Melo 2014
Melo 2014
Melo 2014
12103
© 2014 Blackwell Verlag GmbH
Summary
Moniliophithora roreri, causal agent of cacao frosty pod rot, is considered one of the most devastating pathogens of this crop. In this study,
we report the development of and validation of 28 microsatellite loci from an enriched library. From these, ten loci were demonstrated to
be polymorphic in an Ecuadorian population composed of 27 isolates. The mean number of alleles per locus was 3.2, and the observed
mean and expected heterozygosity were 0.03 and 0.43, respectively. The polymorphic microsatellites described herein may be used to
study the genetic diversity as well as to comprehend other aspects of M. roreri population biology.
1 Introduction
The hemibiotrophic basidiomycete Moniliophthora roreri (Cif.) H. C. Evans, Stalpers, Smason & Benny, infects only fruits of
neotropical trees from the genera Theobroma and Herrania. In Theobroma cacao L. fruit, the major component of chocolate, it
causes frosty pod rot disease, also known as moniliasis, an extremely destructive disease and currently the main yield-limiting
factor in several Latin American countries (Phillips-Mora 2003). Pod losses due to the disease are usually over 30%, but it
might reach 100% depending on factors such as disease management, favourable environmental conditions and time of intro-
duction of the disease in a particular agroecological zone or site (Katip 1994; Krauss et al. 2003). It has been proposed that
M. roreri has its origin in the north-western region of Colombia and is now present in 11 Central and South American
countries (Phillips-Mora et al. 2007). This disease poses a major threat to important cacao-producing areas in Brazil and
Caribbean countries, such as Trinidad and Tobago, Dominican Republic and Haiti that are still free from the pathogen.
It has been shown by AFLP/ISSR data that M. roreri is genetically variable and forms five genetic groups (Phillips-Mora et
al. 2007), being two major ones: (i) the Bolıvar group; comprising isolates from north of Santander in Colombia, all Peruvian
and Venezuelan isolates, and 10 isolates from the periphery of Ecuador and (ii) the Co-West group, including, mainly, isolates
from western Colombia, central Ecuador and Central America. The other groups are all apparently endemic to Colombia
(Co-East and Co-Central groups) or north-western Ecuador. The use of microsatellites or simple sequence repeats (SSRs) will
allow broader inferences regarding the population biology of M. roreri. This knowledge is necessary to establish successful
integrated management strategies for crop breeding. SSRs are considered suitable for genetic studies because they are
co-dominantly inherited, allowing precise discrimination even of closely related individuals as well as allowing the heterozygote
in diploid genomes to be distinguished. In addition, microsatellite analysis is inexpensive, highly reproducible and transfer-
able across related species (Jarne and Lagoda 1996; Goul~ao and Oliveira 2001). SSRs have been the choice in population
genetic studies of plant pathogens (Taylor et al. 1999), including pathogens of cacao such as M. perniciosa (Gramacho et al.
2007), due to their high variability and statistical power (Reusch 2001). Like RAPDs, ISSRs are dominant markers, quick,
cheap, easy to handle. Despite the lower reproducibility, ISSRs are applicable to screening information about the genetic vari-
ation. However, limited statistical analysis and poor cross-experimental reproducibility represent some drawbacks of ISSRs
markers, where the homozygous presence of a fragment is not distinguishable from its heterozygote. In this way, SSRs are
very powerful tools and should be more widely used for population studies of this fungus. The use of SSR allows researchers
to address key questions about the population genetics of M. roreri, to cite a few practical applications recombination, mat-
ing systems, host adaptation and population structure due to host adaptation. Thus, in this study, we characterized 28 micro-
satellite loci for genetic analyses of populations of M. roreri that represent the first SSRs developed for this species.
http://wileyonlinelibrary.com/
Moniliopthora roreri microsatellites 321
Huallaga, San Martin Department, Peru; all maintained in the USDA-ARS fungal Collection (BPI), Beltsville, Maryland, USA.
The isolates were grown in potato dextrose broth for 7 days, and the DNA extracted following the glass powder protocol
(Cambareri and Kinsey 1994). For library construction, the DNA of the three isolates was combined in equal concentrations
and digested separately with RsaI, HaeIII and EcoRV. Each reaction contained 5 lg of DNA and 10 U of restriction enzyme,
and digestions were carried out according to the manufacturer’s instructions (New England Biolabs, Ipswitch, MA, USA).
