RESEARCH ARTICLE Open Access
Genetic diversity of Phytophthora infestans in the
Northern Andean region
Martha C?rdenas1*, Alejandro Grajales1, Roberto Sierra1, Alejandro Rojas1, Adriana Gonz?lez-Almario1,
Angela Vargas1, Mauricio Mar?n2, Gustavo Ferm?n3, Luz E Lagos4, Niklaus J Gr?nwald5, Adriana Bernal1,
Camilo Salazar6,7?, Silvia Restrepo1?
Abstract
Background: Phytophthora infestans (Mont.) de Bary, the causal agent of potato late blight, is responsible for
tremendous crop losses worldwide. Countries in the northern part of the Andes dedicate a large proportion of the
highlands to the production of potato, and more recently, solanaceous fruits such as cape gooseberry (Physalis
peruviana) and tree tomato (Solanum betaceum), all of which are hosts of this oomycete. In the Andean region,
P. infestans populations have been well characterized in Ecuador and Peru, but are poorly understood in Colombia
and Venezuela. To understand the P. infestans population structure in the Northern part of the Andes, four nuclear
regions (ITS, Ras, ?-tubulin and Avr3a) and one mitochondrial (Cox1) region were analyzed in isolates of P. infestans
sampled from different hosts in Colombia and Venezuela.
Results: Low genetic diversity was found within this sample of P. infestans isolates from crops within several
regions of Colombia and Venezuela, revealing the presence of clonal populations of the pathogen in this region.
We detected low frequency heterozygotes, and their distribution patterns might be a consequence of a high
migration rate among populations with poor effective gene flow. Consistent genetic differentiation exists among
isolates from different regions.
Conclusions: The results here suggest that in the Northern Andean region P. infestans is a clonal population with
some within-clone variation. P. infestans populations in Venezuela reflect historic isolation that is being reinforced
by a recent self-sufficiency of potato seeds. In summary, the P. infestans population is mainly shaped by migration
and probably by the appearance of variants of key effectors such as Avr3a.
Background
The potato and tomato late blight pathogen, Phy-
tophthora infestans (Mont.) de Bary [1], has a broad
host range within the Solanaceae family including Sola-
num phureja (yellow potato), S. betaceum (tree tomato),
S. quitoense (naranjilla or lulo), Physalis peruviana (cape
gooseberry), and other wild species [2,3]. Since the Irish
potato famine in the 19th century, this pathogen has
been thoroughly studied because of its severe economic
impact on agriculture, causing billion-dollar losses
annually [4].
Mitochondrial and nuclear DNA regions of P. infes-
tans have been extensively used in different regions of
the world to investigate its evolutionary history and
population structure [5-7]. This has led to the definition
of lineages on the basis of molecular data, which has
allowed the monitoring of populations and resulted in
the generation of important epidemiological inferences
about the pathogen?s migration [5,6]. Additionally,
population studies of P. infestans have been conducted
to elucidate the origin and diversity of isolates from cul-
tivated species [5,8-10] and to determine if there are any
differences between populations from wild and culti-
vated hosts [11-13].
Population studies of P. infestans in the Andean
region have been focused on Peru and Ecuador. These
two countries are considered to be the center of origin
of potatoes, [14] and thus some authors have proposed
that this region is also the center of origin of the
P. infestans pathogen [8,9]. In Ecuador, the pathogen
* Correspondence: martcard@uniandes.edu.co
? Contributed equally
1Universidad de Los Andes, Bogot? D.C., Colombia
Full list of author information is available at the end of the article
C?rdenas et al. BMC Genetics 2011, 12:23
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? 2011 C?rdenas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
has shown low levels of diversity, and its population
structure is strongly influenced by host preference. Each
P. infestans clonal lineage is associated with a different
host group: US-1 lineage with tomato, EC-1 with potato,
EC-2 with wild solanaceous species, particularly with
the Anarrhichomenum section, and EC-3 with S. beta-
ceum [15-17]. Additionally, genetic differentiation is
found among isolates of P. infestans associated with
S. ochranthum [18]. Geographically, no clear subdivision
was found in Ecuador when using allozymes and RFLP
markers [15]. A different pattern was obtained in Peru,
where the lineages are neither structured according to
any host species [19] nor according to the origin of the
hosts, be they cultivated or wild [20]. Other population
studies have documented higher diversity in Peru than
in Ecuador, according to the number of genotypes
found [11]. According to mitochondrial haplotypes,
P. infestans has been classified into four main groups:
Ia, Ib, IIa and IIb [7,21,22]. Each mitochondrial haplo-
type has been used to complement the definition of P.
infestans lineages. The US-1 lineage has been related to
the Ib haplotype, the US-6 to the IIb haplotype, the EC-
1 to the IIa haplotype and the EC-2 and EC-3 lineages
to the Ia haplotype. Recently a new mitochondrial hap-
lotype, Ic, has been also related to lineage EC-2, and has
been correlated with a new species, P. andina [8,23].
Colombia is considered to be the fourth largest potato
producer in Latin America with a production of 1.9 mil-
lion tons and a cultivated surface of 110,000 hectares in
2007 [24]. However, P. infestans population studies are
scarce [25,26]. These studies showed that the P. infes-
tans population revealed low genetic diversity, with no
evidence of sexual reproduction, and consisted mostly of
the A1 mating type. It should be noted that the A2 mat-
ing type was recently detected, but only in one isolate
[26], and a posterior intensive sampling in this site
could not detect the A2 mating type again [26]. To this
date, only one study has investigated the population
structure of P. infestans in Venezuela [27]. Amongst the
sample over this time period, only A1 mating type was
reported.
