Morpho-agronomic diversity and botanical identification of melon accessions from northeastern Brazil

ABSTRACT Melon (Cucumis melo L.) crops are grown in the Semiarid region of Brazil by small, medium, and large farmers, focused on domestic and international markets. However, melon is also grown by family farmers using their own seeds, which are important germplasm for melon breeding programs. Samples of these seeds were collected and stored in the Active Germplasm Bank of Cucurbitaceae from the Northeast Region at the Brazilian Agricultural Research Corporation (Embrapa Semiarid), and require more thorough studies for a better understanding of the existing variability. Thus, the objective of this work was to characterize sub-accessions and their respective endogamic progenies to assess the genetic variability between and among these accessions. Two experiments were conducted in a randomized complete block design, with three replications, using 11 quantitative and 8 qualitative descriptors: the first using seeds from 17 accessions from natural pollination, and the second using seeds from S1 progenies. Morphological data were used for comparisons between generations. The 17 accessions evaluated originated 24 sub-accessions, denoting variability between and among accessions and sub-accessions. A dendrogram developed based on the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) showed the existing variability and, according to the newest melon classification, the groups identified were: makuwa, subgroup nashi-uri; and momordica and cantalupensis, subgroup prescott. The results showed a probable existence of introgression of alleles between different botanical groups, and some sub-accessions were not identified regarding their group by presenting variations in morphological characteristics, indicating the presence of new botanical groups.


INTRODUCTION
Melon (Cucumis melo L.) is a Cucurbitaceae species from the genus Cucumis (BURGER et al., 2010) that has a high economic importance in the world (GUIMARÃES; ARAGÃO, 2019).The Northeast region of Brazil is responsible for 96.84% of the Brazilian production (IBGE, 2020); the yellow melon type is the most grown for commercial purposes, using cultivars that had already been improved through breeding processes (SALVIANO et al., 2017) and present homogeneity and high productivity.
This species has been grown over several generations by family farmers in the Northeast region of Brazil, mainly within the traditional agriculture of the state of Maranhão, where it presents high genetic variability, considering the several natural and artificial selection processes that it underwent over time (QUEIRÓZ;BARBIERI;SILVA, 2015).Thus, melon samples from this region were collected and then stored in the Active Germplasm Bank of Cucurbitaceae (Embrapa Semiarid), in Petrolina, state of Pernambuco, Brazil, which contains approximately 150 accessions of this species and is the holder of very important genes for breeding programs (SILVA et al., 2010).However, despite the Northeast region is rich in melon genetic variability, thorough studies with accessions from traditional agriculture are still scarce and require, firstly, actions for taxonomic identification, characterization, evaluation, and conservation.Torres Filho et al. (2009) and Aragão et al. (2013) are among the few works carried out with samples of this germplasm; they conducted morphological and molecular characterizations of melon accessions and confirmed the existence of a wide genetic variability.However, these works did not estimate variations within accessions and used a classification that establishes only seven and six botanical groups, respectively, based on only one generation.
More recently, Amorim et al. (2016) and Macêdo et al. ( 2017) evaluated two sequential generations of a sample from accessions of the Active Germplasm Bank of Cucurbitaceae from the Northeast Region of Brazil and established the concept of sub-accessions to term the variation within accessions, and used the classification of Pitrat, Hanelt, and Hammer (2000), which considers that the species C. melo is composed of two subspecies (agrestis and melo) and botanical varieties.Amorim et al. (2016) evaluated 15 accessions which unfolded into 26 sub-accessions, and Macêdo et al. ( 2017) also evaluated 15 accessions and subdivided them into 25 subaccessions; both studies described the two subspecies and their respective botanical varieties: agrestis (momordica, conomon and makuwa) and melo (chandalak and cantalupensis).
In addition, Pitrat (2016) conducted a new botanical systematization for melon, considering the previous classification (PITRAT; HANELT; HAMMER, 2000) inconsistent, as the pilosity in the ovary presents variation within a same group, thus, proposing a new classification with 19 botanical groups, and subgroups when required, to represent the variations found within groups.
Therefore, following the new melon classification, the objective of this work was to define botanical groups and subgroups, identify possible sub-accessions, and quantify the morpho-agronomic variability between and among subaccessions from the Active Germplasm Bank of Cucurbitaceae from the Northeast Region of Brazil.