Restriction fragment ends were dephosphorylated with 2U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and
capped with double-stranded SNX-linkers (Hamilton et al. 1999). The linker-ligated fragments were amplified by PCR using
the SNX forward primer according to Hamilton et al. (1999). Enrichment for microsatellites was performed with biotinylat-
ed oligonucleotides specifying either AAG8, GTG8, GTT8 or GAT8 repeats and captured with avidin beads (Vectrex) as
described previously (Rehner and Buckley 2003). The eluted DNA was cloned using the Topo-TA cloning kit, and inserts
obtained from 35 randomly selected transformants were PCR-amplified with M13 forward and reverse primers (Invitrogen,
Carlsbad, CA, USA). PCR products were excised after running in 1.5% Nusieve agarose gels (BioWhittaker, Radnor, PA,
USA), frozen at 80°C for 1 h and centrifuged at 14 000 rpm for 15 min. The supernatant was used for direct sequencing
with the BigDye Terminator cycle sequencing kit v2.0 (Applied Biosystems, Foster City, CA, USA) on an ABI 3100 DNA ana-
lyzer according to the manufacturer’s instructions. The sequence data showed that 31 of the enriched fragments harboured
a microsatellite and were mainly tri-nucleotide.
The obtained sequences were edited using the software SEQUENCHER 4.1 and manually searched for microsatellites. Thirty-
one microsatellite primers were designed with the software DNASTAR (DNASTAR, Inc., Madison, WI, USA).
3 Results
From the 31 microsatellite primers designed, three did not amplify any detectable PCR product due to incomplete flanking
regions, and 28 (90.3%) successfully generated an amplification product in the expected size range. Among these 28 loci,
10 (35.7%) were polymorphic (Table 1) and 28 (64.3%) were monomorphic (Table 2) for the 30 isolates tested in this
study.
A total of 32 alleles were observed in the entire set of isolates used, with an average of 3.2 alleles, ranging from 2 to 5
alleles per locus (Table 1). There was no evidence for null alleles in any of the loci after analysis with Microchecker
software. Moreover, there was no evidence for allele exclusion or naming errors generated by stuttering.
322 B. L. B. Melo, J. T. de Souza, R. M. F. Santos et al.
Table 1. Characteristics of ten polymorphic microsatellite loci from Moniliophthora roreri from Ecuador.
No. of
GenBank accession Name Primer sequence (50 –30 ) Ta (°C) Repeat type (bp) Size range alleles
Gene bank Name Primer sequence (50 –30 ) Ta (°C) Repeat type Size (bp)
Repeat motifs are listed as 50 -30 with respect to the forward and reverse primer. Ta, annealing temperature.
Moniliopthora roreri microsatellites 323
Of the 10 polymorphic SSR loci, eight primers (MR6, MR10, MR11, MR12, MR15, MR22, MR31 and MR33) produced
amplification products within the expected size range and showed higher rates of transferability to M. perniciosa. The two
remaining primers (MR13 and MR30) amplified unspecific PCR products in addition to fragments of the expected size,
indicating that although transferable, adjustments are needed.
4 Discussion
Microsatellites have emerged as powerful genetic markers because of their high polymorphism, the relative ease of scoring
and co-dominance (Estoup et al. 1998). They may be used to make inferences on many genetic parameters of interest such
as migration, population size, bottlenecks and mode of reproduction, for example, to assess the relative contributions of
asexual and sexual reproduction in pathogen populations (Liu et al. 1996).
Dutech et al. (2007) reported that obtaining an acceptable level of polymorphism with microsatellite loci in fungi is gen-
erally more difficult than for other organisms and that the average number of alleles in fungi generally falls between 0.4
and 5.4 alleles per locus, whereas other phylogenetic groups reach higher values. In this study, the average level of genetic
diversity (He=0.43) and the average number of alleles per locus (3.2) found with the developed set of microsatellites are
compatible with the published results and those observed for its closely related species, M. perniciosa (He= 0.06 to 0.79
and average of 2.9 alleles per locus) (Gramacho et al. 2007; Silva et al. 2008).
The cross-amplification of these M. roreri SSR markers in M. perniciosa isolates from cacao indicates that the flanking
regions are conserved across these two species. However, it does not tell anything about the character and structure of the
fragment, and additional sequencing will be needed to clarify the structure of these fragments.
In conclusion, 10 polymorphic microsatellite loci suitable for population genetics studies in natural populations of
M. roreri have been obtained. These loci may also be useful for population studies of the closely related species, M. pernicosa.
5 Conclusions
In this study, we present the first set of microsatellites developed for M. roreri, which could be widely applicable for popu-
lation genetic studies. These primers may be used to make inferences into the evolutionary potential of M. roreri in cacao
and, thereby, would greatly assist cacao breeding programs.
Acknowledgements
The authors thank CFC/ICCO/BIOMOL, Mars Inc. and PROCITROPICS for financially supporting this project and CNPq for providing
scholarships to JTS and KPG. BLBM was funded by Fundo de Amparo a Pesquisa da Bahia (FAPESB; grant # 1077/2010) and Coorde-
nacß~ao de Aperfeicßoamento de Pessoal de Nıvel Superior (CAPES). We thank M. C. Aime for providing isolate C23 in the initial phase of
the study.
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