Introduction

Avocado (Persea americana Mill.) is a fruit species of Mesoamerican origin belonging to the family Lauraceae. This species exhibits high adaptive plasticity to different environments as a result of evolutionary and ecological processes (^{Barrientos-Priego, Muñoz-Pérez, Reyes-Alemán, Borys, & Martínez-Damián, 2007}; ^{Galindo-Tovar, Ogata-Aguilar, & Arzate-Fernández, 2008}). To date, three subspecies or horticultural races have been recognized: the Mexican race (P. americana var. drymifolia), Guatemalan race (P. americana var. guatemalensis) and West Indian race (P. americana var. americana). Genetic diversity among these races has allowed, through hybridization and anthropic selection processes, the development of modern commercial varieties, which have adapted to specific areas with high yields (^{Knight & Campbell, 1999}).

Avocados are naturally distributed from central Mexico to Peru, and are currently grown in more than 50 countries with tropical and subtropical climates around the world. Mexico is the leading producer (2,029,886 t·year^-1), while the Dominican Republic is the country with the highest yields per unit area (43.7 t·ha^-1) (^{FAOSTAT, 2017}). In Colombia, production is estimated at 544,933 t·year^-1 with an average yield of 9.77 t·ha^-1, giving it the fifth highest production volume worldwide, with the departments of Antioquia, Bolívar, Caldas, Huila, Quindío, Risaralda, Santander, Tolima and Valle del Cauca contributing more than 80 % of the national production (^{Agronet, 2019}).

Traditionally, the propagation of avocado plants for crops in Colombia is done by grafting commercial materials ('Hass', 'Lorena', 'Choquette', 'Fuerte', 'Reed', and 'Trinidad', among others) onto rootstocks produced from sexual seed (ungrafted) from Creole and 'Hass' trees (^{Bernal-Estrada & Díaz-Díez, 2020}). Seeds are obtained from local markets, which generally do not know the origin of the material, or from producing areas located in contrasting agroecological regions without any clear selection criteria.

Easy-to-observe morphological characteristics with high discriminant action have been used to determine morphological variety in Creole avocado populations in different regions of the world (^{Acosta-Díaz, Hernández-Torres, & Almeyda-León, 2012}; ^{Acosta-Díaz, Hernández-Torres, & Almeyda-Leó, 2013}; ^{Gutiérrez-Díez et al., 2009}; ^{López-Guzmán et al., 2012}; ^{López-Guzmán et al., 2015}; ^{Montes-Hernández, de la Torre-Vizcaíno, Heredia-García, Hernández-Martínez, & Camarena-Hernández, 2017}; ^{Sánchez-Pérez, 1999}). In Colombia, very little information is available on the diversity of Creole avocados and their effect on productive and phytosanitary behavior when used as rootstocks in commercial crops.

In studies by ^{Cañas-Gutierrez, Galindo-López, Arango-Isaza, and Saldamando-Benjumea (2015)} and ^{Cañas-Gutiérrez, Arango-Isaza, and Saldamando-Benjumea (2019)}, the high morphological and molecular variability in seed-donor Creole avocados from different regions of Colombia is evident; in addition, they relate their genetic compatibility with commercial cultivars such as 'Hass', 'Fuerte' and 'Reed', when used as rootstocks. ^{Burbano-Figueroa (2019)} mentions some West Indian ecotypes for the Montes de María area (Bolívar and Sucre), identified by local producers according to external and internal characteristics of the fruit, and are known as 'Cebo', 'Leche' and 'Manteco'.

There has been little research on the identification of Creole rootstocks with tolerance or resistance to phytosanitary problems. Among these, the selections made by ^{Rodriguez-Henao, Caicedo-Arana, Enriquez-Valencia, and Muñoz-Florez (2017)} in the collection of the National Avocado Germplasm Bank located in the Palmira Research Center of AGROSAVIA (Valle del Cauca) stand out, mainly focused on the identification of materials with resistance to root rot caused by Phytophthora cinnamomi Rands.

Determining the morphological variability existing in the different avocado seed producing regions for rootstock production in Colombia would allow identifying selection attributes for the development of propagation and breeding programs, as well as identifying high diversity refuges that contribute to the implementation of conservation plans. In this sense, the objective of this work was to characterize the phenotypic variability of "Creole" avocado trees as seed donors for rootstock production in three areas of Colombia. This was done with the purpose of expanding knowledge to other regions of the country, which lack information, and providing a basis for the selection of seed-producing trees that meet the needs of nursery operators.

Materials and methods

The work was carried out during the period from March 2015 to November 2016 in three Creole avocado producing areas to obtain rootstocks in Colombia: Norcasia in the department of Caldas (NOR) (05° 36’ North latitude and 74° 52’ West longitude, at 550 m a. s. l.), Alvarado in the department of Tolima (ALV) (04° 37’ North latitude and 075° 01’ West longitude, at 1,250 m a. s. l.) and San José del Palmar in the department of Chocó (SJP) (04° 55’ North latitude and 76° 12’ West longitude, at 920 m a. s. l.).

