RESEARCH ARTICLE

Genetic diversity and association mapping in

the Colombian Central Collection of Solanum
tuberosum L. Andigenum group using SNPs
markers
Jhon Berdugo-Cely1, Raúl Iván Valbuena1, Erika Sánchez-Betancourt1, Luz
Stella Barrero1, Roxana Yockteng1,2*

1 Colombian Agricultural Research Corporation (CORPOICA)-Mosquera, Cundinamarca, Colombia,

2 Muséum National d’Histoire Naturelle, UMR-CNRS 7205, Paris, France
a1111111111
a1111111111 * [email protected]
a1111111111
a1111111111
a1111111111 Abstract
The potato (Solanum tuberosum L.) is the fourth most important crop food in the world and
Colombia has one of the most important collections of potato germplasm in the world (the
OPEN ACCESS
Colombian Central Collection-CCC). Little is known about its potential as a source of genetic
diversity for molecular breeding programs. In this study, we analyzed 809 Andigenum group
Citation: Berdugo-Cely J, Valbuena RI, Sánchez-
Betancourt E, Barrero LS, Yockteng R (2017) accessions from the CCC using 5968 SNPs to determine: 1) the genetic diversity and popula-
Genetic diversity and association mapping in the tion structure of the Andigenum germplasm and 2) the usefulness of this collection to map
Colombian Central Collection of Solanum qualitative traits across the potato genome. The genetic structure analysis based on principal
tuberosum L. Andigenum group using SNPs
components, cluster analyses, and Bayesian inference revealed that the CCC can be subdi-
markers. PLoS ONE 12(3): e0173039. doi:10.1371/
journal.pone.0173039 vided into two main groups associated with their ploidy level: Phureja (diploid) and Andigena
(tetraploid). The Andigena population was more genetically diverse but less genetically sub-
Editor: Xiu-Qing Li, Agriculture and Agri-Food
Canada, CANADA structured than the Phureja population (three vs. five subpopulations, respectively). The
association mapping analysis of qualitative morphological data using 4666 SNPs showed 23
Received: September 25, 2016
markers significantly associated with nine morphological traits. The present study showed
Accepted: February 14, 2017
that the CCC is a highly diverse germplasm collection genetically and phenotypically, useful
Published: March 3, 2017 to implement association mapping in order to identify genes related to traits of interest and to
Copyright: © 2017 Berdugo-Cely et al. This is an assist future potato genetic breeding programs.
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Data Availability Statement: All relevant data are
within the paper and its Supporting Information Solanum tuberosum L. is a herbaceous species that reproduces mainly vegetatively by tubers,
files. distributed from the Southwestern United States to South-central Chile, with centers of diver-
Funding: Support was provided by the Colombian sity located in Central Mexico and in the high Andes from Peru to Northwestern Argentina
Ministry of Agriculture (Ministerio de Agricultura y [1]. Potato is the fourth most important crop food in the world after corn, rice and wheat [2].
Desarrollo Rural de Colombia). It is consumed by people worldwide either as a non-grain staple or as a vegetable. It has high
Competing interests: The authors have declared nutrient value providing carbohydrates, proteins, vitamins and minerals [3]. Solanum tubero-
that no competing interests exist. sum contains two cultivar groups, the Chilotanum group comprising lowland tetraploid

PLOS ONE | DOI:10.1371/journal.pone.0173039 March 3, 2017 1 / 27

 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Chilean landraces [4] and the Andigenum group comprising upland Andean genotypes. Andi-
genum group varies in its ploidy level, going from diploids with 24 chromosomes to hexaploids
with 72 [4]. Within the Andigenum group, the most important potatoes are commonly known
as “Andigenas”, which are autotetraploid (2n = 4x = 48), highly heterozygous with tetrasomic
inheritance, adapted to tuberization under short days and have tuber dormancy [5, 6]. In Andi-
genum, a group of diploids (2n = 2x = 24) known as “Phurejas” can also be distinguished. These
potatoes have a short vegetative period, form small tubers and lack dormancy [5, 7]. They were
cultivated from central Peru to Ecuador, Colombia, and Venezuela [8]. Another group in Andi-
genum, known as “Chauchas”, are triploid potatoes (2n = 3x = 36) generated by natural hybrid-
ization between the species S. tuberosum subsp. andigena and S. stenotonum, and they are
cultivated in Peru, with lower frequency in Bolivia, Ecuador and Colombia [8].
The conservation of cultivated potato species and their wild relatives in germplasm banks
provides long-term availability of crop genetic diversity. The characterization of these collec-
tions are essential to identify alleles/genes associated with traits of interest for plant breeding
such as resistance to pathogens and insect pests, tolerance of abiotic stresses (e.g. salinity and
frost) and tuber quality [9, 10]. In Colombia, part of the diversity of potato genetic resources
(2069 accessions) are maintained in the Potato Germplasm Bank located at the Colombian
Agricultural Research Corporation (CORPOICA). Within this germplasm bank, a subset of
potatoes (826 accessions) known as Colombian Central Collection (CCC), is recognized as one
of the most diverse potato germplasm in the world, after the CIP (International Potato Center)
collection that has over 6000 accessions including cultivated species and potato wild relatives
[11, 12, 13]. The Universidad Nacional de Colombia conserves also a Phureja potato collection
(Colombian Core Collection-CCC). Hence, the CCC-CORPOICA is a potential source of novel
alleles of agronomic value that could help to generate new potato cultivars with increased pro-
ductivity. However, the appropriate use of genetic resources conserved in the CCC, depends on
the understanding of their phenotypic and genetic diversity.
Genetic diversity could be analyzed from agronomic traits data, but the results obtained are
not always robust because the environment often affects phenotypic traits [14]. In addition,
the phenotypic variability would be the result of the interaction and segregation of few major
genes widely distributed in a germplasm collection. Rare alleles cannot be generally detected
or preserved [13]. Therefore, the combination of phenotypic and molecular data could provide
a better estimation of the genetic diversity [15]. Molecular markers have been successfully used
in the analysis of genetic diversity and population structure, linkage disequilibrium and locali-
zation of monogenic or polygenic traits [16]. The genetic diversity in potato has been studied
through different molecular markers as random amplified polymorphic DNA (RAPD), ampli-
fied fragment length polymorphism (AFLP), inter simple sequence repeats (ISSR), and simple
sequence repeat (SSR) [5, 7, 17]. So far, only one study using 42 SSRs, analyzed 97 diploid
accessions (Phurejas) of the CCC-Universidad Nacional de Colombia has been reported [18].
However, the genetic diversity of Andigenum group of the CCC-CORPOICA has not been yet
characterized with molecular markers.
Currently, two beadchips with SNP array technology for genotyping potato at high-density
genome-wide level are available, the Infinium 8K potato SNP array [19] and the 20K SNP
array [20]. The 8K SolCAP array contains a subset of 8303 SNPs selected from transcriptome
data and Sanger EST (Expressed Sequence Tag) database with 69.011 high confidence SNPs
identified among six North American cultivars [21]. The 8K array has been used to study the
genetic diversity of American [9] and European potatoes [22], to infer phylogenetic relation-
ship among species of Solanum section Petota [23] and to identify candidate genes through
linkage mapping [19, 24, 25] and association mapping [26–28].

