Genetic diversity of naturalized cacao (Theobroma cacao L.) in Puerto ...

Tree Genetics & Genomes (2016) 12:88 DOI 10.1007/s11295-016-1045-4

ORIGINAL ARTICLE

Genetic diversity of naturalized cacao (Theobroma cacao L.) in Puerto Rico S . Cosme 1,2 & H. E. Cuevas 1 & D. Zhang 3 & T. K. Oleksyk 2 & B. M. Irish 1

Received: 28 December 2015 / Revised: 24 July 2016 / Accepted: 28 July 2016 # Springer-Verlag Berlin Heidelberg (outside the USA) 2016

Abstract Identification of genetically diverse cacao with disease resistance, high productivity, and desirable organoleptic traits is vitally important to the agricultural crop’s long-term sustainability. Environmental changes, pests, and diseases as well as nation’s sovereign property rights have led to a decrease in accessibility and exchange of germplasm of interest. Having been introduced during colonial times, naturalized cacao in Puerto Rico could serve as an unexplored source of genetic diversity in improvement programs. An island-wide survey was carried out to identify naturalized trees and to determine their genetic associations to reference cacao accessions. Samples were genotyped with Expressed Sequence Tag-derived single nucleotide polymorphism (SNP) markers. Principal coordinate, cluster, and population structure analysis using the genotype data for both local and reference samples assigned individuals into five distinct genetic backgrounds: Criollo, Trinitario, Amelonado, Upper Amazon Forastero (UAF), and Nacional. Puerto Rican cacao fit into four (Criollo, Trinitario, Amelonado and UAF) of the five genetic

Communicated by D. Grattapaglia Electronic supplementary material The online version of this article (doi:10.1007/s11295-016-1045-4) contains supplementary material, which is available to authorized users. * B. M. Irish [email protected] 1

USDA-ARS Tropical Agriculture Research Station, 2200 Pedro Albizu Campos Ave., Mayaguez, Puerto Rico

2

Department of Biology, University of Puerto Rico at Mayaguez, Carr. 108, Barrio Miradero Km 1.3, Mayaguez, Puerto Rico

3

USDA-ARS Sustainable Perennial Crops Laboratory, 1300 Baltimore Ave., Bldg. 001, Rm. 223, BARC-West, Beltsville, MD, USA

backgrounds, being mainly composed of individuals of Criollo ancestry. Based on historical evidence, cacao of Criollo background was probably brought to Puerto Rico from Venezuela and/or Central America during colonial times. Trinitario, Amelonado, and UAF genetic backgrounds are most likely products of more modern introductions. Genotyping cacao in Puerto Rico provides information on the history and possible origin of the naturalized trees on the island. In addition, the assessment has allowed the targeting of material for incorporation and long-term conservation filling gaps in the existing collection and providing new germplasm to be evaluated for agronomic performance. Keywords Chocolate . Germplasm . Cocoa . Tropics . STRUCTURE . SNP

Introduction Cacao (Theobroma cacao L.) is an important perennial agricultural crop in humid tropical regions of the world. The dried and fermented seed or Bbeans^ are the raw product used mainly by the international chocolate manufacturing industry. In the past, much debate surrounded cacao’s center of origin and domestication. This was due to its unclear distribution and dispersion (human and nature) and because of its long history of cultivation (Cuatrecasas 1964; De la Cruz et al. 1995; Motamayor et al. 2002; Loor et al. 2009). Presently, it is widely accepted that cacao has a South American origin where the headwaters of the Amazon River are described as the primary center of diversity for the species (Wood and Lass 1985; Bartley 2005). However, archaeological evidence suggests cacao domestication gradually occurred in Mesoamerica about 3000 years ago (Cheesman 1944; De la Cruz et al. 1995; Motamayor et al. 2002; Ji et al. 2012). Mesoamerican