This is the first study that makes use of nuclear and
mitochondrial regions to conduct a population genetic
analysis of the pathogen in the North Andean region.
Furthermore, sequence diversity at the Avr3a avirulence
(effector) gene was examined to investigate whether
populations are subject to selection at this locus. Only
two alleles have been described for the Avr3a locus
according to virulence and avirulence against the S.
demissum R3a gene [28]. Since effector genes affect
pathogenicity and fitness, they may be under specific
selective pressures after mutation dissemination through
genetic drift, responding to host populations or other
local factors. Local adaptation seems to be playing an
important role in the diversification and distribution of
the effectors alleles/haplotypes in natural and agricul-
tural environments [29]. Thus this locus could be infor-
mative about the P. infestans adaptive history.
Our goals were to conduct sequencing of different
genic regions for populations of P. infestans from the
Northern Andes to describe the geographical distribu-
tion of diversity. We examined genes likely to be selec-
tively neutral or under selection pressure in order to
evaluate the importance of selection, drift and gene flow
in specific genic regions. Additionally, these analyses are
useful in comparing population parameters of the
Northern Andes populations of the pathogen with other
populations previously described in different regions
worldwide.
Methods
Strains and Culture Conditions
A total of 80 strains from Colombia and Venezuela were
included in this study (Additional files 1 and 2). Sixty-
eight Colombian strains were obtained from different
states, including Cundinamarca (N = 23), Antioquia
(N = 18), Nari?o (N = 15) and Boyac? (N = 12), isolated
from S. tuberosum, S. phureja, S. lycopersicum, S. beta-
ceum, S. quitoense, and P. peruviana. All the strains
from Cundinamarca and seven from Antioquia were
previously characterized using mitochondrial haplotype,
metalaxyl resistance, isoenzymes analysis (Gpi and Pep)
and restriction fragment length polymorphism with
probe RG57 (Additional file 1) [26]. Venezuelan strains
(N = 12) were selected from a collection of more than
500 strains isolated from S. tuberosum [27] from the
three Andean states of M?rida, T?chira and Trujillo. All
isolates were collected from commercial crops. All the
isolates were identified as P. infestans sensu lato by ITS
sequencing. Additionally, eight isolates from Ecuador,
kindly provided by Michael Coffey, were also included
for the diversity analysis. Isolates US940480 (mating
type A2) and US940494 (mating type A1), provided by
William E. Fry (Cornell University), were used as refer-
ence strains. In general, all isolates were cultured on rye
medium and incubated at 18?C for eight days in the
dark [30].
DNA Amplification and Sequencing
DNA extraction was performed as previously described
[31]. Four nuclear loci, ITS, b-tubulin, Ras and Avr3a,
as well as one mitochondrial gene, cytochrome c oxidase
1 (Cox1) were amplified and sequenced. For ITS, Inter-
nal Transcribed Spacer 1, 5.8 S rDNA and Internal
Transcribed Spacer 2 were amplified using ITS4 and
ITS5 primers yielding a 950 bp product [32]. Primers
TUBUF2 and TUBUR1 were used to amplify a 989-bp
portion of the b-tubulin gene, excluding introns [33].
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The Ras sequence was obtained after two independent
amplifications. IRF and IRR primers were used to
amplify a 223-bp region corresponding to intron 1.
Additionally, RASF and RASR primers amplified a 600-
bp product corresponding to partial exons 3 and 6,
complete exons 4 and 5, and complete introns 3 to 5
[8,34]. Avr3a was obtained after modification of pre-
viously described primers [28] with an M13 tail to allow
for amplification and sequencing of 453 bp correspond-
ing to the entire gene (M13Pex F: 5?-GTAAAAC-
GACGGCCAGCCATGCGTCTGGCAATTATGCT-3?
and M13Pex R: 5?-CAGGAAACAGCTATGACCTG
AAAACTAATATCCAGTGA-3?). COXF4N and COX
R4N primers were employed to amplify 972 bp of the
mitochondrial gene Cox1 [33].
For all primer combinations the reaction conditions
were the following, in a final volume of 25 ?l: 1? PCR
buffer, 0.5 mM dNTPs, 2.5 mM MgCl2, 0.2 mM of each
primer, 1U Taq DNA polymerase. PCR conditions for
ITS and Ras started with an initial denaturation step of
96?C for 2 min (1 min for Ras), 35 cycles of 96?C for
1 min, 55?C for 1 min (56?C for Ras), 72?C for 2 min, and
a final step of 10 min at 72?C. For b-tubulin and Cox1,
the conditions started with a denaturation step of 2 min
at 94?C, followed by 35 cycles of 94?C for 30 s (1 min for
Cox1), 60?C for 30 s (45 s for Cox1) 72?C for 1 min and a
final step of 10 min at 72?C. For Avr3a, the amplification
conditions included a denaturation step of 2 min at 94? C
followed by 15 cycles of 30 s at 94?C, 30 s at 55?C and
1 min at 72?C. Twenty-five additional cycles of 30 s at
94?C, 30 s at 62?C and 1 min at 72? C were added. The
final extension was performed for 10 min at 72?C. The
amplification products were separated on 1% agarose gels
and visualized by staining with ethidium bromide. Single
band PCR products were sequenced in Macrogen, Korea.
Sequence assembly and editing was performed manually
on the CLC DNA Workbench http://www.clcbio.com.
Sequence alignments were performed using MUSCLE
[35]. Due to the high heterozygosity condition of P. infes-
tans, double peaks were assigned with the IUPAC code
when necessary.