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

By combining molecular and morphological data from the potato germplasm of CCC is
possible to map simple or complex traits and subsequently to identify candidate genes through
Genome-Wide Association Studies (GWAS) or Association mapping (AM). Such studies pro-
vide an efficient way to map quantitative trait loci (QTL) in natural populations or germplasm
collections because they can detect historical recombination events and provide high mapping
resolution [29–31]. The number of molecular markers required for implementing GWAS and
the resolution for QTL mapping, is determined by the rate of LD decay between loci through
the genome [32]. Although the LD decay in potato populations has been previously calculated,
all reports differ: 265 bp (base pairs) [22], 1 cM (centiMorgan) [33], 5 cM [34] and 10 cM [35].
The incongruence between studies is probably due to differences in number, type and origin
of samples and the type and number of molecular markers used. It is then necessary to calcu-
late the LD background in this study.
In the present study, a genetic analysis of the CCC of S. tuberosum Andigenum group was
conducted based on SNPs markers in order to evaluate its population structure and genetic
diversity. Also, the extent of the linkage disequilibrium between pairs of SNPs markers was
estimated in order to determine the utility of this germplasm and the molecular markers used
to implement association-mapping studies. Accordingly, association mapping in tetraploid
potatoes was conducted using morphological traits related with stem, berry, tuber and flower
variables.

Materials and methods

Plant material
A total of 809 accessions (one clone randomly selected from 16 clones grown per accession) of
the CCC-CORPOICA of S. tuberosum group Andigenum conserved under field conditions in
Zipaquira, Cundinamarca, Colombia (5˚ 03” 34.36” N, 74˚ 03” 29.61 W, 2.950 m altitude, aver-
age temperature 15˚C and relative humidity of 75%) were characterized. Six hundred seventy-
five accessions are classified from passport data as Andigena (83.5%), 85 as Phureja (10.5%)
and 49 as Chaucha (6.0%). Six hundred and sixteen accessions were collected from different
Colombia regions (76.1%), 75 accessions from other countries (9.3%) and 118 accessions do
not have passport data (14.6%) (Fig 1, Table 1). The information of each accession is presented
in the S1 Table.

DNA extraction, genotyping and SNP markers selection

Fresh young leaves were collected from one plant randomly selected per accession. The mate-
rial was lyophilized during two days at -50˚C and 0.20 mBar. The genomic DNA was extracted
using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). DNA concentration and qual-
ity were checked by visualization in a 1% (w/v) agarose gel and a NanoDrop 2000 Spectropho-
tometer (Thermo Fisher Scientific, Wilmington, USA). Genotyping was performed using the
array available in 2013, the Infinium 8303 potato SNP array [19, 21]. The array was read in the
Illumina HiScan SQ system (Illumina, San Diego, CA) at CORPOICA. The software GenomeS-
tudio version diploids and polyploids (Illumina, San Diego CA) was used to assign the genotype
to each locus; five possible genotypes (AAAA, AAAB, AABB, ABBB or BBBB) in tetraploid
potatoes and three possible genotypes (AA, AB and BB) in diploid potatoes. The assignation of
samples as diploids through molecular markers was confirmed with the available information
of cytogenetic analysis made in Phureja and Chaucha samples of the CCC reported by Guevara
[36] and Uribe [37]. The SNPs that could not be called or were monomorphic were discarded.
The remaining SNPs were filtered for up to 20% missing data and a Minor Allele Frequency
(MAF) lower than 0.05. Genotypic data is provided in the S2 Table.

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Fig 1. Map of geographical distribution of potato accessions from the Colombian Central Collection with passport data. Each accession is
represented by a circle in which color indicates their classification in a particular population based on the results of software Structure (Red: Phureja, Green:
Andigena).
doi:10.1371/journal.pone.0173039.g001

Population structure and genetic differentiation

The population structure analysis was performed using a Bayesian model implemented in the
software Structure [38] without a priori population information using a tetraploid model

Table 1. Summary of the 809 accessions of the Colombian Central Collection of S. tuberosum Andigenum group used in this study.
CCC Country Department Number of accessions
Andigena Colombia Nariño 201
Boyacá 110
Cauca 63
Cundinamarca 62
Santander 21
N. de Santander 19
V. del Cauca 16
Antioquia 12
Quindio 10
Caldas 8
Tolima 8
Magdalena 1
Unknown 25
Peru - 39
Bolivia - 11
Ecuador - 11
United States - 3
Netherlands - 1
Venezuela - 1
Unknown - 53
Total Andigena 675
Chaucha Colombia Nariño 32
Peru - 1
Unknown - 16
Total Chaucha 49
Phureja Colombia Nariño 40
Cauca 6
V. del Cauca 2
Antioquia 1
Boyacá 1
Cundinamarca 1
Quindio 1
N. de Santander 1
Unknown 1
Peru - 6
Bolivia - 1
United States - 1
Unknown - 23
Total Phureja 85
TOTAL Colombian Central Collection 809
doi:10.1371/journal.pone.0173039.t001

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

(Andigena: 1 = AAAA, 2 = AAAB, 3 = AABB, 4 = ABBB, 5 = BBBB; Phureja: 1 = AA, 3 = AB,

5 = BB). The analyses were conducted by varying the number of possible subpopulations (K)
from 1 to 10, with five independent repetitions, assuming an admixture model with correlated
allele frequencies and a burn-in of 50.000 and 150.000 iterations. The optimal number of subpop-
ulations was established using the Evanno method [39] in Structure Harvester [40]. The number
of subpopulations was confirmed with a Discriminant Analysis of Principal Component (DAPC)
[41] conducted in the package Adegenet [42] in the R software [43] and a Principal Component
Analysis (PCA) in the Tassel software [44]. Coefficients of genetic differentiation among subpop-
ulations (FST) and population inbreeding (FIS) within subpopulations were estimated by an analy-
sis of molecular variance (AMOVA) with 1023 permutations in the Arlequin software [45]. The
gene flow or number of migrants (Nm) was estimated through the equation: Nm = (1-FST)/4FST.

Genetic diversity and cluster analysis

The genetic indexes, Observed Heterozygosity (Ho) and Expected Heterozygosity (He) were
calculated using Genalex software [46]. The Polymorphic Information Content (PIC) was cal-
culated using PowerMarker software [47] and the deviation from the Hardy-Weinberg equilib-
rium (HWE) was calculated using Genepop software [48]. Nei’s distances matrices [49] were
calculated using the package StAMPP [50] in the R software [43] and the dendograms were
constructed using the software PHYLIP [51] selecting the Neighbor-Joining (NJ) method with
1000 bootstrap replicates.