In each study area, producing trees were selected considering the requirements established by the Colombian Agricultural Institute (^{ICA, 2009} and ²⁰¹⁵⁾, in standards 3180 of 2009 and 3168 of 2015 (modified by resolution: 0780006 of 2020). These standards describe the guidelines for the selection of seed-donor trees for rootstock production.

Each tree was georeferenced, marked and characterized by evaluating 39 morphological characteristics of plant, leaves, fruits and seeds according to the standardized descriptors published for avocado by the International Plant Genetic Resources Institute (^{IPGRI, 1995}) (Table 1). Trees were selected during the main harvest periods to obtain information on fruits and seeds.

The sample size used per tree was 15 fruits (with a healthy appearance at physiological maturity), 15 seeds and 5 leaves (fully developed and randomly collected). The morphological characterization of the trees was carried out in situ, while the characterization of leaves, fruits and seeds was done in the AGROSAVIA phytopathology laboratory. The morphometric variables of length and diameter were measured with a Mitutoyo digital caliper (±0.01 mm), and the weight variables with a Mettler Toledo balance (±0.01 g).

Data matrices were constructed with the data obtained and analyzed with descriptive and multivariate statistics. A mixed data matrix was created that included quantitative and qualitative morphological characteristics, on which a multiple factor analysis (MFA) was applied. This statistical method analyzes data tables, where individuals are described by groups of quantitative and qualitative variables, allowing their simultaneous analysis (^{Escofier & Pages, 1994}; ^{Abdi, Williams, & Valentin, 2013}).

The MFA was performed in two stages: 1) principal component analysis (PCA) for quantitative variables and multiple correspondence analysis (MCA) for qualitative variables (ordinal or nominal variables separately), retaining in each procedure the highest eigenvalue, and 2) global PCA with the weights of the original variables obtained in the previous step (^{Escofier & Pages, 1994}). As a result of this analysis, a linear combination of the first component of the PCA and the first dimension of the MCA was obtained, with which the scores for each individual were obtained.

With the scores obtained in the global PCA of the MFA, a cluster analysis was carried out using the squared Euclidean distance and Ward's minimum variance clustering method. The number of groups in the cluster was determined with Hotelling's pseudo t² statistic. To determine the correct allocation of seed-donor trees, discriminant analyses were performed for each group. All analyses were conducted with the SAS ver. 9.4 statistical package (^{SAS Institute Inc., 2013}).

Results and discussion

In the three areas evaluated, the avocado production system was characterized by using low production technology, being associated with other plant species of agricultural interest and having relicts of natural forests. Due to these characteristics, they can be considered as harvesting agroforestry production systems, which is consistent with the descriptions made by ^{Burbano-Figueroa (2019)} for the avocado-producing region of Montes de María located in the departments of Bolívar and Sucre (Colombia).

A total of 80 seed-donor trees that met the technical requirements established by the ^{ICA (2009} and ²⁰¹⁵⁾ were characterized. Of these, 31 trees were selected in the producing area of Norcasia (Caldas), 28 trees in San José del Palmar (Chocó) and 21 trees in Alvarado (Tolima). These trees were characterized by being vigorous with a good phytosanitary appearance (pests and diseases), highly productive with uniform fruit and having had at least three consecutive production periods.

Two periods of fruit production or harvest were identified in the three areas. The first was between the months of March to June, as the main harvest, and the second period was between the months of October to December, with smaller productions known locally as "traviesa” or “mitaca".

The trees in the production area of Norcasia (Caldas) were characterized by variable size, reaching crown heights of up to 15 m and a maximum trunk circumference of 230 cm (Table 2); in addition, most of them were obovate in shape and had ascendant and irregular growth habits. The fruits had an average weight of 459.1 ± 99.9 g, and the fruit length/width ratio was 1.81, with a predominance of narrowly obovate, pyriform and clavate fruits, with a rounded or flattened apex, and asymmetrical pedicel. The skin surface was intermediate, green to dark green, and the fruit flesh was creamy green.

Seeds in Norcasia had an average weight of 65.4 g, representing 14.2 % of total fruit weight. In this area, seeds were recorded as broadly ovate, cordiform, ellipsoid, with a flattened base and a conical and ovate apex. The cotyledon surface was intermediate rough, and creamy yellow or whitish. The position of the seed in the fruit was at the center or to one side of the fruit, and usually the testa or coat was strongly attached to the seed. The free space of the seed cavity was apical, and to a lesser extent absent.

