Melo 2014

Download as pdf or txt
Download as pdf or txt
You are on page 1of 5

For. Path. 44 (2014) 320–324 doi: 10.1111/efp.

12103
© 2014 Blackwell Verlag GmbH

Development of microsatellites for the cacao frosty pod rot pathogen,


Moniliophthora roreri

By B. L. B. Melo1,2, J. T. de Souza3, R. M. F. Santos1, S. A. Rehner4, K. H. Solis5, C. Suarez5, P. K. Hebbar6, L. S. L. Lemos1,2 and


K. P. Gramacho1,7
1
Cacao Research Center, Itabuna, Brazil; 2Universidade Estadual de Santa Cruz/PPGGBM, Ilheus, Brazil; 3CCAAB, Universidade Federal do
oncavo da Bahia, 44380-000 Cruz das Almas, Brazil; 4Systematic Mycology and Microbiology Laboratory, USDA-ARS, BARC-W, Beltsville,
Rec^
MD 20705, USA; 5INIAP, Pichilingue, Ecuador; 6USDA-APHIS-PPQ, Riverdale, MD 20737, USA;
7
E-mail: [email protected] (for correspondence)

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.

2 Materials and methods

2.1 Genomic library and primer design


Three isolates of M. roreri from cacao plants were used for microsatellite isolations: C23 collected from Turrialba, Cartago
Province, Costa Rica, Dis21 collected from Quevedo, Los Rios Province, Ecuador, and Dis160d collected from Juanjui, Rio

Received: 17.4.2013; accepted: 20.2.2014; editor: Jolanda Roux

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).

2.2 Characterization of microsatellite loci


The isolates used to test the microsatellites were obtained from pods collected from the four main cacao-producing regions
of Ecuador that represent the occidental (North, Central and South) and oriental (East) sides of the country. To capture the
most diversity, sampling was carried out by collecting one pod per tree at different representative sites of the Ecuadorian
cacao-growing regions. About 40 pods were taken to the laboratory of Estaci on Experimental Tropical de Pichillingue
(EETP/INIAP), Quevedo, Ecuador, for M. roreri isolation. Pods were washed with water and soap and surface-sterilized by
submerging in 2.5% sodium hypochlorite for 2 min and thereafter distilled water for 2 min. Segments of 0.5 cm2 of fruits
were transferred to plates with acidified potato dextrose agar (PDA) (10 ml de 25% lactic acid per litre of medium) and
incubated at 25°C for 15 days. The isolates obtained from the different regions within the country were characterized
based on morphology to confirm the identity of the species.
DNA was extracted from M. roreri mycelium grown for 12 days in liquid 50% malt extract medium following the proto-
col of Zolan and Pukkila (1986). Only isolates (27) of high-quality DNA was used in the experiment. The polymerase chain
reaction (PCR) (20 ll) was as follows: 0.2 lM of each primer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1x ammonium sulphate PCR
buffer, 1 unit of Taq DNA polymerase and 10 ng of DNA template. All PCR amplifications were performed with an initial
denaturing step at 94°C for 4 min, followed by 10 touchdown cycles of 40 s at 94°C; 40 s at 60°C; and 1 min at 72°C,
decreasing 1°C per cycle down to 48°C; ending with 10 cycles of 4 min at 94°C; 1 min at 48°C; and 1 min at 72°C and a
final 4-min extension at 72°C ending with 20 cycles of 4 min at 94 °C; 1 min at 48 °C; and 1 min at 72 °C and a final
4-min extension at 72 °C. This procedure was carried out with all loci, except for the primers MR10 and MR15 that were
run at an annealing temperature of 56°C. Polymorphism was revealed on 6% denaturing polyacrylamide (29 : 1) gels with
migration at 60W and a temperature of 55°C. The gel was silver-stained as described by Creste et al. (2001). A 10 bp
ladder DNA (Invitrogen) was used to estimate the molecular weight of the SSR fragments.
The amplification of the ten polymorphic SSR markers was tested on eight isolates of Moniliophthora perniciosa from dif-
ferent cacao-producing areas of Bahia State, Brazil, to assess their transferability to this close relative of M. roreri. All loci
were tested under the same PCR conditions used for M. roreri. The PCR products were separated by electrophoresis in 4%
Metaphorâ agarose (Cambrex, Rutherford, NJ, USA) in 1x TBE buffer and visualized by GelRed Nucleic Acid Gel Satingâ
(Biotium, Wembley, WA, Australia) staining.