Haplotype reconstruction was conducted using
DNAsp v4.90.1 [36] implementing the algorithm pro-
vided in PHASE [37]. Basically, PHASE assigns a prob-
ability of the correct inference of haplotype phase at
every heterozygous position. PHASE simulations were
repeated four times for each locus, two without recom-
bination and two with recombination. Each simulation
was run with 5,000 iterations. In all cases, the most
common output inferring haplotypes with >95% confi-
dence was accepted. All the haplotypes sequences were
deposited in GeneBank under accession numbers [Gen-
Bank: GU258154 and GU258156] for Ras, [GenBank:
GU258157-GU258165; GU258167, GU258168] for
b-tubulin, [GenBank: GU258058 and GU258059] for
Cox1 and [GenBank:GU258052-GU258057] for Avr3a.
Additionally, sequences for ITS were deposited for
all the strains employed under accession numbers
[GenBank:GU258061-GU258072, GU258074-GU258077,
GU258080-GU2580125, GU2580127-GU2580152].
Gene Networks and Population Genetic Analyses
Different datasets were generated for each DNA region.
Additional sequences of ITS, b-tubulin, Ras and Cox1,
from P. infestans isolates collected worldwide were
retrieved from GenBank and included in the analyses
(additional file 3). The genealogical relationships among
haplotypes were established by statistical parsimony
with a 95% connection limit as is implemented in the
TCS software version 1.21 [38].
Nucleotide diversity (?), nucleotide substitution rate
(?) and haplotypic diversity (HD) were calculated for
each data set from the Northern Andean region, includ-
ing Colombia and Venezuela (NA) and for the rest of
the world, corresponding to available sequences from
other geographical origin (R) using DnaSP v.4.90.1 [36].
For the worldwide data set (W) the combination of the
NA and R data sets was used (W = NA +R. The popula-
tion genetics analyses were performed only with the NA
dataset.
To test the hypothesis of genetic structure (complete
Panmixia vs. geographically structured populations)
among these populations, a permutation test for Hud-
son?s statistics [39] was performed with SNAP Work-
bench [40]. Sequences were collapsed into haplotypes
recoding indels and excluding infinite-sites violations
using MAP TOOL [40]. Then, a distance matrix was
generated with SEQTOMATRIX [40]. This matrix was
used to perform a non-parametric permutation test with
PERMTEST [40] under default parameters.
Tajima?s D [41] was used as a test of neutral evolution
for b-tubulin, Ras, Avr3a and Cox1 with DnaSP v.4.90.1
[36]. A mismatch distribution analysis was carried out
with the statistic R2 [42] to establish population size
changes as is implemented in DnaSP v.4.90.1 [36]. To
assess a confidence interval for R2 value observed, a
coalescent simulation with 1000 replicates was run with
theta (?) and no recombination as starting parameters.
The software SITES [43] was used to estimate the
number of fixed differences or shared polymorphisms
among the defined populations in the NA dataset, using
b-tubulin, Ras and Cox1. Fixed differences would be
indicative of isolation, while shared polymorphisms
would indicate gene flow. MIGRATE-n [44] was used to
estimate theta and the direction and amount of gene
flow among these populations, with nuclear (b-tubulin,
Ras) and mitochondrial (Cox1) genes. In each case, 20
short chains with 5000 sampled genealogies and five
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short chains with 50000 genealogies were run. Heating
was set to be active with four temperatures (1.0, 1.5, 2.5
and 3.0).
A pairwise comparison of the rates of nonsynonymous
nucleotide substitutions per nonsynonymous site (dN)
and synonymous nucleotide substitutions per synon-
ymous site (dS) were estimated for Avr3a using the
approximate method of Nei and Gojobori (1986) [45]
implemented in the YN00 program in PAML [46].
CODEML was employed to identify the amino acids
under strong diversifying selection. Avr3a sequences
were translated into amino acids and aligned using
P. sojae [GenBank: EF463064] as an outgroup. Details of
these tests are explained elsewhere [47].
Results
Summary statistics and Gene Networks
Tables 1 and 2 contain the summary statistics for the
analyzed sequences and haplotype information. The
number of sequences analyzed differed for each gene,
due to challenges with the amplification/sequencing of
specific isolates or availability of previously published
data (Table 1). The ITS region showed no polymorph-
ism in any of the 755 sites analyzed and was excluded
from further analyses (and from Table 1). The Northern
Andean region (Venezuela and Colombia) showed low
levels of nucleotide diversity and genetic variability
(Table 1). The regional distribution of the haplotypes is
shown in Figure 1. The highest number of haplotypes
was observed for b-tubulin. The haplotype most fre-
quently found (H1) was present in Colombia and Vene-
zuela. Venezuelan P. infestans showed only two
haplotypes, H1 and H8. The Central Andes showed four
haplotypes, one of them restricted to this zone (H10). In
the Eastern Andes three haplotypes were found for
b-tubulin, and H11 was only found in this region. The
Southwestern Andes showed the highest number of dif-
ferent haplotypes for this gene (9), five of them (H2, H3,
H4, H5 and H6) restricted to this area. For Ras, haplo-
type the same nomenclature was used as the one in
Gomez-Alpizar et al., 2007 [8] for comparison purposes.
The Ras region showed two haplotypes (H2 and H13)
with only one haplotype (H2) found in the Southwes-
tern, Central and Eastern Andes. Venezuelan isolates
were heterozygotic (H2/H13) and the H13 haplotype
was exclusive to this country. Cox1 showed two haplo-
types (H1 and H5), and H1 was present in all regions.