In San José del Palmar (Chocó), trees with heights greater than 20 m and a trunk circumference of 128.9 cm were recorded. Most of the trees were vigorous, columnar in shape, with axial or irregular branch distribution. The fruits were characterized by being large, with an average weight of 542.8 g and a maximum weight of 976.9 g. The fruit length/width ratio was 1.77, and fruits were pyriform, ellipsoid, narrowly obovate, obovate, clavate or rhomboidal. Fruit apex and pedicel position were frequently asymmetrical. The surface of the fruit skin was intermediate or smooth, which varied from green to light green. The texture of the flesh was generally watery, and ivory or light green in color.

Seeds from the San José del Palmar producing area were large, with an average weight of 107.3 ± 35.6 g, representing 19.7 % of total fruit weight. The most frequent seed shapes were ovate, broadly ovate and a flattened base with conical apex. The cotyledon surface was mostly intermediate rough. The location of the seed in the fruit was central, and the free space of the seed cavity was apical.

In the Alvarado (Tolima) producing area, the trees were of variable size, with maximum crown heights of 15 m and an average trunk circumference of 81.5 cm. Most of them had a columnar or pyramidal crown, with an axial branch distribution habit. Fruits were characterized by being of medium size (x- = 427.2 ± 87.4 g), and the fruit length/width ratio was 1.92. The most frequent fruit shape was pyriform, followed by narrowly obovate, clavate, ellipsoid, and obovate. Fruits had a rounded or asymmetrical apex, and the pedicel position was asymmetrical. The skin surface was intermediate, and light green to yellow. The texture of the flesh was buttery or pastose, with a good taste, and a creamy green to deep yellow color.

The average weight of seeds in Alvarado was 87.1 ± 21.3 g, representing 20.3 % of total fruit weight. In this area, seeds were predominantly broadly ovate, with a flattened base and a conical and ovate apex. They have cotyledons with an intermediate rough surface and detachable testa, centrally positioned and to one side of the fruit; it can be ivory, cream yellow or cream pink. The free space of the seed cavity was apical.

The quantitative morphological characters evaluated showed a wide range of variation in the three producing areas. In 58.3 % of the descriptors considered, coefficients of variation (CV) greater than 20 % were recorded (Table 2). According to ^{Hidalgo (2003)}, attributes with a higher percentage may indicate high variability. The characteristics with the greatest variation in the three areas were FW, FPP, SW, FSW and FST.

According to the simple correlation analysis presented in Table 3, 16.6 % of the associations between quantitative variables were significant (P ≤ 0.05). Therefore, coefficients with significant values >0.40 were considered to represent associations with natural patterns of variation (^{Rojas, 2003}). Among the variables with the highest correlation coefficients were: FW with FPP (r = 0.93), followed by SW with SD (r = 0.91), LA with LL (r = 0.88) and SW with SL (r = 0.70).

The morphological characteristics observed in the three producing areas are consistent with the descriptions made for fruits and seeds of avocados of the West Indian race (^{Bernal-Estrada & Díaz-Díez, 2020}; ^{Cañas-Gutiérrez et al., 2015}; ^{Sánchez-Pérez, 1999}), and conform to the size and shape characteristics indicated by ^{López-Guzmán et al. (2012)}, which are appreciated in the consumer market. FW ranged from 256.5 to 976.9 g, and the most common shapes were pyriform, obovate and clavate. Seed weights ranged from 29.7 to 220.6 g, and the predominant shapes were obovate and those with a flattened base and a conical apex.

SW was variable in the three areas, being higher in San José del Palmar (Chocó) (CV = 33.2 %), followed by Alvarado (Tolima) (CV = 24.4 %) and Norcasia (Caldas) (CV = 24.2 %). SD and the SL were characters of moderate variability, although positively correlated with SW. Seeds from the three study areas can be categorized as large, if compared to that cited by ^{Acosta-Díaz et al. (2012)} (x- = 35.3 g), ^{Acosta-Díaz et al. (2013)} (x- = 36.0 g) and ^{Gutiérrez-Díez et al. (2009)} (max. weight = 69.76 g) for Creole avocados from the state of Nuevo León (Mexico), and by ^{Montes-Hernández et al. (2017)} in Mexican avocado (P. americana var. drymifolia) germplasm from the Bajío Experimental Field in the state of Guanajuato, Mexico (x- = 38.1 g).

The PCA generated in the MFA allowed determining the relationship between the quantitative variables evaluated and the avocado samples from the three producing areas. According to Table 4, and according to the Kaiser-Gutman criterion (^{Palacio, Apodaca, & Crisci, 2020}), the first four components with eigenvalues greater than 1 were able to explain 92.2 % of the total variability. Table 5 shows the greatest contribution of variability explained by each variable that makes up the four principal components, where fruit and seed characteristics have the greatest weight in describing the level of variation in the three populations.