MATERIAL AND METHODS
Seventeen melon accessions from traditional agriculture in the states of Piaui (1 accession), Bahia (3 accessions), and Maranhão (13 accessions), collected from 1991 to 1998, were evaluated (Table 1 The first experiment started in April 2018, using seeds from accessions from natural pollination; and the second in January 2019, using seeds from the S 1 generation (endogamic progenies).Twenty seeds from each accession were sown in polyethylene trays filled with a commercial substrate (Plantmax ® ) in a greenhouse covered with a 50% screen shade, and irrigated twice a day.
The seedlings were transplanted to the experimental field at 20 days after sowing.The soil was previously prepared with plowing, harrowing, furrowing, and soil fertilizer application, carried out based on the soil analysis and recommendations for growing melon crops in the Northeast region of Brazil.Both the experiments were conducted in a randomized complete block design, with three replications, five plants per plot, spacings of 2.5 m between rows and 0.8 m between plants, under surface irrigation (infiltration furrows) in the first experiment, and localized irrigation (drip irrigation) in the second.Cultural and phytosanitary practices were carried out according to the recommendations for melon crops in the region.Parental and S 1, generations were obtained by subjecting, on average, nine plants of each accession to the self-fertilizing process.
The fruits were harvested between 30 and 45 days after the self-fertilizing process and characterized based on 11 quantitative and 8 qualitative descriptors (4 binary and 4 multicategory, using the list of descriptors of the IPGRI ( 2003).The quantitative descriptors evaluated were: fruit weight (kg); pulp thickness on the right, left, upper, and lower sides of the fruit (cm); fruit length and diameter (cm); fruit cavity length and diameter (cm); and total soluble solids in the lateral part of the fruit and in a composite pulp sample (°B rix).The qualitative descriptors evaluated were: fruit shape (globular, flat, elliptical, piriform, oval, elongated, ball, and poor-formation); epidermis color (yellow, intense yellow, orange, cream, light-green, dark-green, greenish-yellow); grooves (absent, light, medium, deep); cracks in the epidermis (absent, present); pulp color (white, cream, orange, greenish); peduncle dehiscence (absent, present); pilosity in the ovary and in young fruits (short or long trichomes); and sexual expression (monoecious or andromonoecious).
Focused on reaching a better representativeness, systematized photographs of fruits of each accession of the two generations (paternal and S 1 ) were developed to assess the segregation between generations and, thus, identify variations within accessions and identify sub-accessions, as described by Amorim et al. (2016) and Macêdo et al. (2017).
Regarding the statistical analyses, the quantitative data were firstly subjected to descriptive statistics.Normality and homogeneity tests were then applied, followed by a multivariate analysis based on the quantitative and qualitative descriptors.A dissimilarity matrix between accessions and sub-accessions for each type of data was developed, in which quantitative data were obtained through the Mahalanobis distance, and qualitative data were obtained using the index described by Cruz (2013), which represents the mean Euclidean distance: where: a = the number of type 1-1 agreements for the j-th variable; b = number of type 1-0 disagreements for the j-th variable; c = number of type 0-1 disagreements for the j-th variable; and d = number of type 0-0 agreement for the j-th variable.
These matrices were then summed, forming a sum matrix.Joint of the distance and dissimilarity matrices was carried out, firstly, by the standardization of the d and D 2 values, obtaining (D 2 )' and d' values, followed by simple sum.The sum dissimilarity matrix was used for the clustering analysis, using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), and the cut point was based on the method of Mojena (1977).The coefficient of cophenetic correlation (CCC) (SOKAL; ROLF, 1962) was estimated to validate the groups generated by the UPGMA based on the Pearson's coefficient of correlation between the distance matrix and the cophenetic matrix (CRUZ; CARNEIRO, 2003).All procedures were carried out using the Genes (CRUZ, 2013) and R (R DEVELOPMENT CORE TEAM, 2019) programs.