Morphological characterization and correlations among morphological,

geographical and genetic data
Phenotypic data from fifteen qualitative characteristics of stem, berry, tuber, and flower were
used for the morphological analysis (Table 2). The Plant Genetics Resources team of COR-
POICA recorded this information in eight different years (1995, 1996, 1997, 2004, 2006, 2009,
2010 and 2012) in 624 Andigena accessions using the descriptors of the CIP to characterize
native potatoes [52]. The collection was evaluated in three different locations over field condi-
tion in Zipaquira (5˚ 03” 34.36” N, 74˚ 03” 29.61 W), Tibaitata (4˚ 41” 43.2” N, 74˚ 12” 13.3
W), and San Jorge (6˚ 01” 50.74” N, 74˚ 02” 40.65 W). Sixteen plants per accession were grown
during eight months, time required to present structures to characterize. One plant per acces-
sion was randomly selected, and data was registered for each descriptor in five different ber-
ries, stems and flowers. Finally, tuber descriptors of five tubers per accession were registered
after harvest. Phenotypic data for 624 Andigena accessions are presented in the S3 Table. The
mode values of all variables for each accession were used to conduct a Multiple Correspon-
dence Analysis (MCA) and a cluster analysis based on the Gower’s distance and the Ward
method implemented in the software InfoStat [53]. The available passport data of 691 acces-
sions of the CCC was used to generate the geographical distances between accessions in the
Geographical Distances Matrix Generator software (http://biodiversityinformatics.amnh.org).
The correlation between geographical, morphological and genetic distances was estimated
by a Mantel test [54] with 1000 permutations in the software Genalex [46]. The correlations
between morphological and genetic data were independently estimated for each variable. Sub-
sequently, the global correlation was first calculated using the total of variables, and then using
only positive and significant correlated variables.

Linkage disequilibrium
The linkage disequilibrium (LD) was calculated in each inferred population. The SNPs used
presented the physical position (mapped) on the potato genome version 4.03 [55]. To include

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Table 2. Results of morphological analysis of qualitative characters of Andigena population of the CCC.
Variable Variable Coding Multiple Correspondence Analysis (MCA) Correlation with
abbreviation genetic distance
Dimension 1 Dimension 2 Dimension 3 Percentage p-value
(4.59%) (4.18%) (3.53%) (%)
Primary Flower Intensity Color PFIC 0–3 0.081 0.155 0.019 18 0.001*
Primary Flower Color PFC 1–8 0.084 0.159 0.016 12.9 0.001*
Primary Tuber Skin Intensity PTSIC 0–3 0.166 0.023 0.001 12.8 0.001*
Color
General Tuber Shape GTS 1–8 0.035 0.018 0.027 9.4 0.001*
Distribution of Secondary Flower DSFC 0–9 0.075 0.119 0.059 9 0.01*
Color
Primary Tuber Skin Color PTSC 1–9 0.128 0.089 0.086 8.5 0.001*
Distribution of Secondary Tuber DSTSC 0–7 0.097 0.023 0.269 7.5 0.001*
Skin Color
Secondary Tuber Skin Color STSC 0–9 0.094 0.059 0.271 7.1 0.002*
Berry Color BC 1–7 0.014 0.056 0.022 5.7 0.001*
Distribution of Secondary Tuber DSTFC 0–7 0.024 0.064 0.023 -5.3 0.026*
Flesh Color
Stem Color ST 1–7 0.094 0.04 0.112 -6.4 0.001*
Secondary Tuber Flesh Color STFC 0–8 0.029 0.082 0.006 -8.4 0.003*
Primary Tuber Flesh Color PTFC 1–8 0.005 0.002 0.033 3.5 0.114
Berry Shape BS 1–7 0.003 0.007 0.014 0.4 0.431
Secondary Flower Color SFC 0–8 0.071 0.103 0.04 -0.8 0.425

* Significance at p < 0.05

doi:10.1371/journal.pone.0173039.t002

all SNP dosage (heterozygous genotypes), diploid and tetraploid data were analyzed following
the report by Vos et al. [56], using the Pearson correlation coefficient between each pair of
SNP marker. The LD decay was estimated using a combination of SNP markers in significant
correlation (p < 0.001) with a threshold of r2 that corresponded to 90th percentile [56] of pair-
wise correlations of each population.

Association mapping analyses

Phenotypic data corresponding to 15 qualitative variables (Table 2, S3 Table) of 466 tetraploid
accessions of Andigena accessions were used to identify marker-trait association using Mixed
Linear Model (MLMs) analyses accounting for the population structure and kinship as fixed
effects using the package GWASpoly [27] in the R software [43]. Additionally, the Andigena
genotypic data was filtered with the default parameters of GWASpoly [27] (5% of missing data
and a MAF of 0.10). To identify the SNPs with significant associations, the p values were cor-
rected with the Bonferroni method [57] at p values of 0.05, 0.01 and 0.001.

Results
Genetic molecular analyses
The 809 accessions of the CCC were genotyped with 8303 SNPs using the Infinium SolCAP,
1584 markers were removed from the dataset since 1174 were monomorphic (14.1%), 405 SNPs
could not be called (4.9%), and five presented more than 20% of missing data (0.1%). Genotype
calling inferred 6719 high confidence SNPs (81%), from which 751 SNPs presenting a MAF less
than 0.05, were also excluded, giving a total of 5968 useful markers (72%) (Table 3). Of these

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Table 3. Genetic diversity statistics of the Colombian Central Collection of S. tuberosum group Andigenum.
Population Subpopulation N Polymorphic markers Ho (Mean +/- SD) He (Mean +/- SD) PIC (Mean +/- SD) HWE
CCC Phureja 133 3950 (66.2%) 0.194 (0.003) 0.167 (0.002) - 266 (7.88%)
Andigena 676 5951 (99.7%) 0.516 (0.004) 0.337 (0.002) - 73 (1.22%)
Total 809 5968 (100%) 0.355 (0.003) 0.252 (0.002) 0.437 (0.191) -

Phureja Phureja_1 90 2775 (99.86%) 0.387 (0.004) 0.339 (0.003) - 5 (0.18%)

Phureja_2 19 1516 (54.55%) 0.545 (0.009) 0.272 (0.005) - 0 (0%)
Phureja_3 24 1064 (38.29%) 0.380 (0.008) 0.190 (0.005) - 0 (0%)
Total 133 2779 (100%) 0.437 (0.005) 0.267 (0.002) 0.279 (0.090) -

Andigena Andigena_1 24 3742 (63.41%) 0.628 (0.006) 0.315 (0.003) - 50 (0.84%)

Andigena_2 64 5144 (87.17%) 0.498 (0.005) 0.304 (0.003) - 1 (0.03%)
Andigena_3 170 4670 (79.14%) 0.471 (0.005) 0.285 (0.003) - 1 (0.03%)
Andigena_4 138 5900 (99.98%) 0.601 (0.003) 0.389 (0.002) - 14 (0.32%)
Andigena_5 280 5758 (97.58%) 0.476 (0.004) 0.304 (0.002) - 7 (0.15%)
Total 676 5901 (100%) 0.535 (0.002) 0.319 (0.001) 0.269 (0.108) -

N: Number of samples, Ho: Observed Heterozygosity, He: Expected Heterozygosity, PIC: Polymorphic Index Content, HWE: Hardy-Weinberg equilibrium:
SNPs not in HWE, SD: Standard deviation.

doi:10.1371/journal.pone.0173039.t003

markers, 5790 were mapped on 12 chromosomes of the potato genome and 97 mapped on
unanchored scaffolds (Chr. 0). Therefore, an average of 483 markers mapped on potato chro-
mosomes ranging from 347 markers for Chr. 12 to 646 for Chr. 4.