All isolates from the Central Andes belonged to only
this haplotype, which was also the most common one in
the Southwestern and Eastern Andes. H5 was the most
common haplotype in Venezuela (Figure 1). For Avr3a,
four haplotypes were found. The most frequent allele,
H1, was present in Colombia and Venezuela. H5 and
H6 were only found in the Southwestern Andes. H2 was
only found in the Eastern Andes. All isolates from
S. tuberosum corresponded to haplotype H1, which was
the most common haplotype. In S. betaceum the main
haplotypes were H1, H2, H5 and H6 (Additional file 1).
Gene Networks were constructed for each Northern
Andean region and for Colombia and Venezuela when
more than one haplotype was found, in order to deter-
mine the evolutionary relationship among them
(Additional files 4 and 5). As a general pattern, nuclear
and mitochondrial haplotypes present in the Eastern and
Central Andes as well as in Venezuela were only sepa-
rated from each other by one or two steps. However, in
the Southwestern Andes a different pattern was observed
for b-tubulin and Avr3a. Three haplotypes were found
for Avr3a: H1, H5 and H6. H5 and H6 were six and nine
steps distant from H1, respectively. For b-tubulin, nine
haplotypes were found and only five of them were related
to each other by one step. More than two steps separated
the other four haplotypes from each other.
Nucleotide diversity (?) and substitution rate were low
in all genes analyzed in the R and W datasets (Table 1).
Again, datasets were designated NA for Colombia and
Venezuela, R for the rest of the world, which corre-
sponds to available sequences from other geographical
origins, and W for worldwide, corresponding to the
combination of the two previous datasets (W = NA +R).
The Northern Andean region showed the same pattern
of low nucleotide diversity and genetic variability as the
rest of the world population (Table 1). Haplotypic diver-
sities were high as a consequence of heterozygous indi-
viduals showing alleles with low frequency. b-tubulin
showed the highest number of haplotypes, followed by
Avr3a, Ras and Cox1 (Table 1 and Figure 2). The
Northern Andean region shared with the rest of the
world three haplotypes for b-tubulin (H1, H7, H9), two
haplotypes for Cox1 (H1 and H5), and just one haplo-
type for Ras (H2) and Avr3a (H1).
The estimated gene networks for the W dataset
(Figure 2) showed as a general pattern a dominant hap-
lotype separated by few steps from the others. The net-
work representing the b-tubulin haplotypes showed a
dominant haplotype, H1, present in Central America
and South America (Figure 2A). H7 was present in Cen-
tral America, South America, North America, and
Africa. H9 was found in North and South America, as
well as in Europe (The Netherlands). The remaining
haplotypes were restricted to South America. The most
divergent clade was represented by isolates from S. beta-
ceum (data not shown). For Cox1, two haplotypes (H1
and H5) were widely distributed among regions and
connected directly in the network. The three remaining
haplotypes were derived from H5; two of them were
restricted to Central America (Mexico) and one, H4, to
South America (Ecuador) (Figure 2B). For the Ras gene,
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a dominant haplotype H2 was present in North Amer-
ica, Central America and South America, as well as in
Europe (Ireland) (Figure 2C). H2 was mainly associated
with S. tuberosum, although this species also hosted
other haplotypes. H13 was only found in South America
(Venezuela).
Population Genetic Analyses
To test the hypothesis of population subdivision, isolates
were first assigned to populations following their
geographical distribution on a regional scale. Then, per-
mutation tests for Hudson?s statistics were performed
for all combinations among regions (Table 3). Signifi-
cant genetic differentiation was found for Ras and Cox1,
between the Eastern Andes and Venezuela and between
the Central Andes and Venezuela (Table 4). For b-
tubulin, population subdivision was found between the
Central and Eastern Andes and between the Southwes-
tern and Eastern Andes (Table 3). Avr3a showed no
differentiation according to geographical location.
Tajima?s D test failed to reject the null hypothesis of
neutral evolution in Ras, b-tubulin and Cox1 (Table 4).
This indicates that, for these loci, the Northern Andean
population has not been subject to selection pressure.
Table 1 Summary statistics for loci Cox1, Ras, ITS, b-tubulin and Avr3a from P. infestans in the Northern Andean region
and worldwide
GEOGRAPHIC
REGION
LOCUS Sequences
(N)a
Length
(bp)
Segregating
SITES
GENETIC
VARIABILITY
(Segregating Sites/
Length)
?
b
?
SITEc
N
Haplotypes
HDd
NORTHERN ANDEAN (Colombia and
Venezuela)
Cox1 73 669 1 0.15 0.0004 0.0003 2 0.3
Ras 85 765 3 0.39 0.0008 0.0008 2 0.21
b-tub 90 815 14 1.72 0.0015 0.0036 11 0.47
Avr3a 80 444 12 2.70 0.0017 0.0055 4 0.12
Central Andes (Antioquia) Cox1 17 669 0 0 0 0 1 0
Ras 18 765 0 0 0 0 1 0
b-tub 20 815 4 0.49 0.0012 0.0014 4 0.56
Avr3a 16 444 0 0 0 0 1 0
Eastern Andes (Boyac?