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The data of morpho-agronomic descriptors found for the two generations enabled to identify the variability within the accessions evaluated, and the multivariate analysis enabled to separate groups with morphological similarities.The dendrogram (Figure 1) showed a coefficient of cophenetic correlation (CCC) of 0.86 for the parental generation and 0.88 for the generation S 1 (Figure 2), denoting a good representativeness (CRUZ; CARNEIRO, 2003).The parental generation (Figure 1) showed the formation of three main groups: the first dendrogram included the accessions BGMEL 22,16,156,104,117,113,and  The morpho-agronomic data of the two generations and the comparison of them showed the formation of three data sets, in which group I was formed by nine accessions (Figure 3, Table 2), with fruits presenting the same phenotype, in both generations, for flower and fruit characters, in addition to present characters that enable to identify botanical groups and subgroups, namely: mormordica (BGMEL20.0)(Figure 3A), makuwa subgroup nashi-uri (BGMEL22.0,104.0, 113.0, 114.0, 116.0, 117.0, and 156.0) (Figures 3B the 3H), and the group cantalupensis subgroup prescott (BGMEL30.0)(Figure 3I).These accessions presented no variations within accessions from one generation to another and, thus, were identified with zero in their identification code from the Active Germplasm Bank of Cucurbitaceae from the Northeast Region of Brazil, as described by Amorim et al. (2016).The correlation of the results found for group I with the multivariate analysis showed that the groups in the dendrograms reinforced this separation and the respective botanical identifications, as the parental (Figure 1) and S 1 (Figure 2) generations showed approximations of subaccessions of the botanical group makuwa subgroup nashi-uri, and indicated that momordica and cantalupensis subgroup prescott remained close to other sub-accessions (BGMEL71.2,74.2,28.1,T6COD98.1,27.2,T5COD5.1,71.1,42.1,T5COD5.2,27.1,74.1,T6COD98.2,23.0,and 28.2) with similar morpho-agronomic characteristics.
According the new systematization for melon (PITRAT, 2016), the subgroup prescott of the group cantalupensis is characterized by presenting deep grooves on the fruit surface; however, the sub-accession BGMEL30.0 presented a variation, with grooves varying from medium to deep (Table 2).This character is controlled by a recessive gene (DOGIMONT, 2011), and the gradual variation observed in the present work may have been originated from introgression of alleles that are responsible for expression of different types of fruit surfaces in melon genotypes that are grown by family farmers in the Northeast region of Brazil (MACÊDO et al., 2017).
Group II was composed of six sub-accessions (Figure 4, Table 2), with the parental generation presenting variation within the accessions evaluated.Thus, these accessions with variations were separated and grown in the field to be analyzed (generation S 1 ) and compared to data of the parental generation.The analysis showed that the two generations expressed the same characteristics previously presented, with no segregation for flower and fruit descriptors (PITRAT, 2016).Thus, this uniformity allowed for the identification of botanical groups and subgroup: momordica (BGMEL27.2,28.1, 42.1, 74.1, and T6COD98.1)(Figures 4A to 4D), and cantalupensis subgroup prescott (BGMEL42.2) (Figure 4F).The multivariate analysis (Figures 1 and 2) grouped closely the sub-accessions identified as momordica.The subaccession BGMEL42.2 (cantalupensis) in generation S 1 was isolated in the dendrogram (Figure 2) in relation to BGMEL30.0, which was also identified for the same botanical group.It was probably because, in addition to qualitative, quantitative characters were analyzed, such as fruit weight (BGMEL42.2:2.290 kg; BGMEL30.0:0.700 kg), fruit length and diameter (BGMEL42.2:21.95 cm and 16.25 cm, respectively; BGMEL30.0:13.50 cm and 10.90 cm, respectively), fruit cavity length and diameter (BGMEL42.2:16.45 cm and 9.85 cm, respectively; BGMEL30.0:9.90 cm and 5.40 cm, respectively) and pulp thickness (EPD -BGMEL42.2:2.80 cm and BGMEL30.0:2.60 cm; EPE -BGMEL42.2:3.30 cm and BGMEL30.0:2.0 cm; EPS: BGMEL42.2:3.45 cm and BGMEL30.0:1.90 cm; EPI -BGMEL42.2:2.10 cm and BGMEL30.0:1.70 cm), which showed higher values and may have caused this separation, The variations found for color of pulp (white, cream, and orange) and epidermis (yellow, yellow-intense) in the momordica group (Table 2) are consistent with the group description presented by Pitrat (2016), i.e., pulp colors from light-green, sometimes white, that may reach orange.This variation is also expressed in the epidermis color, which may present uniform color or spots and stripes (PITRAT, 2016).Melon pulp color is controlled by two genes that involve the colors green, orange, and white; this species may present variations in epidermis color according to the different combinations of three main pigments: chlorophyll; carotenoids; and naringenin chalcone, which is a flavonoid pigment responsible for yellow colors in mature fruits (DOGIMONT, 2011).Therefore, crossed forms of alleles may result in different colors for botanical groups.
