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Analysis of Population Genetic Diversity and Differentiation
in Hemileia vastatrix by Molecular Markers
D. BATISTA1*, L. GUERRA-GUIMARÃES1, P. TALHINHAS1, A. LOUREIRO1,
D.N. SILVA1,2, L. GONZALEZ1, A.P. PEREIRA1, A. VIEIRA1,2, H.G. AZINHEIRA1,
C. STRUCK3, M.C. SILVA1, O.S. PAULO2, V. VÁRZEA1
1Centro de Investigação das Ferrugens do Cafeeiro (CIFC)/ Instituto de Investigação
Científica Tropical (IICT), Oeiras, Portugal. E-mail: *dccastro@fc.ul.pt
2Computational Biology and Population Genomics Group (CoBIG2), Centro de Biologia
Ambiental (CBA), Faculdade de Ciências da Universidade de Lisboa (FCUL),
Lisboa, Portugal
3Faculty of Agricultural and Environmental Sciences, University of Rostock,
Rostock, Germany
SUMMARY
The present study intends to assess H. vastatrix population genetic diversity and
differentiation, migration/dispersal patterns and gene flow among populations using a
comprehensive and across-time coverage of isolates from different geographical origins, as
well as to get some insights on the pathogen evolution. Here we report the initial stage of this
study with the analysis of a first set of 31 H. vastatrix isolates from 12 coffee-growing
countries using AFLPs and gene sequences. Several loci, including the universally used
rDNA-ITS region,
β
-tubulin 1, TEF1 and candidate genes, were tested for marker informative
value. Some level of variability could be detected in ITS and
β
-tubulin 1 loci providing some
insights on the discrimination among H. vastatrix populations and a first step to better
understand the underlying population structure. On the other hand, among a set of AFLP
selective primers tested for sample screening, two AFLP primer combinations generated
distinctive fragment patterns among different isolates. Based on both datasets, population
structure and diversity parameters will be discussed, as well as inferences on the spatial
distribution of the genetic variability. These first results open the way to unravel the
molecular differentiation and the dynamics of H. vastarix populations.
INTRODUCTION
Coffee leaf rust (CLR) caused by the biotrophic fungus Hemileia vastatrix Berk. & Br. has
long gained a world-class status, reaching almost all coffee growing countries with severe
economical damages. Breeding for rust resistance has proven successful over the years to
control the disease, but the highly adaptable nature of the fungus shaped by the dynamic
system of host-pathogen co-evolution has shown to be a critical limitation for achieving
durable CLR resistance (Várzea and Marques, 2005). As a consequence, gradual breakdowns
of resistance have been observed in many of the improved varieties in several countries
(Várzea and Marques, 2005; Prakash et al., 2005). In fact, the emergence of new evolving
pathotypes under a strong selective pressure and the potential for these new races to become
epidemically spread on a continental scale is a serious and constant threat. Thus, a better
understanding of the genetic variation of H. vastatrix populations across large geographic
areas and periods of time, and their phylogenetic relationships is a priority. High molecular
diversity among rust isolates has been documented using RAPD markers (Gouveia et al.,
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2005; Nunes et al., 2005), but patterns of differential population genetic structure has not been
yet demonstrated. It is most probable that increasing the resolution of the molecular analyses
with a more comprehensive rust sampling and higher informative markers will bring detailed
information about the genetic variation observed among isolates of H. vastatrix and the
pathogen evolution.
MATERIALS AND METHODS
Rust isolates and DNA extraction
A group of 31 isolates of H. vastatrix from the collection of CIFC/IICT were analyzed,
comprising 12 geographical origins, different collection years and different virulence profiles.
DNA was extracted as described by Kolmer et al. (1995).
Gene sequence data
Four nuclear gene regions were amplified in this study: the internal transcribed spacer (ITS)
region (ITS1-5.8S ribosomal gene-ITS2), using primers ITS1Ext and ITS4Ext (Brown et al.,
1996); β-tubulin 1; translation elongation factor 1-α (TEF1) and a hexose transporter (HXTp1)
from a H. vastatrix haustoria-rich cDNA library (Talhinhas et al., 2010). For these latter loci,
primers were designed using PerlPrimer v1.1.17. Sequencing reactions were performed in
both directions with BigDye v3.1 (Applied Biosystems) and run on an ABI Prism 310
automated sequencer. Since clean sequences could not be obtained for the ITS region, cloning
was performed for 10 isolates using the Fermentas CloneJET PCR cloning kit according to
manufacturer’s instructions. Multiple alignment of sequence data was performed using
MAFFT v6.717b (Katoh et al., 2009). The phylogenetic tree was generated using the
Maximum Likelihood (ML) method in PAUP* v4.0d99 (Swofford, 2002) with the best fit
model of nucleotide evolution, under the Akaike Information Criterion (AIC) from ModelTest
(Posada and Crandall, 1998). Heuristic searches with 100 random addition replicates were
executed. Nonparametric bootstraping was also conducted using 100 pseudoreplicates with 10
random additions.