Cundinamarca)
Cox1 35 669 1 0 0.00009 0.0004 2 0.057
Ras 35 765 0 0 0 0 1 0
b-tub 36 815 2 0.24 0.0002 0.0006 3 0.16
Avr3a 35 444 1 0.22 0.0001 0.0005 2 0.057
Southwestern Andes (Nari?o) Cox1 10 669 1 0.15 0.0005 0.0005 2 0.36
Ras 12 765 0 0 0 0 1 0
b-tub 21 815 12 1.47 0.004 0.004 9 0.843
Avr3a 17 444 11 2.48 0.007 0.007 3 0.412
Venezuela Cox1 11 669 1 0.15 0.0003 0.0005 2 0.182
Ras 20 765 3 0.39 0.0021 0.0011 2 0.526
b-tub 13 815 1 0.13 0.0002 0.0004 2 0.154
Avr3a 12 444 0 0 0 0 1 0
REST OF THE WORLD Cox1 69 669 4 0.59 0.0097 0.0012 5 0.574
Ras 166 766 13 1.69 0.0031 0.0029 12 0.583
b-tub 13 815 2 0.24 0.0011 0.0008 3 0.59
Avr3a 8 444 4 0.9 0.0046 0.0035 3 0.607
Cox1 142 669 4 0.59 0.00078 0.0011 5 0.473
WORLDWIDE Ras 251 766 13 1.69 0.0024 0.0028 13 0.482
b-tub 103 815 14 1.71 0.0016 0.0033 11 0.544
Avr3a 88 444 12 2.7 0.002 0.0054 6 0.174
aNumbers represent sequences and can exceed the numbers of strains due to heterozygous individuals.
b
? = Nucleotide Diversity.
c
? SITE = Watterson?s Theta per site.
dHD = Haplotype diversity.
Colombia and Venezuela are designated NA; the rest of the world, corresponding to available sequences from other geographical origins, is designated R; and
worldwide, corresponding to the combination of the two previous designations, is labeled W (W = NA +R).
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Table 2 Haplotype distribution of P. infestans for Avr3a, Ras, b-tubulin and Cox1
Avr3a Ras b-tubulin Cox 1
Position 1 2 2 2 2 2 2 2 3 4 1 1 1 2 3 3 4 4 4 5 6 1 3 4 4 5 5 7 8 8 8 4 4 6
4 5 8 0 0 2 3 4 6 8 0 1 6 9 0 1 6 0 1 4 6 7 9 8 3 3 6 3 5 2 7 2 4 7 0 0 1 1 2 4 2
3 5 3 0 2 0 8 2 4 4 9 5 3 8 5 2 2 3 2 8 2 1 7 7 5 6 0 3 2 5 0 4 8 9 4 1 2 5 1 5 6 0
Site 1 1 1 1 1 1 1 1 1 1 1
Number 1 2 3 4 5 6 7 8 9 0 1 2 1 2 3 4 5 6 7 8 9 0 1 2 3 1 2 3 4 5 6 7 8 9 0 1 2 3 1 2 3 4
Consensus A T A A C A A G G A T C C G A A A G G G G T G T T C C C A C T G C T T C A T G A G C
Site Type v v v v t t t t v t v v t v v t t t v t v t t t v t t t t t t t t v t t v v v t v t
Character
Type
_ i _ _ _ _ i _ _ _ i i i - i i i i i i i i i i i i i i i i i i i i i - - i - - - -
H1 (80) . A . . . . G . . . G A H1 (19) T T C G . A . . . . A C A H1 (68) . . . . . . . T . C . . . H2 (1) C . . .
H2 (1) T A . . . . G . . . G A H2 (178) . . . . . . . . . . . . . H2 (1) T T T G T . . . G . . . . H1 (94) . . . T
H3 (2) . . . . . . . . . . . . H3 (2) . . . . . . T A . C . . . H3 (1) T T . . . . . . . C . . G H5 (43) . . . .
H4 (1) . . . . . . . . . . . A H4 (14) . . . . . . . A . . . . . H4 (1) T T T G T C A . G . . . G H4 (1) . . C .
H5 (2) . . T C . . . . . . . . H5 (1) . . . . . . . A T C . . . H5 (1) . . . G . C A . . . . . . H3 (3) . G . .
H6 (2) . . . . T G . A T G . . H6 (20) . . . . . . . A . C . . . H6 (1) . . . . T . . . . . . . .
H7 (1) . . . . . . . . . . . C . H7 (11) . . . . . . . . . . . . .
H8 (1) T . C G . A . . . . A C A H8 (3) . . . . . . . . . C . . .
H9 (1) . . . . G . . . . . . . . H9 (11) . . . . . . . T . . . . .
H10 (1) . . . . G . . A . C . . . H10 (3) . . . . . . . T . C T C .
H11 (1) . . . . G . . A T . . . . H11 (2) . . . . . . . T . C . . G
H12 (2) . . . . . . T . . . . . .
H13 (10) . . . . . . . . . . A C A
t: transitions; v: transversions; i: informative sites; -: uninformative sites; H: Haplotype.
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However, the Avr3a gene was found to be subject to a
selection pressure. A negative tendency was observed in
Avr3a and b-tubulin indicating a possible population
expansion or a recent bottleneck effect. The R2 test failed
to reject the null hypothesis of constant population size
in the NA populations. The L shape pattern found in the
mismatch distribution graphs of all genes (Additional file
6) is also indicative of a constant population size. No
fixed differences were observed in any of the defined
populations in the NA dataset (data not shown).
The analyses of gene flow were in general concordant
between nuclear and mitochondrial genes (Table 5).
Nuclear and mitochondrial genes showed low levels of
genetic flow between Colombia and Venezuela. Both
nuclear and mitochondrial genes showed that the Cen-
tral and Eastern Andes are interchanging migrants.