Population structure and genetic diversity in the Colombian Central

Collection
The population structure analysis of the CCC using the software Structure discriminated two
main populations (K = 2) (Fig 2A, S1A Fig). The previous result was supported by Neighbor-
Joining clustering analysis (Fig 2B) and the Principal Component Analysis in which 25.5% of
variability was explained by the three first components (Fig 2C). The first population, named
as Phureja, contains 133 accessions (16.4% of the CCC) from which 82 accessions have pass-
port data and are classified as Phureja, two as Andigena (And_4 and And_183) and 49 as
Chaucha. The majority of accessions of the CCC (83.6%) constituted the second population,
named as Andigena, which regrouped 673 accessions with passport data of Andigena and
three of Phureja (Phu_47, Phu_119 and Phu_122) (Table 3, S1 Table). The percentage of poly-
morphic SNPs was 66.2% and 99.7% for Phureja and Andigena populations, respectively
(Table 3).
The genetic differentiation between Phureja and Andigena populations was high (FST =
0.203, p = 0.000), and the percentage of genetic variation was higher within populations (81%)
than among populations (19%) (Table 4). High values of genetic variation within populations
imply high genetic diversity. The CCC presented an excess of heterozygosity (FIS = -0.517,
p = 1.000) and a low gene flow (Nm = 0.98) (Table 4). High genetic diversity was found in the
CCC (Ho CCC = 0.355, He CCC = 0.252), where the genetic diversity was higher in Andigena
(Ho = 0.516, He = 0.337) than Phureja (Ho = 0.194, He = 0.167) (Table 3). Observed popula-
tion structure supported the passport data that differentiates two main groups, Andigena and
Phureja. Samples included in the Phureja population were characterized by presenting a

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Fig 2. Population structure of 809 accessions of S. tuberosum group Andigenum. (A) Clustering Structure analysis. (B) NJ-tree based on Nei´s
genetic distances. (C) Principal Component Analysis.
doi:10.1371/journal.pone.0173039.g002

chromosome number of 24 (2n = 2x) determinate previously by Guevara [36] and Uribe [37].
Because of the difference in ploidy level, the two populations were analyzed independently.
Phureja population. In the Phureja population, 2779 SNPs had a MAF value higher than
0.05 and passed missing data filters. The accessions of Phureja were clustered in three subpop-
ulations (K = 3) (Phureja_1, Phureja_2 and Phureja_3) (Fig 3A, S1B Fig). The simulations
from the software Structure were consistent with the NJ-tree (Fig 3B) and the DAPC analysis
(Fig 3C), where 43.5% of the variation was explained by the three first components of the PCA.
The three Phureja subpopulations differed genetically among them (FST = 0.225, p = 0.000),
and were characterized by presenting an excess of heterozygotes (FIS = -0.342, p = 1.000) and
low gene flow (Nm = 0.86) (Table 4). The genetic differentiation was supported by significant
FST values (p = 0.000) observed among the subpopulations that ranged from 0.161 (Phureja_1
vs. Phureja_2) to 0.435 (Phureja_2 vs. Phureja_3) (S4 Table). The distribution of genetic varia-
tion within and among subpopulations estimated by AMOVA indicated that 77% of the total
genetic variation was found within subpopulations and 23% among subpopulations (Table 4).
The population Phureja presented high genetic diversity with an average Ho of 0.437, He of
0.267 and PIC of 0.279 (Table 3).
Andigena population. A total of 5901 SNPs (MAF > 0.05) were polymorphic in Andi-
gena population and the analyses conducted on these data subdivided the Andigena popula-
tion in five groups (K = 5) (Andigena_1—Andigena_5) (Fig 4 and S1C Fig). The inferred
groups in the structure analysis were not clearly separated by the cluster analysis (Fig 4B) and
the DAPC, where the three first components of the PCA only explained the 20.7% of the

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Table 4. Analysis of Molecular Variance (AMOVA) based on SNP markers for each population of S. tuberosum of the Colombian Central
Collection.
Population Source of variation Degrees of Sum of squares Mean square Variance Percentage of variation F-statistics p-value Nm
freedom component (%)
CCC Among 1 99887.3 99887.3 94.295 19 FST = 0.203 0.000 -
populations
Within populations 1616 1569442.1 917.1 1011.791 81 FIS = -0.517 1.000 -
Total 1617 1669329.4 1106.086 100 - - 0.98
Phureja Among 2 17322.3 8661.1 126.446 23 FST = 0.225 0.000 -
populations
Within populations 263 114539.9 435.5 435.513 77 FIS = -0.342 1.000 -
Total 265 131862.3 561.959 100 - - 0.86
Andigena Among 4 66274.5 15639.3 65.7 6.5 FST = 0.06 0.000 -
populations
Within populations 1161 1240882.0 990.0 938.7 93.5 FIS = -0.59 1.000 -
Total 1165 1307156.5 1004.4 100 - - 3.91

Nm: Gene flow or Number of migrants.

doi:10.1371/journal.pone.0173039.t004

variation. An unique subpopulation (Andigena_1) was genetically differentiated of the other

four subpopulations (Fig 4C; S2 Table). The AMOVA showed that genetic variation was
higher within subpopulations (93.5%) than among subpopulations (6.5%), with a population
with low genetic structure (FST = 0.06, p = 0.000), excess of heterozygosity (FIS = -0.59,
p = 1.000) and high gene flow (Nm = 3.91) (Table 4). The FST values (p = 0.000) of Andigena_2
to Andigena_5 subpopulations were low, ranging from 0.031 (Andigena_3 vs. Andigena_5) to
0.080 (Andigena_2 vs. Andigena_3), and high among these subpopulations with Andigena_1
that ranged from 0.122 (Andigena_1 vs. Andigena_5) to 0.216 (Andigena_1 vs. Andigena_3)
(S4 Table). The Andigena population presented a high genetic diversity with averages of
Ho = 0.535, He = 0.319 and PIC = 0.269 (Table 3).

Morphological characterization of Andigena population

The MCA based on morphological traits among 624 Andigena accessions showed that the total
morphological variation was distributed in 73 dimensions, from which the three first dimensions
explained the 12.3% of the variation (Table 2). The first dimension was provided by tuber variables
as shape (GTS), color (PTSC) and primary skin intensity color (PTSIC). The second by berry color
(BC), secondary color (STFC) and distribution of tuber flesh (DSTFC) and all variables related
with flower (PFC, PFIC, SFC and DSFC). Finally, the primary tuber flesh color (PTFC), secondary
color (STSC) and distribution of skin tuber (DSTSC) and variables related to stem color (SC) and
berry shape (BS) contributed to the variation of the third dimension (Table 2).
The cluster analysis discriminated six morphological groups within the Andigena popula-
tion (Fig 5). Although all the groups presented flesh tubers cream, in every group the largest
proportion of accessions was characterized by specific tuber traits (S5 Table). Group 1 (108
accessions) is characterized to present compressed tubers with pale yellow skin and purple
dots. Group 2 (59 accessions) had compressed tubers with dark purple skin, sometimes with
scattered yellow spots and flesh cream color with secondary purple color distributed in narrow
vascular ring. Group 3 (119 accessions) had compressed tubers with dark red skin. Group 4
(32 accessions) had compressed tubers with pale purple skin. Group 5 (159 accessions) had
round tubers with dark purple skin with scattered yellow spots. Finally, compressed tubers
with pale purple skin and yellow scattered spots are characteristics of group 6 (147 accessions).
Group 4 presented white flowers while the other groups presented dark purple flowers.

