 mg/g).
Additionally, Antonious and Jarret [60] studied different species of Capsicum, finding low concentration levels of capsaicin (0.0009 to 0.002 mg/g). Estrada et al. [61] reported increasing levels of capsaicinoids as maturation progresses, finding total capsaicinoid concentrations of between 0.15 to 0.70 mg/g (ps). However, these values are lower than those reported in the accessions studied here, where values between 5.59 to 0.20 mg/g were found (Table 8). The Biplot from the principal component analysis of the 15 selected accessions shows the first two principal components explaining variance greater than 92% (Figure 2). The contents of polyphenols, antioxidants and flavonoids are highly correlated with each other and these in turn are correlated with humidity. In the case of capsaicin, its content is independent of the amounts of polyphenols, antioxidants and flavonoids. Of the four selected accessions with the best nutritional content, two of them belong to the genus C. chinense (17,750 and 10,757) and the accession (15,661) belongs to the genus C. annuum, accession 9892 belongs to C. frutescens. Regarding the classification by conglomerates, these four accessions belong to groups G2, G3 and G9. The highest capsaicin content was recorded in accession 16,209 belonging to the species C. baccatum.

Discussion and Conclusions
The most discriminant qualitative characteristics were colour of the corolla, the colour of the anthers and position of the flower; while the most discriminant quantitative characteristics were leaf length/width ratio, width mature leaf, fruit length, fruit width, fruit wall thickness and plant height. This is similar to previous studies by Medina et al. [62], Pardey et al. [18] Ortiz et al. [20] and Castañón et al. [63] who have also identified colour of the corolla, corollar spot colour, fruit width and fruit length as discriminant variables within other Capsicum spp. collections. A practical morphological characteristic found in our C. annuum and C. frutescens accessions is the persistence of the pedicel with the fruit, According to Sreelathakumary et al. [41] this characteristic, along with the length of the placenta, is correlated with the mass of the fruits and, therefore, longer shelf life.
The agronomic characterization allowed classifying the genetic variability of the Capsicum germplasm collection into two large groups and nine subgroups. The two large groups are formed by the subgroups G7, G8, G5 and G3 represented by C. annuum, and the second group formed by subgroups G4, G9, G6, G2 having mostly C. frutescens accessions plus G1, represented mainly by C. baccatum. Subgroup 1 (G1) differentiates from the other subgroups because of the presence of spots on the corolla (Corollar spot colour) similarly observed by García [48], Palacios & García [51]. and Walsh & Hoot [64]. It is important to notice that within all subgroups (except G8) there are few intercalated accessions of C. baccatum, C. pubescens, C. chinense and C. spp. (Table 4). Capsicum phylogeny determined closer relation among C. annuum, C. frutescens and C. chinense which is known as C. annuum complex Pickersgill, [43], Vallejo et al. [49], Pardey et al. [18] and Palacios and García [51].
The description of CATIE's Capsicum spp. international collection permitted to identify promising materials e.g., after the morphological and participatory characterization, the following accessions were identified as promising: C. chinense-10,757 and 17,750; C. frutescens-9892; C. annuum-15,661 and 7816; and C. baccatum- 16,209. Also, four accessions were selected with the highest chemical concentration (polyphenols, flavonoids, and capsaicinoids), two of them belong to the species C. chinense (17,750 and 10,757), the accession (15,661) belongs to C. annuum, and accession 9892 is referred to C. frutescens. These selected materials or the collection as a whole could be used by interested scientist as well as farmers.