Avr3a Amino Acid Analysis
Six different amino acid sequences were obtained after
the translation of the nucleotide sequences from the W
dataset (Figure 3). H1 was characterized by amino acids
E80 and M103 and is associated with a virulent pheno-
type. H3, characterized by amino acids K80 and I103
and associated with an avirulent phenotype, was exclu-
sively found in Ecuador, as was H4. To identify which
amino acid positions were under diversifying selection,
three pairs of ML models of amino acid substitutions
were tested. Each pair consisted of one model that
allowed sites to be under diversifying selection with ? >
1 (M2, M3 and M8) and a second model that did not
allow sites to be under selection (M0, M1 and M7) with
?= dN/dS. The likelihood ratio tests failed to reject the
models of neutral evolution. However, model M8
accounted for a small percentage (2%) of amino acids
under selection pressure (? >1). These amino acid posi-
tions were S19C, E80K and M103I.
Discussion
This is the first time that a detailed population genetic
analysis of P. infestans in the northern Andean region
Figure 1 Cox1, b-tubulin, Ras and Avr3a haplotype distribution of Phytophthora infestans in the Northern Andean region. Colors
indicate the different haplotypes for each locus analyzed.
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was conducted. Until now, the pathogen population
structure was unknown in this portion of the continent
(Colombia and Venezuela). Colombia is situated in a cri-
tical geographical position, acting as a bridge between
Central and South America. According to FAO statis-
tics, this country has a commercial history in trading
potatoes with several countries in Europe, Asia, North
and South America, and the Caribbean [24]. This sug-
gests there have been opportunities for migration of the
pathogen, along with its host, towards and within the
northern Andean region.
The low genetic diversity found on global and regional
scales in the Northern Andean region (Colombia and
Venezuela), in both mitochondrial and nuclear regions,
is consistent with previous reports in other countries of
South America [8]. Even for microsatellite markers low
levels of genetic diversity have been reported in
Colombia and Venezuela [25-27]. Specifically, for Ras,
only 2 out of 13 haplotypes found globally could be
observed in the region. The A2 mating type was discov-
ered in Ecuador and Colombia [16,26], and sexual
reproduction is expected to produce new allele combi-
nations. However, even though the A1 and A2 mating
types converge in the same geographical region, as
occurred in Cundinamarca Department (Colombia), sex-
ual recombination is apparently not prevalent. This is
probably due to a process of host adaptation [11,16,48],
or because the presence of the A2 mating type is too
recent to have resulted in recombination. Additionally,
the low frequency of the A2 mating type described in
Colombia diminishes the chances for sexual reproduc-
tion to occur [26]. Indeed, in the nuclear genes few indi-
viduals were heterozygous, suggesting that genetic
interchange occurs but at extremely low frequency.
Figure 2 Haplotype networks of genes studied in the Phytophthora infestans populations of the Northern Andean region. A. b-tubulin;
B. Cox1; C. Ras; D. Avr3a. Respective haplotypes are identified with the letter H, followed by a corresponding number. Sizes of the circles reflect
haplotype frequencies in the population and were constructed with statistical parsimony as is implemented in TCS [38]. For Ras, South America:
Colombia, Venezuela, Ecuador, Bolivia, Brazil, Peru; Central America: Mexico and Costa Rica; North America: USA; Europe: Ireland. For b-tubulin,
South America: Colombia, Venezuela and Ecuador; Central America: Mexico; North America: Canada; Europe: The Netherlands; Africa: Uganda and
Kenya. For Cox1, South America: Colombia, Venezuela, Ecuador, Bolivia, Brazil and Peru; Central America: Mexico and Costa Rica; North America:
USA; Europe: Ireland.
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Because of this, the presence of heterozygous individuals
might be the result of ancestral polymorphisms or gene
flow and not the result of sexual reproduction.
The gene networks for the different regions showed,
as a general pattern, that the relationships between the
haplotypes in each population of the Northern Andean
region are consistent with variation within a clonal
population and that haplotypes are discriminated by one
or two steps. However, in the Southern Andes, at least
for b-tubulin and Avr3a, more than two steps separated
four and two haplotypes, respectively. The more diver-
gent haplotypes were found in samples from S. beta-
ceum and corresponded to the mitochondrial haplotype
Ia. Recently it has been suggested that a new species,
P. andina, with an Ia mitochondrial haplotype, could be
associated with S. betaceum [8,23,49]. However, its taxo-
nomic status has not been clarified yet, and continues to
be considered as P. infestans sensu lato. The presence of
this new species in the population of the southwestern
Andes could explain the different pattern observed in
Avr3a and b-tubulin.
The genetic variation of P. infestans is mainly explained
by the variability present within each population that is
generated by the existence of different low-frequency
alleles, particularly for b-tubulin and Avr3a in the South-
ern Andes. Nevertheless, the variation detected in this
study was enough to show some genetic differentiation.
At the regional level, the P. infestans population in Vene-
zuela appeared to be isolated from the Central and East-
ern Andes populations. Indeed, at the mitochondrial level
Venezuelan isolates belonged largely (11 out of 12) to the
Ia haplotype, and just one corresponded to IIa, the most
common haplotype in the North Andean region. This
and the presence of a particular haplotypic composition
for the nuclear genes may suggest that the Venezuelan
population probably has been structured by different
population events. Reasons for the apparent genetic isola-
tion of the Venezuelan isolates of P. infestans have been
discussed elsewhere [27]. The estimates of genetic flow
support the idea that the population of Venezuela is not
donating or receiving migrants from any other region
(Table 5). However, a large area has not been sampled on
the Colombian side close to the Venezuelan border.
More sampling is therefore needed in this region in order
to detect possible recent gene flow.
Additionally, two significant patterns were observed.