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Fig 3. Population structure of 133 diploid accessions (Phureja population) of Colombian Central Collection of S. tuberosum. (A) Clustering
Structure analysis. (B) NJ-tree based on Nei´s genetic distances. (C) Discriminant Analysis Principal Component.
doi:10.1371/journal.pone.0173039.g003

Correlations among morphological, geographical and genetic data

The Mantel test showed no correlation between geographical distribution and morphological
(1.2%, p = 0.311), and geographical distribution and genetic data (4.2%, p = 0.111). However, a
low but significant correlation (13.2%, p = 0.001) was identified between all morphological var-
iables analyzed and the genetic data (Table 5). Additionally, the correlation analysis was imple-
mented for each morphological variable, independently. Within the 15 variables used, three
(BS, PTFC and SFC) were not correlated (p > 0.05), three (SC, STFC and DSTFC) were nega-
tively correlated (p < 0.05) and the remaining nine were positively correlated (p < 0.05). The
variables with higher correlation were those related with flower variables (PFIC: 18.0%, PFC:
12.9%, DSFC: 12.8%), general tuber shape (GTS: 9.4%) and primary (PTSIC: 12.8%, PTSC:
8.5%) and secondary color of skin tuber (DSTSC: 7.5%, STSC: 7.1%) (Table 2). The global cor-
relation between morphological and genetic data using only variables significantly correlated
was of 21.6% (p = 0.001) (Table 5). Although this correlation was low, the subpopulations iden-
tified using molecular markers were characterized by presenting tuber traits in common. For
instance, the tuber skin primary color of Group 1 and group 2 is dark purple, group 3 is pale

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Fig 4. Population structure of 676 tetraploid accessions (Andigena population) of Colombian Central Collection of S. tuberosum. (A)
Clustering Structure analysis. (B) NJ-tree based on Nei´s genetic distance. (C) Discriminant Analysis Principal Component.
doi:10.1371/journal.pone.0173039.g004

yellow, group 4 is pale purple and group 5 is dark red. However, morphological and genetic
groups did not completely match.

Linkage disequilibrium
The linkage disequilibrium between pairwise SNPs was estimated for Phureja and Andigena
populations; the analysis showed that the amount of SNPs in LD and the extent of LD differed
among these.
Linkage disequilibrium in Phureja. The LD in Phureja was estimated using data from
the entire population (133 accessions) and separately for the subpopulation Phureja_1. The
analysis was not conducted in the subpopulations Phureja_2 and 3, because they presented a
low number of samples. In this analysis the 2555 markers used, mapped on the 12 chromo-
somes of the genome, with a mean distance between markers of 22.7 Mb, ranging from 11.5
Mb (Chr. 2) to 34.2 Mb (Chr. 1). The Pearson r2 values for the 133 Phureja accessions were
0.463 for linked markers with 49.8% of the markers in significant LD. The r2 values ranged
from 0.440 (Chr. 1) to 0.496 (Chr. 12) (Table 6). The pairwise correlations among linked mark-
ers in significant LD (p < 0.001) were used to assess the extension of LD decay. The threshold
for r2 was 0.45 representing the 90th percentile of all pairwise correlations in the Phureja popu-
lation. Using this threshold, the LD declined to 3.5 Mb for linked markers in the population
Phureja. For each chromosome of the potato genome the LD decay was estimated and ranged
from 2 Mb (Chr. 1, 4, 11) to up to 9 Mb (Chr. 3, 12) (Table 6).

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Fig 5. Dendrogram generated by the Ward method and Gower distances between 624 tetraploid accessions (population Andigena) of the
Colombian Central Collection of S. tuberosum based on qualitative morphological data.
doi:10.1371/journal.pone.0173039.g005

Table 5. Correlations between genetic, geographical and morphological distances in populations of the Colombian Central Collection of S.
tuberosum.
Analyses Percentage of correlation (%) p-value
Phenotypic_distance_Andigena/Geographic_distance_Andigena 1.20 0.311
Genetic_distance_CCC/Geographic_distance_CCC 4.20 0.111
Genetic_distance_Andigena/Phenotypic_distance_Andigena (All variables) 13.2 0.001*
Genetic_distance_Andigena/Phenotypic_distance_Andigena (variables correlated) 21.6 0.001*

* Significance at p  0.05 at 1000 permutations.

doi:10.1371/journal.pone.0173039.t005

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Table 6. Linkage disequilibrium in populations of the Colombian Central Collection of potato.

Phureja population Andigena population
ChrA Number of Mean r2 Significant LD LD decay Number of Mean r2 Significant LD LD decay
Markers distance (Mb) (%)B (Mb)C markers distance (Mb) (%)B (Mb)C
1 340 34.2 0.440 49.4 2 553 31.6 0.242 50.5 0.8
2 192 11.5 0.464 49.2 4 403 9.5 0.257 50.3 1.8
3 167 21.7 0.484 49.1 9 351 11.9 0.247 50.7 1
4 354 24.9 0.446 49.4 2 537 17 0.234 50.7 0.3
5 156 21.6 0.487 50.1 5.5 350 16.5 0.252 50.3 0.4
6 225 23.5 0.486 50.2 8 411 18.6 0.280 50.0 4
7 141 28.2 0.477 49.0 4.5 432 23.5 0.277 49.9 4
8 186 19.2 0.469 49.2 6 349 15.1 0.283 50.4 8
9 252 24.8 0.468 48.7 7 439 20.3 0.239 50.1 0.4
10 202 23.3 0.468 49.9 3.5 318 16.6 0.263 50.3 1.7
11 192 17.8 0.466 49.8 2 309 13.1 0.267 50.8 0.8
12 148 21.4 0.496 49.7 9 291 21.6 0.255 50.0 0.5
Linked 2555 22.7 0.463 49.8 3.5 4743 17.9 0.256 50.1 0.8
markers
A
Chromosome Number
B
Significant threshold is set to p < 0.001
C
Threshold in 90th percentile (Phureja: r2 = 0.45; Andigena: r2 = 0.25).

doi:10.1371/journal.pone.0173039.t006

Linkage disequilibrium in Andigena. The LD of the Andigena population was calculated

using 4743 molecular markers distributed over the 12 chromosomes identified in 652 acces-
sions corresponding to the Andigena subpopulations except for subpopulation Andigena_1.
In Andigena population the SNPs mapped on the chromosomes had a mean distance between
markers of 17.9 Mb ranging from 9.5 Mb (Chr. 2) to 31.6 Mb (Chr. 1). The LD was not esti-
mated for each subpopulation independently, because these groups did not differ genetically.
The subpopulation Andigena_1 was excluded of the analyses because it presented a high
genetic differentiation from the others. In addition, the LD in this subpopulation was not inde-
pendently assessed because it was represented by a low number of samples. The average Pear-
son r2 values obtained was 0.256 for linked markers with 50.1% combinations of markers in
significant LD. The mean r2 value in the 12 chromosomes ranged from 0.234 (Chr. 4) to 0.283
(Chr. 8). To estimate the LD decay in the Andigena population, the r2 threshold was 0.25 rep-
resenting the 90th percentile of the all pairwise Pearson correlations. The extent of LD was 0.8
Mb in linked markers and every chromosome ranged from 0.3 Mb in chromosome 4 to 8 Mb
in chromosome 8 (Table 6).