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natives revered cacao as sacred, and it played an important role in economic, ritual, and political systems (Willson 1999; Grivetti and Shapiro 2009). Today cacao remains important in many tropical regions of the world where, it is grown mostly by small farmers as a cash crop. Global cacao production in 2012 was close to five million metric tons with West African countries such as Ivory Coast, Ghana, Nigeria, and Cameroon providing 70 % of total global production (FAOSTAT 2014). Environmental changes, pests and diseases, and nation’s sovereign property rights are among some of the primary reasons for a decline in accessibility to valuable plant genetic resources for conservation and utilization efforts (Gepts 2006). In addition, genetic erosion of cultivated agricultural crops by selection and utilization of limited numbers of improved varieties has become a serious problem worldwide (Ouborg et al. 1991; Gepts 2006). Therefore, the acquisition and long-term preservation of genetically diverse plant germplasm with useful horticultural and agronomic traits (e.g., productivity, disease resistance, and quality) is essential for agricultural sustainability (Glaszmann et al. 2010). In cacao, diversity assessment of genetic resources in the wild has aided in the identification of unique phenotypes and genotypes. Distinct germplasm is then targeted for inclusion into ex situ national and international genebanks, where it can be further characterized, utilized, and conserved in perpetuity. Evidence shows that cacao was disseminated expansively from its center of origin during pre- and colonial American times (Motamayor et al. 2002, 2008). During Spanish colonization, Puerto Rico was a gateway to the empire and ships passed through from Central and South America. Chronological records indicate that by 1625 cacao was introduced and became established on the island of Puerto Rico (Bartley 2005). During colonial times, cacao and ginger were two of the most important export crops (Henshall and Richardson 2009). The importance of cacao as a crop in Puerto Rico would drastically change in the early 1700s, when a series of climate disturbances (reputed hurricanes) destroyed much of the island’s agriculture. The food shortages that ensued caused cacao plantations to be abandoned (Miner-Sola 1995). However, vestiges of cacao plantations have persisted and can be found throughout the island. Cacao trees can be found with greater frequency in the rural areas of the central mountains. Here, they have been Brescued^ by families for generations who used the cacao trees as companion crop for coffee and the pods for artisanal chocolate production. Since Puerto Rico’s cacao trees are of unknown provenance and because they have never been systematically characterized, they might serve as a valuable source of genetic diversity. Diversity identified in these naturalized cacao trees and/or unique recombinants derived from early introduced cultivars could be conserved and possibly used in selection and/or breeding programs.

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Single nucleotide polymorphism (SNP) genotyping is one of the latest technologies developed and is considered an ideal co-dominant marker system for assessing genetic diversity. SNPs are the most common type of sequence variation between alleles and can be used as simple genetic markers to show associations between the allelic forms of a gene and/or phenotype (Rafalski 2002). Advantages of SNP markers include their abundance throughout the genome, low error rate compared with SSRs (Jones et al. 2002), ease of analysis, and consistency in scoring across platforms (Rafalski 2001; Livingstone et al. 2010; Yang et al. 2011; Fang et al. 2014). Application of SNP markers in cacao research include construction of linkage maps (Micheletti et al. 2011; Allegre et al. 2011), genetic diversity analysis (Ji et al. 2012; Fang et al. 2014), marker trait association studies (Argout et al. 2008), and marker assisted selection studies (Kuhn et al. 2012). The main objectives of the present study were (1) identify and collect naturalized cacao in Puerto Rico through an islandwide survey, (2) assess relationship and genetic diversity in surveyed island cacao by SNP genotyping and by comparison with known accessions held in international and USDA-ARS Tropical Agriculture Research Station (TARS) cacao collections, and (3) acquire and incorporate unique germplasm identified in the island survey into the existing collection at the TARS.

Materials and methods Island-wide survey Leaf and pod (when available) samples from identified naturalized seedling cacao trees were collected throughout the island of Puerto Rico (Fig. 1). Use of several different media to reach out to the local population for identifying cacao trees and samples were employed. A Facebook page, a Website, an interactive blog site (https://es-es.facebook. com/pages/Puerto-Rican-Cacao-Project/130060173817371; http://www.ars-grin.gov/may/prcacao/index.html; http://www.puertoricancacaoproject.blogspot.com), as well as radio and television interviews were all developed and used as part of the project’s outreach and survey efforts. Passport information as well as records of relevant phenotypic traits for collected trees was stored in an electronic database. A biased strategy was taken when surveying for cacaonaturalized trees. Special care was taken in selection of plant material, avoiding individuals with a Bmodern^ introduction history and/or individuals with known recent provenances from USDA-ARS sites, Puerto Rican agricultural experimental stations, or local nurseries. A questionnaire was developed and filled for each sample collected and consisted of relevant phenotypic traits for genetic diversity assessment (e.g., pod shape, seed color). Other collected information for samples


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Fig. 1 Map of the island of Puerto Rico showing cacao tree pod and leaf sampling sites. In some cases, multiple trees were sampled and collected at a particular site/area

included latitude and longitude coordinates. A total of 180 samples (Fig. 1; Supplementary Table 1) were collected for the evaluation, including several cacao-related species and genera from the TARS station grounds to serve as internal references and outgroups in the analysis.