The sub-accessions BGMEL42.1 and 42.2 (Table 2, Figures 4C and 4F) were previously from the same accession; however, after analyzed for variations in the two generations, they were separated as different botanical groups (momordica and cantalupensis subgroup prescott, respectively).This variation factor was probably caused by seed exchanges between family farmers, which is common practice; in addition, simultaneous handling of seeds from different melon groups occurs within the sites where these accessions were collected, which probably led to mixtures of germplasms from different botanical groups (AMORIM et al., 2016).
The botanical identification carried out for the two first groups showed that the characteristics found in the present study are consistent with results found in other works (MANOHAR;MURTHY, 2012;MALIK et al., 2014), defining marked characteristics for the group momordica.The fruits presented shapes varying from elliptical to elongated, weights reaching 2.380 kg, and cracks in the epidermis when mature, which is not a satisfactory morphological trait, as decreases in post-harvest durability hinder the fruit marketing; the plants were monoecious, with a smooth fruit peel, floury pulp with white, creme, or orange colors, and low sugar contents (Table 2).The group momordica is highly marketed in street markets in the Northeast region of Brazil, where it is popularly known as melão-de-cheiro, pepino, or caxixi (MACÊDO et al., 2017).
The characteristics presented by the botanical group makuwa are also consistent with results of other works (BURGER et al., 2010;XIAO-HANG et al., 2014) and denote that their fruits usually present low weight (fruits had weights of up to 0.100 g in the present study).However, variations in this characteristic may be found within the group, reaching higher weights (fruits with up to 0.900 g were found in the present work).In addition, the fruits presented elliptical to oval shapes, epidermis with cream color, and fruit pulp with crisp texture, cream color, and high sugar contents, reaching 10.2 °Brix, which is considered sweet, and small seeds (Table 2).
Studies on fruits of the group cantalupensis found in the literature have classified them as medium-sized fruits, which is true for most of them.However, variations in this character may be found, with fruits classified as large (IPGRI, 2003), as the fruit weight in the present work reached 2.870 kg.The cantalupensis fruits presented peduncle dehiscence, elliptical shape, and light-orange pulp with a juicy smooth texture; fruits with green pulp were not found.In addition, the fruits were aromatic when mature, confirming the findings of other works (PITRAT, 2016;VALADARES et al., 2018).
The groups makuwa and cantalupensis present subgroups, including nashi-uri and prescott, respectively, as identified in the present work; nashi-uri is characterized by presenting uniform cream to white epidermis color and no presence of grooves, whereas the subgroup prescott is characterized by presenting deep grooves, pulp exclusively orange, and being monoecious (PITRAT, 2016), as found in the present work.
Group III was composed of nine sub-accessions (BGMEL23.0,27.1, 28.2, 71.1, 71.2, 74.2, T5COD5.1,T5COD5.2, and T6COD98.2) (Figure 5), for which botanical groups and subgroups were not possible to identify, as they presented introgression of characters or characteristics different from those present in groups and subgroups already defined, making it difficult to separate them based on Pitrat (2016) (Table 2).The sub-accession BGMEL23.0 (Figure 5A) presented uniform flower and fruit characteristics in the two generations studied; however, classifying it into a group based on the classification of Pitrat (2016) was not possible, which denotes that it is probable from a new subgroup, or even from a new botanical group.This sub-accession presented, in the two generations, fruits with a piriform shape; yellow to orange epidermis color; no stripes; no grooves; no cracks in the epidermis; no reticulated peel; no roughness; cream to orange pulp color; no peduncle dehiscence; and monoecious flowers (Table 2).The sub-accessions BGMEL27.1,T5COD5.2, and T6COD98.2 (Figures 5B, 5H and 5I) presented similar characteristics to those of the momordica group, but with no cracks in the fruit epidermis, which is a marked character of fruits of this group when mature.In addition, T5COD5.2 and T6COD98.2 (Figures 5H and 5I) presented andromonoecious flowers (Table 2), diverging from the classification of Pitrat (2016), which characterize these flowers as monoecious.Dhillon et al. (2015) confirmed the existence of cracks in the epidermis or only a peeling in momordica fruits, which makes it a characteristic for identification of the group.Thus, the different characteristics that make them segregating were considered as introgression of characters between botanical groups in the present work, thus hindering the definition of the groups.