Association mapping analyses

The marker-phenotype association analysis was implemented using 4666 polymorphic SNPs
of 463 tetraploid accessions of the CCC. A complete dataset of the phenotypic variables was
available for these accessions. A total of 23 markers with log10 (p-value) ranging between 4.6
for STFC (solcap_snp_c1_12945) and 9.36 for PFC and PFIC (solcap_snp_c2_43970), were
significantly associated with 9 of the 15 evaluated variables (Table 7). In addition, seven mark-
ers presented significant p values less than 0.01 and four had p values less than 0.001. Of these
four markers, three (solcap_snp_c2_45693, solcap_snp_c2_23347 and solcap_snp_c2_43970)
were associated with PFC and PFIC and one (solcap_snp_c2_45235) with STFC (Table 7).

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Table 7. List of SNPs associated to qualitative data in tetraploid accessions of S. tuberosum of the Colombian Central Collection.
Variable Variable Marker Chromosome Position log10(p- Model1 Significance
abbreviation (bp) value)
Stem Color ST solcap_snp_c2_46710 7 4874743 5.16 SD *
solcap_snp_c2_36061 4 58752313 5.09 DD *
Primary Tuber Skin Intensity Color PTSIC solcap_snp_c2_21750 2 25053972 5.29 AD *
solcap_snp_c2_51533 7 53656225 5.05 DD *
Distribution of Secondary Tuber DSTSC solcap_snp_c2_45235 10 58437496 6.62 AD ***
Skin Color
Primary Tuber Flesh Color PTFC solcap_snp_c2_12578 7 53077509 4.75 SD *
Secondary Tuber Flesh Color STFC solcap_snp_c2_35705 2 47327646 5.33 SD **
solcap_snp_c1_12945 4 57899405 4.6 SD *
Distribution of Secondary Tuber DSTFC solcap_snp_c2_15070 2 45695772 4.64 SD *
Flesh Color
General Tuber Shape GTS solcap_snp_c2_26014 7 50155390 4.74 DD *
Primary Flower Color PFC solcap_snp_c2_24563 12 243727 4.76 SD *
solcap_snp_c1_5388 3 3161621 4.94 DD *
solcap_snp_c2_23355 7 42119766 5.5 DD **
solcap_snp_c2_46329 7 48224240 6.36 DD **
solcap_snp_c1_9878 7 50960978 5.86 DD **
solcap_snp_c2_45693 10 51357092 6.53 DD ***
solcap_snp_c1_16169 1 44194124 5.29 DD *
solcap_snp_c2_23347 7 42120645 7.01 DD ***
Primary Flower Intensity Color PFIC solcap_snp_c1_4464 5 32104744 5.53 DA **
solcap_snp_c2_45701 3 43326802 5.19 DD *
solcap_snp_c2_42348 6 36881223 5.88 DD **
Primary Flower Color / Primary PFC/PFIC solcap_snp_c2_36468 3 38153740 5.87/4.85 DA/SD **
Flower Intensity Color solcap_snp_c2_43970 1 13545189 9.36/5.29 DD ***
1
Model with the most significant marker is listed. AD = additive, SD = simplex dominant, DD = duplex dominant, DA = diplo—additive. Significant
* < 0.05
** < 0.01
*** <0.001

doi:10.1371/journal.pone.0173039.t007

Discussion
The growth in food demand and climate change raised the necessity to generate crop varieties
having higher yield and adapted to a changing environment [58]. It is fundamental to plant
breeding to characterize the genebank collections because the genetic improvement of eco-
nomically important traits depends on the genetic diversity available within the crop species
and its wild relatives [59, 60]. Modern elite gene pools could be created exploring the genetic
resources conserved in large ex situ germplasm collections to identify genes of interest and
allelic diversity [61, 62]. Highly polymorphic molecular markers could be identified in diverse
germplasm that could be effectively used for mapping genes or QTLs [62] to assist plant breed-
ing programs.
In Colombia, the CCC contains potato accessions coming from different Colombian
regions and several countries. Researchers from CORPOICA had selected accessions from the
CCC presenting valuable traits such as resistance to drought, to several diseases and to insect
pests. Information about the genetic diversity and population structure of the CCC and the
identification of molecular markers related to traits of interest for potato breeding could speed

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

up the selection for desirable traits. So far, only one study of the genetic diversity and popula-
tion structure of the CCC-Universidad Nacional de Colombia has been published [18]. The
analysis included only 97 diploid accessions, from which few are in common with the CCC-
CORPOICA [18]. The accession numbers of the CCC-Universidad Nacional de Colombia
were modified and do not correspond to the accessions numbers of the CCC-CORPOICA, dif-
ficulting the comparison between studies. The present study is the first report using the major-
ity of accessions of the CCC to assess its genetic variability, population structure and linkage
disequilibrium. The information obtained will allow the implementation of association-map-
ping studies to this collection.

Genetic analyses
The development of SNP arrays using high-throughput technology has allowed to genotype
germplasm of crops such as potato [20, 19], tomato [63], barley [64], rice [65] among others.
In this study, the Infinium SolCAP 8K was used to genotype accessions of the CCC, providing
informative data with 72% of polymorphic loci. Previous studies in potato germplasm of other
collections reported similar level of polymorphism using the same array: 77% [9], 74% [22],
61% [23], 67% [25], and 76% [66]. A degree of ascertainment bias could be expected when the
SolCAP 8K is used to analyze populations such as the Colombian potato germplasm because it
was designed based on transcriptome data and EST databases of North American cultivars [19,
21]. However, the high percentage of polymorphism suggested that the array provided enough
markers representing the allelic composition of the CCC compared to previous works in other
germplasm using the same array [22, 23]. A high number of polymorphic markers was
expected due to the significant number of samples included [20].
This paper presents a robust analysis of the genetic diversity of CCC using a high number
of molecular markers distributed on the 12 chromosomes of the potato genome. A previous
genetic study using only 97 diploid accessions and 42 SSR covered a small amount of the
potato genome, with a mean coverage of three markers per chromosome [18]. In general, the
highest proportion of genetic studies in potato have used techniques that produced few molec-
ular markers such as SSR [67–69], AFLPs [34, 59, 70] and RAPDs [71–73]. Each type of molec-
ular marker provides information not always comparable because some have a biallelic and
others a multiallelic nature [7, 74]. However, the estimation of the genetic variability of a popu-
lation improves as the number of markers increase [75]; the SolCAP 8K could then provide a
better assessment of the genetic variability of the CCC.