DNA isolation Young, visually disease-free, fully expanded leaves were collected for DNA extraction. Leaf samples were freeze dried using a FreezeZone 4.5® lyophilizer1 (Labconco, Kansas City, MO) for 48 h and stored at 4 °C. The GeneJet Plant Genomic DNA Purification Mini Kit® from Fermentas was used for DNA extraction. Approximately, 20 mg of lyophilized leaf tissue was placed in a 2.0-ml tube already containing 350 μl of lysis buffer A and 50 μl of lysis buffer B. Prior to use, polyvinyl-poly-pyrrolidone (PVPP) at a 2 % (w/v) was added to lysis buffer A in order to reduce the amount of polysaccharides and other organic DNA contaminants present in leaf tissue. Tissue was homogenized using a Tissue Rupture® homogenizer (Qiagen, Germantown, MD) in one 60-s cycle at 30.0 Hz. Immediately after disrupting, samples were put on ice and 20 μl of RNAse A was added and vortexed thoroughly. Samples were incubated for 30 min at 65 °C, 350 rpm in a benchtop Thermomixer® (Eppendorf, Hauppauge, NY). Subsequent steps in the DNA extraction protocol were performed according to the manufacturer’s instructions. DNA was quantified and assessed for purity using a 1 Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

NanoPhotometer® spectrophotometer (Implen, Westlake Village, CA). SNP genotyping SNP loci were identified in previous experiments from expressed sequence tags (ESTs) from a wide range of cacao plant parts that displayed differences in the transcriptome (Argout et al. 2008, 2011). The selection of 96 SNP loci for the panel used in the current study was chosen based on the screening of 1536 SNPs using Illumina’s GoldenGate Assay (Michel Boccara, unpublished data) and their application in previously reported cacao research (Ji et al. 2012; Fang et al. 2014; Lukman et al. 2014). Marker selection criteria also included SNP call rate, representativeness across the ten chromosomes, and heterozygosity based on the Illumina genotyping results. In addition, sequences flanking the candidate SNP loci needed to be at least 60 bp. The protocol for SNP genotyping of cacao used the Fluidigm 96.96 Dynamic Array™ (Fluidigm, San Francisco, CA). One key feature of this protocol is its specific target amplification (STA), which allows the enrichment of template molecules for each individual Integrated Fluidic Circuit® (IFC) reaction that will facilitate the multiplexing during genotyping. As cacao leaf tissues contain high levels of polysaccharides and polyphenolic compounds that can potentially inhibit PCR amplification, the STA step was implemented. The STA master mix was composed of 2.5 μl of TaqMan® Taq polymerase (Life Technologies, Carlsbad, CA), PreAmp Master Mix (2×), 1.25 μl of pooled assay mix (0.2×), and 1.25 μl of genomic DNA for a total reaction volume of 5.0 μl. PCR was performed with an initial denaturation step of 95 °C for 10 min, followed by 14 cycles of a two-step