The sub-accession BGMEL28.2 (Figure 5C) did not express the character cracks in the epidermis, presenting light grooves in the epidermis (Table 2), which is not consistent with momordica, which usually presents a smooth surface with no grooves (PITRAT, 2016).This also denotes introgression of characters between momordica and, probably, cantalupensis, which has grooves.The sub-accession BGMEL71.2 (Figure 5E) presented superficial grooves and green epidermis and pulp, which are characteristics of the group cantalupensis; however, it was not possible to identify it, as it presented non-dehiscent peduncle in the two generations studied, which also is a characteristic of the group cantalupensis.
Regarding these sub-accessions that were not identified, the dendrogram of generation S 1 (Figure 2) showed the formation of a main cluster with all genotypes identified as momordica and cantalupensis subgroup prescott, in addition to grouping all sub-accessions that were not identified but were morphologically similar to cantalupensis or momordica, denoting an introgression of characters that may have easily occurred, as there are no barriers for the occurrence of cross-pollination between different botanical groups (VALADARES et al., 2017) and the management of farmers facilitates this process between neighboring crops with different botanical groups.
The findings of the present work are consistent with those of Burger et al. (2010), who reported that C. melo exhibits high diversity for fruit characteristics, varying in size, shape, external color, aroma, sugar contents, pulp color, and other characters, which often hinders the intraspecific classification that has been carried out since the first half of the nineteenth century (PITRAT, HANELT;HAMMER, 2000).However, despite all existing variation found in the present work, 62.50% of the sub-accessions were identified regarding their group and respective subgroup (Table 2, Figure 6).Similarly, Trimech et al. (2013) had not succeeded in identifying some groups when studying variability in melon, which may be attributed to the high heterogeneity and introgression of characters that did not allow for a clear identification of groups.The morpho-agronomic data found showed the existence of a high genetic variability and denoted the importance of knowing the existing variation in melon species and how it is distributed through a throughout study of variations between and among accessions and sub-accessions.It was also shown in other studies evaluating melon germplasm from family farmers in the Northeast region of Brazil (TORRES FILHO et al., 2009;DANTAS;HOLANDA;2015;AMORIM et al., 2016;MACÊDO et al., 2017;ANDRADE et al., 2019), which confirms the existence of high variability, denoting the wealth present in farming production units, which has been rescued and stored in the Active Germplasm Bank of Cucurbitaceae from the Northeast Region of Brazil.Although these throughout analysis had been carried out with botanical classifications different from those used in the present study, they are very important by presenting detailed information that can be used in future breeding programs for these melon types studied, as the + )/( +  +  + ) Table2.Morphological characterization of melon (Cucumis melo) accessions from the Active Germplasm Bank of Cucurbitaceae from the Northeast Region at the Brazilian Agricultural Research Corporation (Embrapa Semiarid), in parental (P) and S 1 generations (G), with identification and classification of sub-accessions and their respective botanical group (BG) and subgroup (SG), in study carried out in 2018 and 2019.

Figure 1 .Figure 2 .
Figure 1.Dendrogram of parental generation of melon (Cucumis melo) accessions, generated by joint analysis of quantitative and qualitative data, using UPGMA. 1
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Figure 6 .
Figure 6.Percentage of identified and non-identified botanical groups and subgroups of melon (Cucumis melo) accessions from the Active Germplasm Bank of Cucurbitaceae from the Northeast Region at the Brazilian Agricultural Research Corporation (Embrapa Semiarid), in the study carried out in 2018 and 2019.

Table 1 .
Passport data of melon (Cucumis melo) accessions from the Active Germplasm Bank of Cucurbitaceae from the Northeast Region at the Brazilian Agricultural Research Corporation (Embrapa Semiarid), used in the study in 2018 and 2019.
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