Population structure and genetic diversity in the Colombian Central

Collection
In this study, the molecular markers were useful to identify mislabeled accessions [7, 23]; some
accessions of Andigena and Phureja did not clustered according to their passport data. The
impossibility to identify two different populations of Phureja and Chaucha suggested an error
of classification in the CCC. According to Guevara [36], accessions of the CCC labeled as
Chaucha are not triploids as expected but diploids (2n = 2x = 24) as Phureja accessions [37].
Hence these accessions were probably misclassified as Chaucha, being in fact Phureja. The
misclassification of accessions and errors in the assignment of samples to corresponding
group in the CCC could have several explanations. The common name used by farmers for the
same type of potato probably changes from region to region. For example, in the state of Nar-
iño in Colombia farmers use the name Chauchas for potatoes similar to Phurejas. Another
explanation could be a hybrid origin of these accessions; natural hybridization occurs between

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

varieties in cultivated areas because potato farmers do not cultivate the varieties separately [4,
76].
Population structure and genetic diversity in Phureja and Andigena populations. The
two inferred populations of CCC present high genetic diversity and were genetically differenti-
ated with low gene flow among them, probably due to the difference in ploidy level [35]. The
SolCAP array was also able to differentiate European [22] and American [23] potatoes by their
ploidy level. The diploid population (Phureja) had high genetic differentiation, all the multi-
variate analyses supported the presence of the three subgroups and genetic admixture was no
identified. In fact, the results showed a low gene flow, suggesting a strong genetic differentia-
tion, given that Nm is inversely proportional to the genetic differentiation among populations
[77]. Human selection (e.g. breeders, farmers) to color and quality of tuber probably played an
important role shaping the current population structure of group Phureja. However, it is nec-
essary to conduct a morphological evaluation of Phureja potatoes of the CCC in order to sup-
port this hypothesis. The results obtained from Phureja population contrasted to the reported
in the study of Juyó et al. [18], who identified a moderate population structure (FST = 0.09), a
high gene flow (Nm = 1.61) and only 9.64% of the variation among populations in diploid
accessions of CCC-Universidad Nacional de Colombia. These two studies differed in the
molecular markers (number and type) and samples (number and origin) evaluated. Samples
analyzed in the two works were not exactly the same. Although the CCC-Phureja from the
Universidad Nacional de Colombia conserves part of the accessions of the CCC-CORPOICA,
the ID numbers did not match. In addition, some accessions of the CCC-Universidad Nacio-
nal were recently collected. Juyó et al. [18] used SSR markers, which are considered more effi-
cient than SNP markers to identify subpopulations, because they are neutral and more alleles
can be identified [78–79]. However, the high number of SNPs markers used in this study
allowed to identify three populations in Phureja accessions. The population structure is influ-
enced by the joint effects of many factors including the mating system, natural and artificial
selection, mutation, migration and dispersal mechanism, drift, etc. [80, 81]. In potato, the
selection of potatoes by farmers and breeders presenting characteristics such as high yield,
large tubers, low glycoalkaloid levels, desirable flavor, short cooking times and high nutritional
value could affect the genetic structure [82–84].
Andigena population presents a genetic admixture supported by a high gene flow among
populations [85]. The lack of population structure in tetraploid potatoes has been previously
reported in other studies [35, 86–88] and has been explained by sexual polyploidization, inter-
varietal introgressive hybridization and long-distance dispersion [5, 89]. Although the whole
Andigena population did not show a population structure, a cluster (Andigena_1) with sam-
ples probably belonging to the Tuberosum group could be identified. The S. tuberosum group
tuberosum of CCC were probably originated from landraces and breeding material from
United States and Europe [12]. Tuberosum potatoes differentiate from other Andigena pota-
toes by the formation of tubers in long days and by their adaptation of medium altitudes and
subtropical weather from Europe, United States and Asia [8, 90].
High genetic diversity was found in both populations according with other studies [18, 68,
89]. In this work, the observed heterozygosity was higher than expected heterozygosity. Potato
is an outcrossing species thus the proportion of inbreeding is expected to be low, thus the het-
erozygosity is higher than expected. The high diversity in potato is explained by its evolution
shaped by selection, migration, mutation, hybridization, polyploidization and introgression.
In the case of diploid potatoes, wild and cultivated species are often self-incompatible (SI) [91,
92]. Thus, potato genetics allow the production of heterozygote plants increasing the genetic
variability [1, 35, 81]. The PIC values suggested that the SNPs of SolCAP are useful to analyze
diploid and tetraploid accessions and could support the suggestion that genetic diversity in

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

tetraploid potatoes has not been narrowed in spite of the commercial breeding efforts [10, 34].
Based on PIC values, the CCC (PIC = 0.437) is more diverse than European potatoes
(PIC = 0.35), supporting the idea that South American potato populations are more diverse
than European potatoes reported by Bornet et al. [93] and Esfahani et al. [94]. According to
these results, the CCC has a broad genetic basis with alleles that could be profitable for plant
breeding [21]. In fact, studies in diploid accessions of the CCC-Universidad Nacional de
Colombia have already detected markers related to resistance to Phytophthora infestans [95],
sugar content and frying color [96].

Morphological characterization of Andigena population

The accessions of Andigena population showed wide phenotypic diversity based on fifteen
morphological traits, in which shape, skin color and color intensity of tuber and flower
attributes were the most informative variables to discriminate the six groups of Andigena.
Previous works reported that the same variables were useful to differentiate potato acces-
sions [97–99]. Variables describing the tuber are the most useful descriptors to select pota-
toes for breeding programs [100]. The dark color in skin and flesh tuber is an indicator of
the presence of phenolic compounds which are considered health-promoting phytochemi-
cals because of their antioxidant properties [101]. The CCC presents a wide variability in
tuber colors indicating a potential source of accessions with high phenolic compounds lev-
els; further characterization of content of biochemical compounds of the CCC is needed.
Previously, Bernal et al. [97] analyzed morphologically 464 accessions of the CCC of potato.
They found seven different groups instead of six groups and they identified higher morpho-
logical variability than the present study. However, the same traits were reported as infor-
mative in the two studies, and the samples were regrouped based on the same characters of
tuber and flower. The difference in results between studies could be due to a smaller num-
ber of variables used in this study. Additionally, the data analysis made by Bernal et al. [97]
was based on one year of morphological records. In contrast, the present study used mor-
phological data recorded on eight different years. Our analyses identified that some descrip-
tors changed over the years such as color and intensities of tubers and flowers. The lack of
stability of morphological characters has been also reported in the evaluation of the CIP
collection [5] suggesting that the selection of potato materials could not be only based on
morphological data. The characterization and selection of potato accessions should be com-
plemented with molecular data, reported to be more informative and neutral than pheno-
typic traits in establishing potato relationships [62].

Correlations among morphological, geographical and genetic data

In this work, geographic distance was not correlated between genetic and morphological dis-
tances. Similar results were obtained in previous studies using potato collections for morpho-
logical data [102, 103] and molecular data [7, 104–106]. The lack of correlation is probably the
result of tuber transportation by humans [107], caused by historical migrations of wild potato
germplasm away from their regions of origin [23]. Morphological and genetic data were
weakly correlated; similar results were found in other populations of potato [108–110]. The
low correlation between genetic and morphological data is probably due to differences in
selection pressure. Non-adaptive molecular markers are usually not subjected to natural or
artificial selection while phenotypic characters are subjected to selection pressure and influ-
enced by the environment [106, 111]. This result could explain why groups identified through
molecular and morphological markers did not match.