First, subdivision was found between the Eastern Andes
with the rest of Colombia for b-tubulin. This could be
the result of a long history of self-sufficiency in terms of
potato seed tuber supply in the eastern Andes as well as
avoiding movement of the pathogen on plant tissue. Sec-
ond, results obtained with the mitochondrial gene Cox1
and nuclear genes suggested that historically the South-
western and the Eastern P. infestans populations have
been isolated, but recent gene flow could be taking place.
The selection imposed on a gene with a known func-
tion in host recognition as Avr3a may have resulted in a
different pattern of sequence polymorphisms in the
sampled population in comparison to the other nuclear
regions analyzed. However, low genetic diversity was
found for this gene. No genetic subdivision could be
detected in the Northern Andean region, in contrast
with what was found for b-tubulin and Ras. At the
amino acid level interesting patterns emerged. Previous
Table 3 Permutation tests for Hudson?s statistic
Gene Subdivisions Kst Ks Kt P valuea
Avr3a A vs B/C -0.023100 0.040122 0.039216 NS
A vs V nan 0.000 0.000 NS
B/C vs V -0.030565 0.043854 0.042553 NS
N vs V 0.037646 1.844118 1.916256 NS
N vs A 0.065266 1.589757 1.700758 NS
N vs B/C 0.127264 0.999764 1.145551 NS
Cox1
A vs B/C -0.021429 0.039286 0.038462 NS
A vs V 0.856818 0.068182 0.476190 <0.001
B/C vs V 0.774561 0.083859 0.371981 <0.001
N vs V 0.487489 0.263577 0.514286 NS
N vs A 0.131826 0.123671 0.142450 NS
N vs B/C 0.093524 0.115370 0.127273 NS
Ras
A vs B/C -nan 0.000 0.000 NS
A vs V 0.300420 0.835913 1.194879 0.001
B/C vs V 0.386997 0.557276 0.909091 <0.001
N vs V 0.237184 1.015038 1.330645 NS
N vs A -nan 0.000 0.000 NS
N vs B/C -nan 0.000 0.000 NS
b-tubulin
A vs B/C 0.071097 0.443943 0.477922 0.001
A vs V 0.022594 0.662711 0.678030 NS
B/C vs V 0.002664 0.161134 0.161565 NS
N vs V 0.082367 1.938315 2.112299 NS
N vs A 0.075742 1.999553 2.163415 NS
N vs B/C 0.178038 1.170111 1.423559 <0.001
Phytophthora infestans population subdivision was tested in the Northern
Andean region for Avr3a, Cox1, Ras and b-tubulin.; V: Venezuela; A: Antioquia;
N: Nari?o; B: Boyac?; C: Cundinamarca.
Table 4 Neutrality test using Tajima?s D
Locus Tajima?s-D
Northern Andean Region Cox1 0.54 (NSa)
Ras 0.1 (NS)
b-tub -1.64 (NS)
Avr3a -1.89 (<0.05)
Cox1, Ras, b-tubulin and Avr3a from Phytophthora infestans in the Northern
Andean region were tested for neutral evolution employing Tajima?s D [42].
aNS: Not significant.
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studies reported only three polymorphic positions in a
sampling of isolates from S. tuberosum from different
locations worldwide. Two alleles were characterized
based on those amino acid positions, C19 K80 I103 and
S19 E80 M103, with avirulent and virulent phenotypes,
respectively [28]. Here we report four new allelic var-
iants. Three were only present in the isolates from the
southwestern Andes. The natural selection analysis at
the amino acid level showed that three out of the nine
polymorphic positions were under diversifying selection
and two of these were located in the C-terminus region
as previously reported [28]. According to the model of
interaction of the virulence genes with the host cell, at
least two mechanisms could be affected by amino acid
substitutions. The first one is the direct or indirect
recognition by the resistance protein from the host. Bos
et al. [50] have shown that some mutations leading to
specific amino acid substitutions at the position E80K of
Table 5 Migration and theta estimates for the Northern Andean populations of Phytophthora infestans
Theta Number of Migrants per generation
Source population
Recipient population Central
Andes
Eastern
Andes
Southwestern
Andes
Venezuela
Central Andes 8.2e-3
(4.3e-3 - 0.019)
- 364.08
(147.23-1071.6)
8.4e-12
(3.85e-12 - 25.9)
8.4e-12
(3.85e-12 - 25.9)
2e-4
(1e-4 - 3e-4)
- 4.08
(1.22 - 9.54)
6.8e-7
(3e-7 - 1.28e-3)
6.8e-7
(3e-7 - 1.28e-3)
Eastern Andes 8.8e-3
(7.6e-3 - 0.0102)
1.43e-14
(1.08e-14 - 0.81)
-
-
1.43e-14
(1.08e-14-0.81)
1.43e-14
(1.08e-14-0.81)
2e-4
(2e-4 - 2e-4)
2e-7
(3.2e-7 - 2.4e-4)
1.72e-1
(4.08e-2 - 4.6e-1)
1.68e+1
(1.49e+1 - 1.9 e+1)
Southwestern Andes 0.0048
(0.0023-0.0125)
19.97
(4.15 - 100.5)
43.97
(12.33-181.5)
5.14e-10
(2.16e-10 - 14.11)
0.001
(0.0007-0.0018)
2.14
(1.16-7.92)
1e-6
(7e-7 - 2.38e-3)
- 2.62
(6.2e-1 - 9.22)
Venezuela 0,0057
(0.0033-0.0108)
24.39
(8.02-74.73)
2.76e-15
(1.41e-15 - 6.27)
4.89
(0.67-24.6)
-
0.0002
(0.0001-0.0003)
2e-7
(1.6e-7 - 3.72e-4)
20
(8.94 - 34.1)
1.07e-1
(5.65e-3 - 5.86e-1)
Migrations estimates correspond to migrants per generation (Nm) and 5% and 95%, confidence intervals are reported as are implemented in MIGRATE [45]. For
each population the estimates correspond to the nuclear genes (above) and the mitochondrial gene (below).