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amplification profile consisting of 15 s at 95 °C and 4 min at 60 °C. The resulting amplified DNA was then diluted 1:5 in TE buffer in order to reduce the concentration of any remaining PCR by-products. Samples were then genotyped using the nanofluidic 96.96 Dynamic Array™ IFC (Fluidigm, San Francisco, CA). The procedure of a 96.96 Dynamic Array IFC for SNP genotyping was described by Wang et al. (2009) and was carried out at the USDA-ARS Sustainable Perennial Crops Laboratory (SPCL) in Beltsville, MD. Endpoint fluorescent images of the 96.96 IFC were acquired on an EP1™ imager, and the data was recorded with Genotyping Analysis Software (Fluidigm, San Francisco, CA). Genotype data was sorted according to call rate percentage in order to establish a threshold for genotyping success. For duplicate identification, pairwise multi-locus matching was applied among individual accessions, using computer program GenAlex 6.5 (Peakall and Smouse 2006, 2012). For Puerto Rican samples, accessions which fully matched at the genotyped SNP loci were declared duplicates or synonymous accessions and used as one accession for subsequent analysis. In addition, 100 DNA samples representing different cacao germplasm groups were included in this experiment to serve as references (Supplementary Table 2). Genetic identity of these clones have been analyzed and reported in previous publications (Zhang et al. 2009; Ji et al. 2012). Genetic diversity and population structure Summarizing descriptive statistics measuring informativeness of the 87 successfully amplified SNP loci were calculated for naturalized and reference cacao samples. Allele frequencies, observed (HObs) and expected (HExp) heterozygosity and polymorphic information content (PIC) were estimated using Cervus v3.0.0 (Kalinowski et al. 2007). Pairwise Euclidian genetic distance (Rogers 1972) was calculated to assess the associations among Puerto Rican cacao samples and their relations to reference samples using Power Marker v3.25 (Liu and Muse 2005). Principal coordinate analysis (PCoA) was generated based on pairwise genetic distance matrix using GenAlEx v6.4 (Peakall and Smouse 2006). Using Power Marker v3.25 (Liu and Muse, 2005) a neighbor-joining dendrogram was produced and viewed with the Interactive Tree of Life Software (iTOL) v2.2 (Letunic and Bork 2006, 2011). Population structure analysis was carried out for Puerto Rican and reference samples using the model-based Bayesian cluster analysis software STRUCTURE v2.3.4 (Pritchard et al. 2000). An admixture model was used with 200,000 iterations after a burn-in period of 100,000. The number of clusters (K) tested was from 1 to 10 and each K had ten iterations (or replicated runs). The optimal cluster (optimal K) number for the dataset was determined using the Evanno method (Evanno et al. 2005) as implemented in the STRUCTURE HARVESTER software (Earl and von Holdt

2012). The ten replicated runs for the determined number of clusters were subsequently matched by permutations using CLUMPP v1.1.2b (Jakobson and Rosenberg 2007). The genetic variation among and within clusters was determined by analysis of molecular variance (AMOVA) using GenALEx v6.4 (Peakall and Smouse 2006). Phenotype assessment In an effort to try to identify associations between population structure (identified by the SNP markers) and observed traits in pods/seed, eight highly discriminating phenotypic traits, traditionally used for identifying cacao germplasm, were recorded for cacao pod samples collected from naturalized trees in Puerto Rico. These descriptor traits, previously reported by Bekele et al. (2006), were gathered as part of the sample collection questionnaire or assessed directly and included pod color, pod basal constriction, pod apex shape, pod rugosity, pod furrow depth, pod length, overall pod shape, and cotyledon color (i.e., seed color). The association between population structure and each phenotypic trait was performed by Chisquare tests using InfoStat v2014e (Di Rienzo et al. 2011).

Results Plant and DNA samples In all, 32 Puerto Rican municipalities were visited during the field survey with a total of 180 leaf samples collected from identified trees (Fig. 1). Pods were obtained for 135 of the 180 sample sites. Locality information including latitude, longitude (GPS coordinates) and altitude were recorded for the 180 tree sample sites (Supplementary Table 1). Some of the leaf samples were provided by collaborators only with municipality location information and with no corresponding pods. Eight leaf samples, collected from trees on the TARS station grounds, of T. cacao-related genera (Herrania spp.) and T. cacao-related species were included as outgroups in the experiments (Supplementary Table 1). SNP genotyping and duplicate identification From the initial 96-SNP panel chosen to study genetic diversity of Puerto Rican naturalized cacao, 87 SNPs generated high call rates (>90 %) across T. cacao samples (Supplementary Table 3). For the 87 markers that generated consistent results, a total of 145 (from the 180) local samples had a SNP call rate percentage higher than the threshold (>90 %). SNP markers did not amplify well for outgroup T. cacao-related genera or species and the genotypes for these outgroup samples were thus removed from further analysis.