2.3 Data analyses


Ecuadorian population was composed of 27 isolates. In all analyses, it was assumed that M. roreri is a functional diploid
(dikaryon). All data were consistent with this assumption. The number of alleles per locus was obtained with FSTAT 2.9.3.2
(Goudet 2002), and null alleles were examined using MICRO-CHECKER software, version 2.2.3 (Van Oosterhout et al. 2004).

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

FN252882 MR6 Forward:GCCGTCTCCAGCCATAGTAGG TD 60–48 [AGA]6 127–130 2


Reverse:TGAAGCCCAGAGACCCCATCC
KF317678 MR10 Forward:CAGGCGCAACAAGAGGAGTGGAT 56 [AAC]6 121–133 4
Reverse:GCTGCAGCGACGTCATTTGTG
FN252883 MR11 Forward:TCGGGTGGATGGTCTAAATGG TD 60–48 [GAGAA]12 139–149 3
Reverse:AATCCAAATCCCCCAAATCCCAGTC
FN252884 MR12 Forward:CTAAGGCCTTGCTAGCAGAAG TD 60–48 [TCT]5 132–138 2
Reverse: CAATCAAATCGGGTTCGCCA
FN252885 MR13 Forward:AGAAGCCCCAAGCCGAG TD 60–48 [GAA]5 124–139 4
Reverse:CAAAAGAGTCCGGTGCCG
KF317680 MR15 Forward:AATGAGGATGAAGCGGAAGTGTAT 56 [GTG]6 154–160 3
Reverse:AAGGGGCTACACAAGACACCACT
FN252886 MR22 Forward:AGTGGATGCGTCAACAGATG TD 60–48 [CAA]3CAG[CAA]3[CAG]3 141–150 3
Reverse:CAGAAGCCCCAAAACCAGTA
FN252887 MR30 Forward:ATGATGGGTGTAGCGATGGT TD 60–48 CAA[CAG]6CAA[CAG]3 134–149 5
Reverse:TACTCGTCGTAGCGGATGA
FN252888 MR31 Forward:CAGGATACGAGAGGCTGAGG TD 60–48 [AGA]5 146–149 2
Reverse:CTCGCTGCTCTTCTTCTTCC
FN252889 MR33 Forward:CACTCATCAGCAGCACCAAT TD 60–48 [GAAGGA]4 101–125 4
Reverse:AGAAGAGGGAGCCGAAAAAG
Mean – – – – – 3.2
0 0
Repeat motifs are listed as 5 –3 with respect to the forward and reverse primer . Ta, annealing temperature.

Table 2. Characteristics of twenty monomorphic microsatellite loci from Moniliophthora roreri.

Gene bank Name Primer sequence (50 –30 ) Ta (°C) Repeat type Size (bp)