Figure 3 Alignment of the Amino acid haplotypes observed at the Avr3a locus. The six AVR3a haplotypes are shown with the shared
amino acid positions represented by dots and replacements in IUPAC code.
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this protein can be associated with a loss of recognition
by the R3a gene product. On the other hand, the repla-
cement of lysine by arginine at the same position of the
protein does not affect the cell-death suppression activ-
ity of AVR3a [50]. It has been recognized that the var-
iant AVR3aEM produces significantly less hypersensitive
response than the other known variants of the gene,
producing a virulent phenotype [28], while the AVR3aKI
variant is the most effective in suppressing cell-death in
the host [51]. This scenario leads to the possible advan-
tage of maintaining polymorphic residues, which may
give the pathogen population different adaptive path-
ways. The remaining polymorphic positions detected in
this study were not under strong positive selection.
However, their effect on avirulence on S. tuberosum
R3-expressing plants remains to be determined in order
to define their potential effect on host resistance at the
subspecific level.
Polymorphisms of the Avr3a gene may be related to
the plant species from which the isolates were collected.
Armstrong et al. [28] reported that 55 isolates of
S. tuberosum from different locations in the world
contained only two alleles. In this study we found both
previously reported haplotypes AVR3aEM (H1) in
S. tuberosum as well as in other Solanum species, and
AVR3aKI (H3) restricted to S. muricatum. The other
four reported variants were not present in S. tuberosum.
Because of this, it is required to establish the correlation
of each of these variants and their host range. Addition-
ally, the taxonomic status of the P. infestans-related
species P. andina should be clarified in order to under-
stand the real sympatry of the two species and their
respective contribution of alleles to the population.
Furthermore, a more intensive sampling is needed in
order to correlate polymorphisms with the host. Finally,
the wider picture of the population structure in this
region suggests that the low steps in the networks,
together with the low genetic diversity of this pathogen
and the evidence of the presence of selection in the
effector gene, might be consistent with a scenario of
random dispersion of new mutations by drift that in
some cases might be fixed by selection. We need further
research in other populations and more data from effec-
tor genes and neutral loci to really understand the
Phytophthora infestans diversification process in South
America.
Conclusions
The Northern Andean P. infestans population genetic ana-
lysis suggests that a clonal polymorphic population inha-
bits the region with population subdivision between
Colombia and Venezuela as a result of potato seed self-
sufficiency. Given the observed variation at an avirulence
locus, the study of this type of loci could be useful to
understand the epidemiological behavior of this pathogen
on different hosts and in response to other ecological vari-
ables. Avirulence genes are thought to coevolve in an arms
race with corresponding resistance genes in the host plant.
Thus avirulence genes or other effectors could be excellent
candidates for understanding the evolutionary forces
responsible for the diversity observed, particularly for
clonally reproducing populations of this pathogen. As was
said before, comparisons of other populations and neutral
vs. avirulence loci are needed to understand the popula-
tion dynamics of Phytopthora infestans.
Additional material
Additional file 1: Phytophthora infestans isolates analyzed in this
study.
Additional file 2: Geographical distribution of the sampling sites.
Additional file 3: Sequences retrieved from the GenBank public
database employed in this study for Ras, Cox1, b-tubulin and ITS.
Additional file 4: Gene networks showing relationships among
haplotypes for each gene in each North Andean region.
Additional file 5: Gene networks showing relationships among
haplotypes for Colombia and Venezuela.
Additional file 6: Mismatch distribution for all analyzed regions.
Acknowledgements
In Colombia this work was financed by the Facultad de Ciencias of the
Universidad de Los Andes. In Venezuela, the study was financed by FONACIT
(Grant 2004000490-26140) and CDCHT-ULA (Grant C-1260-04-01-B). NJG was
supported by funds from USDA Agricultural Research Service CRIS Project
5358-22000-034-00 D.
Author details
1Universidad de Los Andes, Bogot? D.C., Colombia. 2Universidad Nacional,
Medell?n, Antioquia, Colombia. 3Universidad de Los Andes, La Hechicera,
M?rida, Venezuela. 4Universidad de Nari?o, Pasto, Nari?o, Colombia. 5USDA
Agricultural Research Service, Corvallis, OR, USA. 6Smithsonian Tropical
Research Institute. Apartado 0843-03092, Panam?, Rep?blica de Panam?.
7Department of Zoology, University of Cambridge, Downing Street,
Cambridge, CB2 3EJ, UK.
Authors? contributions
MC acquired data, carried out and analyzed the molecular genetic studies,
and drafted the manuscript. AG carried out the molecular genetic studies
and drafted the manuscript. RS participated in acquisition of data and
drafted the manuscript. CS participated in the molecular genetic analyses,
and drafted and critically revised the manuscript. AR, AG, AV, MM, GF and
LEL participated in acquisition of data and in revising the manuscript. NJG,
AB, CS, and SR conceived the study, participated in its design and
coordination, and helped to draft the manuscript. All authors read and
approved the final manuscript.
Received: 5 October 2009 Accepted: 9 February 2011
Published: 9 February 2011
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doi:10.1186/1471-2156-12-23
Cite this article as: C?rdenas et al.: Genetic diversity of Phytophthora
infestans in the Northern Andean region. BMC Genetics 2011 12:23.
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