According to the MCA developed in the MFA for the qualitative variables, the first two dimensions were identified as those with the greatest contribution to the observed variability (41.04 %). Dimension 1 contributed 24.5 % of the variability, and dimension 2, 16.4 %. The characteristics with the highest polymorphism with respect to the highest inertia values were LS (0.2094), FS (0.1572), SS (0.0810) and SCC (0.0829) (Table 6). The characteristics TS, TBD, TTS, SP, FSSC, FSP, GLP, LAS and LAS had low polymorphism, so they were not included in the MCA.

According to the descriptive and multivariate analyses, the characteristics that presented the greatest morphological variability were those related to fruits and seeds, which confirms that these traits constitute the most important morphological criteria to explain the phenotypic variability in the three avocado populations. ^{López-Guzmán et al. (2012)} reported similar results for Creole avocados from the state of Nayarit, and ^{Gutiérrez-Díez et al. (2009)} and ^{Acosta-Díaz et al. (2012)} for Creole avocados of the Mexican race in the state of Nuevo León (Mexico).

Avocado is a highly heterozygous cross-fertilizing species that produces fruits with monoembryonic seeds, so its progeny can be highly variable (^{Bernal-Estrada & Díaz-Díez, 2020}; ^{Alberti, Brogio, da Silva, Cantuarias-Avilés, & Fassio, 2018}). In this sense, it is to be expected that, in the three agroecological regions evaluated, where repopulation processes occur spontaneously with little selection interference by producers, there is high morphological variability, especially in fruit and seed characteristics, which are those with the greatest natural variability (^{IPGRI, 1995}).

Based on the global PCA scores, the cluster analysis obtained in the MFA (^{Escofier & Pages, 1994}) allowed us to identify, at a cut-off height of 0.08 units (semi-partial R²), three groups fully formed from quantitative attributes and similar qualitative ones, which indicates that it was not possible to discriminate groups of trees by areas of origin (Figure 1). According to the discriminant analysis, no reclassification of trees between groups was observed, so it is assumed that the groupings formed are correct.

Figure 1 Dendrogram of quantitative and qualitative morphological data generated by global principal component analysis of 80 seed-donor Creole avocado trees in Colombia. NOR = Norcasia; ALV = Alvarado; SJP = San José del Palmar. 

Group I was made up of 20 trees representing 25 % of the population, 14 of which belong to the producing center of San José del Palmar (SJP), four to Norcasia (NOR) and two to Alvarado (ALV). This group was characterized by having trees with higher FW, FPP, FD, SW, SL, SD, LL and LA. In addition, they are trees with elongated fruits (clavate), flesh with a pastose texture (doughy), skin with intermediate adherence to the flesh, ivory-colored seeds and narrowly obovate leaves (Table 7).

Group II consisted of 44 trees (55 % population) with different centers of origin (NOR = 19 trees, ALV = 15 trees and SJP = 10 trees). This group included trees with intermediate quantitative characteristics (leaves, fruits and seeds), where the most determinant qualitative characters were trees with pyriform fruits, seeds with a flattened base and conical apex, and trees with oval leaves. In contrast, group III was made up of the smallest number of trees (16, 20 % population) (NOR = nine trees, ALV = five trees and SJP = two trees) and with the lowest quantitative characteristics, with the FST variable being the character with the highest weight. This group consisted of trees of intermediate vigor, with obovate fruits and oblong-lanceolate leaves.

^{Alberti et al. (2018)} and ^{Ramírez-Gill (2016)} recommend that the selection of seed-donor trees be carried out based on vigor, productivity, adaptation to local climatic conditions and compatibility with the grafted variety. However, in Colombia, seed size and weight are important attributes for avocado rootstock production. This is due to the fact that nursery operators prefer large seeds for plant multiplication, as they consider that these induce better vigor and generate grafting diameters in less time, as confirmed by studies conducted by ^{Adjei, Banful, and Idun (2011)} and ^{Ndoro, Anjichi, Letting, and Were (2018)} in other regions of the world.

Seeds obtained from trees in the three evaluated areas, belonging to groups I and II (formed in the cladogram and corresponding to 80 % of the analyzed population), produced large and medium-sized seeds, which meet the physical characteristics required by nursery operators for rootstock production in nurseries in Colombia. However, when selecting seed-donor trees, additional aspects of the seed such as physiological, sanitary and genetic factors should be considered in order to determine their quality, affinity and productivity with commercial markets of interest for Colombia (^{Reyes-Herrera et al., 2020}).

Conclusions

The high phenotypic divergence in fruits and seeds found in the three avocado populations implies a broad genetic base, in which each agroecological niche can harbor a high variability of shapes and sizes. This explains why it was not possible to identify characteristic variation patterns for each production center.

Eighty percent of the avocado trees identified as seed donors in the three producing areas presented outstanding physical characteristics for the production process of planting material (rootstocks), since in them it is possible to guarantee seeds of good weight and size for nursery activities.