Tree Genetics & Genomes (2016) 12:88 Table 1 Eleven synonymous groups (i.e., identical multi-locus genotypes) corresponding to 66 samples identified from SNP genotyping of 145 naturalized cacao trees sampled in Puerto Rico

Page 5 of 13 88 Table 1 (continued)

SYN group

Accession

1 1 2 2 2

PRCP52 PRCP53 PRCP135 PRCP136 PRCP137

3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4

PRCP04 PRCP23 PRCP37 PRCP88 PRCP85 PRCP79 PRCP86 PRCP118 PRCP120 PRCP121 PRCP122 PRCP128 PRCP129 PRCP131 PRCP132 PRCP139 PRCP160 PRCP163

5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7

PRCP119 PRCP123 PRCP124 PRCP125 PRCP130 PRCP134 PRCP40 PRCP41 PRCP42 PRCP116 PRCP144 PRCP145 PRCP146 PRCP147 PRCP153 PRCP155 PRCP77 PRCP165

8 8 8 8 8 9 9 9

PRCP07 PRCP18 PRCP53 PRCP92 PRCP111 PRCP03 PRCP10 PRCP14

SYN group

Accession

9 9 9

PRCP15 PRCP19 PRCP66

9 9 9 9 9 9 9 10 10 11 11 11 11 11

PRCP70 PRCP71 PRCP72 PRCP74 PRCP96 PRCP170 PRCP173 PRCP12 PRCP98 PRCP09 PRCP13 PRCP69 PRCP97 PRCP171

PRCR, Puerto Rican Cacao Project

Individual genotype matching (pairwise comparisons) based on the 87 SNPs identified 11 synonymous groups including 66 accessions (Table 1). The members within each group shared identical multi-locus SNP profiles, thus met our definition of duplicates or synonymous accessions. After consolidation of synonymous genotypes into 11 groups, a total of 90 local samples were combined with reference samples (100) for the analysis of genetic diversity. Genetic diversity analysis Summary statistics for the combined naturalized Puerto Rican cacao and reference samples showed HObs values that ranged from 0.057 (for the TcSNP1392 locus) to 0.835 (for TcSNP277 locus) and an overall HObs average of 0.248 (Supplementary Table 4). HExp ranged from 0.093 for the TcSNP1392 locus to 0.501 for several loci and averaged 0.413. Polymorphic information content (PIC) ranged from 0.089 for TcSNP1392 to 0.375 for several loci and averaged 0.321. Minor allele frequency ranged from 0.049 for TcSNP1392 to 0.500 for the TcSNP645 and TcSNP723 loci and averaged 0.337. Population structure Analysis of population structure assigned and separated (based on high membership coefficients) the 245 samples (100 Reference, 145 Puerto Rico) into three populations (Fig. 2; Supplementary Fig. 1). Although ΔK is highest at K = 2 in Supplementary Fig. 1, a more suitable and

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Fig. 2 Population assignment tests for naturalized Puerto Rican (145) cacao (Theobroma cacao L.) and references (100) using STRUCTURE software. Numbers 1–245 represent individuals. Colors red, green, and blue represent a specific population (K = 3). Individuals with color

combinations are denoted as admixed. A total of five clusters were identified: (a) Criollo, (b) Trinitario, (c) Amelonado, (d) Upper Amazon Forastero, and (e) Nacional. R reference accessions, PR Puerto Rican cacao samples

consistent number of clusters as suggested by PCoA analysis and the use of characterized reference accessions was K = 3. Fixation index (FST) values for the three populations ranged 0.74–0.79 (data not shown). Additionally, two clusters with high admixture were also apparent. Similar population structure was observed in the PCoA (Fig. 3) and in the

dendrogram (Supplementary Fig. 2). The five clusters represent the Criollo, Amelonado, Nacional, and the hybrid Trinitario and UAF cacao genetic backgrounds. The Criollo cluster was mainly composed of ‘Ancient’ Criollo samples which exhibited extremely low within-group diversity (Fig. 2 and 3; and Supplementary Fig. 2). A

Fig. 3 Principal coordinate analysis (PCoA) for naturalized Puerto Rican (145) cacao (Theobroma cacao L.) and reference samples (100). Clustering results show three main clusters (Criollo, Amelonado, and Nacional) and two admixed clusters (Trinitario and Upper Amazon

Forastero [UAF]). Puerto Rican samples are indicated by circles (notice no Puerto Rican samples in the Nacional cluster) and reference samples are indicated by triangles