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Linkage disequilibrium and association mapping analyses

The linkage between molecular markers and phenotypic polymorphisms is required for the
association mapping of genes or QTLs underlying traits of interest [112]. The extent of LD can
be affected by factors such as genetic drift, population structure and selection [113]. In associa-
tion mapping studies, a key factor is to know the population structure in order to improve the
statistical power and decrease the false positive rate in gene discovery [76]. The analysis of LD
was independently conducted for Phureja and Andigena, where the LD levels varied between
these. High levels of r2 and SNP pairs with significant LD in Phureja and Andigena were iden-
tified. These results contrasted with the study of Juyó et al. [18] in diploid potatoes in which no
molecular markers in significant LD were detected, probably due to the number and type of
markers used (SSRs). Additionally, the number of linked markers in LD was higher than
unlinked markers as expected, thus physical linkage strongly influences LD. The results indi-
cate that molecular markers found in CCC in this study are suitable for an association analysis
[114].
To estimate the LD decay in Phureja and Andigena populations, a r2 threshold of 0.45
(Phureja) and of 0.25 (Andigena) were used. Those values corresponded to the 90th percentile
of the distribution of all pairwise Pearson correlation in each population. Vos et al. [56] found
that percentiles of 90 or 95 are useful to estimate the LD in potato. The difference in cutoff
used in previous studies (r2 = 0.1) did not allow the comparison among studies [22, 34, 35, 95].
However, the LD decay values obtained in this work in tetraploid potatoes were similar to the
reported in the potato germplasm (0.6–1.5 Mb) analyzed by Vos et al. [56]. The r2 values and
extent of LD through the genome differ among studies because of differences in population
size, number and type of markers [115] and the regression methods used to measure the LD
[116]. The polyploidy and outcrossing species generally exhibit low LD because of the recom-
bination events, which occur more frequently in large and highly heterozygous populations
[117]. In contrast, the self-pollinated crops usually display LD over larger distances as a conse-
quence of their mating system [34]. Based on its LD value, potato behaves as a self-pollinated
crop even if it is an outcrossing species. The clonal propagation of potato limits the number of
meiotic generations and in consequence the recombination events [33–35, 118]. The LD in
Andigena and Phureja decayed slowly, previous works also reported a slow LD decay for
potato populations: 1 cM [33], 10 cM [35] and 5 cM [34]. It is not rare to found differences in
values of LD decay among populations that have suffered different breeding history and
human selection [119, 120].
The LD decay value is useful to design future GWAS studies; it makes possible to estimate
the minimum number of SNPs required to have a successful GWAS [115]. Since Phureja and
Andigena populations have a long range LD through the genome, with a physical genome
length of 844 Mb [121, 122] and a genetic map length of 800 cM [123], association studies can
be performed with a modest number of markers per unit of genetic distance, this inference in
potato has been reported previously by D’hoop et al. [34] and Simko et al. [35]. The inferences
about the association mapping in the CCC of potato were validated with the identification of
molecular markers associated with the morphological traits. In a GWAS analyzing North
American potatoes using the same array, molecular markers with minor effects were identified
to be related to morphological data such as total yield, eye depth, tuber shape and tuber length
[27]. In the present work, four of 23 associated markers presented p values less than 0.001. Of
these four markers, three were associated with flower primary color and one with secondary
color distribution in tuber skin. The marker solcap_snp_c2_45235 (Chr. 10, position:
58437496) was associated to secondary color and was mapped to the gene Sotub10g021050.1.1
(PGSC0003DMG400008137) which has a glucosyltransferase function. Some

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 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

glucosyltransferase enzymes are implicated in the production of anthocyanin, pigment com-

pound of skin and flesh tubers [124]. In addition, the same SNP (solcap_snp_c2_45235) is
located closed to two genes (PGSC0003DMG400013965, PGSC0003DMG400012891) associ-
ated to skin and flesh color of potato tuber, reported recently by Endelman and Jansky [125].
The SNP dataset produced in this study and the germplasm analyzed would allow the
implementation of association-mapping studies and to detect markers or genes associated to
traits of interest useful for potato breeding such as resistance to pathogens and insect pests, tol-
erance of abiotic stresses and tuber quality. The function of the associated markers should be
validated through genetic transformation. Additionally, conventional potato plant breeding
programs could be supported using the genetic information through marker-assisted selection
(MAS) and genomic selection (GS), and thus to accelerate the selection of potato materials and
reduce the cost and time to develop new potato varieties.

Conclusion
The present study is the first report of phenotypic and genotypic evaluations of the Colombian
Central Collection of Solanum tuberosum using morphological and SNP molecular markers.
The study identified high levels of genetic diversity and genetic differentiation in diploid and
tetraploid potatoes. CCC constitutes a potential source of variable traits useful for a genetic
breeding program. Additionally, the linkage disequilibrium study of the CCC indicated that
the genomes of Phureja and Andigena presented an elevated number of SNP pairs in signifi-
cant LD and a slow LD decay, suggesting that with a modest number of molecular markers, a
marker-phenotype association could be detected. The information obtained in this work
allowed to conclude that the CCC is a germplasm with a broad genetic base and is useful to
conduct association mapping studies suitable for the identification of QTLs/genes associated
to quality traits and biotic and abiotic stress tolerance traits.

Supporting information
S1 Fig. Delta K inferred in each analyzed population. (A) Overall Colombian Central Collec-
tion. (B) Phureja population. (C) Andigena Population.
(TIF)
S1 Table. List of accessions of the Colombian Central Collection of S. tuberosum group
Andigenum and information of sample collection sites.
(DOC)
S2 Table. Genotypic data of 809 accessions of Colombian Central Collection of S. tubero-
sum group Andigenum obtained through Infinium technology.
(XLSX)
S3 Table. Phenotypic data of Colombian Central Collection of S. tuberosum group Andi-
genum (Andigena population).
(XLSX)
S4 Table. Pairwise genetic differentiation (FST) values between populations of S. tuberosum
in the Colombian Central Collection.
(DOC)
S5 Table. Summary of characteristics for the six groups identified by the morphological
analysis in tetraploid accessions of the Colombian Central Collection of S. tuberosum.
(DOC)

PLOS ONE | DOI:10.1371/journal.pone.0173039 March 3, 2017 20 / 27

 Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum

Acknowledgments
We thank the Potato Germplasm Bank of CORPOICA for providing the germplasm material
and associated information of the CCC used in this study. The authors thank Ivania Cerón for
assistance in revising the final version of the manuscript. This study was funded by the Colom-
bian Ministry of Agriculture.

Author Contributions
Conceptualization: JB-C L-SB RY.
Data curation: JB-C RIV.
Formal analysis: JB-C.
Investigation: JB-C RY RIV.
Methodology: JB-C ES-B.
Project administration: JB-C RY L-SB.
Supervision: RY.
Validation: JB-C.
Visualization: JB-C.
Writing – original draft: JB-C RY.
Writing – review & editing: JB-C RY RIV ES-B L-SB.

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