Amplified Fragment Length Polymorphism (AFLP) data
A subset of 28 rust isolates was used in this study. AFLP was performed according to Vos et
al. (1995) modified and adapted to the ABI system, using PstI and MseI restriction enzymes, a
preamplification step with no selective nucleotide and a specific amplification step with +2/+3
selective nucleotides. A prescreening test was carried out using 22 PstI/MseI combinations.
Two informative AFLP combinations were selected and used for further characterization of
all studied isolates: P-TG/M-CTA (PM105) and P-TG /M-AG (PM109). Selective PCR
products were separated by capillary electrophoresis using an ABI Prism 310 automated
sequencer. Factorial Correspondence Analysis (Benzécri, 1973) of the individual multilocus
scores, as implemented in the program GENETIX v. 4.01 (Belkhir et al., 2004), was used as
an exploratory tool to assess the similarity/dissimilarity between isolates. The original dataset
is converted into a new three state matrix for each isolate in each allele of each locus (1 for
absence and 2 for presence). The algorithm finds independent eigenvectors of the matrix and
determines the factorial axes. New coordinates of each individual are recalculated in each
factor and can then be plotted.
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RESULTS AND DISCUSSION
From the DNA sequence datasets, nucleotide diversity among isolates was only found on ITS
and
β
-tubulin 1 loci. Analysis of ITS sequences revealed the presence of at least two different
copies in all the isolates investigated, as confirmed by inference from the double peak pattern
in chromatograms as described by Sousa-Santos et al. (2005) in comparison with cloned
sequences. However, all isolates shared the same pattern of heterozygous indels, except for
one isolate which differed in one nucleotide site. Also some cloned genotypes suggested the
existence of more than two ITS copies. Multiple ITS copy sequences is not a rare
phenomenon in fungi and have been reported in other rusts (Morin et al., 2009). Moreover,
detection and analysis of different copies from unlinked nuclear gene regions can uncover
events of hybridization, which is now believed to be an important mechanism for rapid
evolution and speciation in fungi (Morin et al., 2009). Further investigation is required to
ascertain the number and type of ITS copies present in H. vastatrix populations, representing
a promising tool to explore and understand the extant genetic variability. The
β
-tubulin 1 loci
dataset presented seven neutral polymorphic sites, with a nucleotide diversity (Pi) of 0.00149,
revealing four diverging genotypes. The slight genetic structure revealed in the present
phylogeographical frame shows no apparent correlation with geographical location or
virulence profile (Figure 1).
Figure 1. Maximum likelihood tree obtained for the
β
-tubulin 1 dataset following 100
replicates. The tree was rooted using Puccinia graminis as outgroup taxa.
In contrast, AFLP markers revealed to be highly polymorphic and informative. A very
preliminary factorial analysis based on the AFLP data generated by 2 selective primer
combinations clearly clustered the H. vastatrix isolates studied in three distinctive groups (G1,
G2 and G3, Figure 2). In these data projection, where the first four axes explain 55% of total
variation, in particular the first axe, these groups are very well separated and delimited. A
higher similarity of G2 with G3 is suggested, being the former only constituted by 3 isolates
which seem genotypically more distinct. On the other hand, this apparent structuring seems
tendentiously correlated with geographical origin since in a total of 14 African isolates, 12 are
clustered together between G2 and G3. Although the AFLP data is still very limited and these
results can only be interpreted as potential real relationships, it shows the existence of
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underlying genotypic affinities and population structure whose significance we hope to
unravel in the future. Together with the higher resolution power of AFLP markers for genome
coverage and ability to discriminate small differences and identify potential relationships,
searching for additional polymorphic gene loci is on progress aiming to improve information
on H. vastatrix phylogeographical patterns. In particular, ITS region seemed to constitute a
source of variability worth exploring for the reconstruction of a more complete scenario
towards a possible evolutionary history of H. vastatrix.
Figure 2. Factorial correspondence analysis of 28 H. vastatrix isolates based on the
polymorphic AFLP loci generated by primer combination PM105 and PM109. G1, G2
and G3: Groups distinguished by the present analysis.
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