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Trinitario and UAF clusters. AMOVA showed strong genetic differentiation (FST) between populations when Puerto Rican (0.32) and reference (0.30) samples were analyzed separately (Table 2). The strong differentiation was also true when the combined analysis was performed. Inbreeding coefficient among samples within subpopulations, denoted as FIS, was considerable for Puerto Rican individuals (0.05). In contrast, little inbreeding (0.34) was shown for individual samples within subpopulations for the reference accessions. The dendrogram (Supplementary Fig. 2), generated from the cluster analysis, divided samples, and reference accessions into five main groups (Criollo, Amelonado, Nacional, Trinitario, and UAF) and generally agreed with clustering in the STRUCTURE and PCoA figures.

significant number of samples from Puerto Rico (71) were part of this genetic background, and many of the samples with identical genotypes belonged to this group. A second admixed cluster included locally sampled individuals (25) with Trinitario genetic backgrounds. Puerto Rican samples within the Trinitario cluster had a tendency to group closer to the Criollo samples. This was evident visually (Fig. 3) and from the lower FST values between the Criollo and Trinitario samples (FST 0.302) from Puerto Rico compared with the reference samples from the same two groups (FST 0.322). As expected, the majority of the samples in the Trinitario group were admixed with allelic contributions from both Criollo and Amelonado progenitor genetic backgrounds. The third cluster was composed of individuals with Amelonado genetic background with a relatively small number of local samples (9) grouping here. The local samples exhibited high similarity with reference accessions and many of the samples exhibited low heterozygosity values. The fourth cluster corresponded to the samples with UAF genetic backgrounds, and a considerable number of local samples belong to this group (40). The UAF cluster also contained samples with a high degree of admixture. The fifth group of samples was the Nacional cluster and no naturalized Puerto Rican cacao samples were part of this genetic background. Population structure analysis indicated a major Amelonado admixture contribution in Trinitario and UAF clusters (Fig. 2). In addition, results showed that the Amelonado ‘contribution’ was lower for Puerto Rican samples when compared with references within the

Table 2 Analysis of molecular variance for cacao samples belonging to naturalized Puerto Rican cacao (Theobroma cacao L.) trees, for reference samples and for the combined Puerto Rican and reference samples

Source of variation Puerto Rican samples Between subpopulations Between individuals within subpopulations Within individuals Total Reference samples Between subpopulations Between individuals within subpopulations Within individuals Total

Phenotype assessment A group of eight highly discriminating phenotypic descriptor traits were used to characterize the collected pods and seed from naturalized Puerto Rican cacao. The pods and seeds were grouped based on the different genetic backgrounds (clusters) identified during genotyping. Phenotypic discrimination between assessed genetic backgrounds was evident for all of the traits evaluated (Table 3). Cotyledon color (p < 0.0001), pod color (p < 0.0427), basal constriction (p < 0.0001), apex shape (p < 0.0001), and overall pod shape (p < 0.0001) showed significant differences between assessed genetic backgrounds. Cotyledon color for the Criollo background is mainly white,

df

Variance components

Variation (%)

Fixation indices

3 86

6.85 0.67

32.4 3.2

FST = 0.32 FIS = 0.05

90 179

13.63 21.15

64.4

FIT = 0.36

4 95

6.98 5.48

30.4 23.9

FST = 0.30 FIS = 0.34

100 199

10.49 22.95

45.7

FIT = 0.54

7.00 3.76

30.8 16.5

FST = 0.31 FIS = 0.24

11.98 22.74

52.7

FIT = 0.47

Puerto Rican and references samples Between subpopulations 4 Between individuals within 185 subpopulations Within individuals 190 Total 379

FST genetic differentiation among subpopulations, FIS inbreeding coefficient of subpopulations, FIT inbreeding coefficient in the total sample

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Table 3 Observed frequencies for each of the traits are displayed for assessed genetic backgrounds for naturalized Puerto Rican cacao

Trait

Criollo

Trinitario

Amelonado

UAF

X2

p value

Cotyledon color White Purple Mixed

34 (69 %) 4 (8 %) 11 (13 %)

2 (17 %) 7 (58 %) 3 (25 %)

1 (13 %) 6 (75 %) 1 (13 %)

3 (9 %) 21 (66 %) 8 (25 %)

44.22