KF317673 MR4 Forward:AGTGTCCTTTTAGTCTCCTTTGAA TD 60–48 [CTT]5 112


Reverse: GATGATCTCAGTAGCAACAG
KF317674 MR5 Forward:CTAAGGCCTTGCTCGCAGAAGCCCGA 56 [GAA]18 283
Reverse:GAGCACGTTCTTGTTCTTCGGCAACGGGC
KF317675 MR7 Forward:GCGGCGAAGGGCTCATTACTC T 56 [CCA]7 142
Reverse:TTACTCCGCAAATCCAAGG
KF317676 MR8 Forward:AACGCCTCGAGCCACACCAT 56 [CCA]7 124
Reverse:CGGACGAGACTGCGAAAAAT
KF317677 MR9 Forward:TTCAGTATCGCGCAAAAAGACCA 56 [CAG]6 135
Reverse:CTAGCAGAAGCACCACCTC
KF317679 MR14 Forward:GAGCCAACATACCAAATCCATAGG 56 [CTT]6 98
Reverse:AAGCAGGAGGAGAAGACGATGATT
KF317681 MR16 Forward:GGTGGCCTTGCTCCGCTATTTG TD 60–48 [TGATGC]6 106
Reverse:GGGCACCCACCACCTCGTCA
KF317682 MR17 Forward:GCTGTGCCGATGAATGGGAATACG 56 [GGT]10 136
Reverse:ACTCCACCAGCGGCAGCAACACC
KF317683 MR18 Forward:GTAAATCAGGCGGCGGCGGTG 56 [TGG]8 159
Reverse:ATTCGCCTCCTCCTCCGCCTCAAC
KF317684 MR19 Forward:GTGGAGAAAGGGGGATGACT 56 [TGG]6 110
Reverse:GCACCAATGCCTGCTCACAGTTACTCT
KF317685 MR21 Forward:GGAGAGTGTGACAGCGATGA 56 [TGA]8 93
Reverse:ATCTCCTTCCTCCCACT
KF317686 MR24 Forward:AGAGATTGGCCAACTTCCTG 56 [CAT]12 133
Reverse:GACGAGCTTTCGGAATTGAG
KF317687 MR25 Forward:CCCTGCTATGGCTCCTGTAG 56 [TGA]10[TGG]6 144
Reverse:CCGACTCTTCTTCTTCCCAGT
KF317688 MR26 Forward:CCTGTGTTGTTGCTGTTTGG TD 60–48 [TTC]6 130
Reverse:AAGAAACCAGAGGACCAGGAA
KF317689 MR27 Forward:CCCTACCCGTCATCATCATC TD 60–48 [CCA]8[CAA]10 142
Reverse:AGGAGGAGGAATACGACGAA
KF317690 MR28 Forward:TACGGGCGAAGGTAGTTGAG TD 60–48 [GGT]6[GAT]2 126
Reverse:TCATCACGAGCATCAGTTCC
KF317691 MR29 Forward:ACTTCCTCCTCTGCTGGTGA TD 60–48 [CTT]13 131
Reverse:CTGGAAGGCTGGGTTAATGA
KF317692 MR32 Forward:GTTCCGGATTCCGAAGGTAT 56 [CAA]8 137
Reverse:GAGGGGTTTGGTGAAGTGAA

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.

References
Cambareri, E. B. J.; Kinsey, A., 1994: A simple and efficient system for targeting DNA to the analysis locus of Neurospora crassa. Gene 142,
219–234.
Creste, S.; Tulmann, N. A.; Figueira, F., 2001: Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing
gels by silver staining. Plant Mol. Biol. Rep. 19, 299–306.
Dutech, C.; Enjalbert, J.; Fournier, E.; Delmotte, F.; Barres, B.; Carlier, J.; Tharreau, D.; Giraud, T., 2007: Challenges of microsatellites isolation
in fungi. Fungal Genet. Biol. 44, 933–949.
Estoup, A. F.; Rousset, Y.; Michalakis, J. M.; Cornuet, M.; Guyomard, R., 1998: Comparison analysis of microsatellite and allozyme markers, a
case study investigating microgeographic differentiation in brown trout (Salmo trutta). Mol. Ecol. 7, 339–353.
Goudet, J., 2002: FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.3.2). http://www2.unil.ch/pop-
gen/softwares/fstat.htm.
Goul~ao, L.; Oliveira, C. M., 2001: Molecular characterization of cultivars of apple (Malus x domestica Borkh.) using microsatellite (SSR and
ISSR) markers. Euphytica 122, 81–89.
Gramacho, K. P.; Risterucci, A. M.; Lanaud, C.; Lima, L. S.; Lopes, U. V., 2007: Characterization of microsatellites in the fungal plant pathogen
Crinipellis perniciosa. Mol. Ecol. Notes 7, 153–155.
Hamilton, M. B.; Pincus, E. L.; Di Fiore, A.; Fleischer, R. C., 1999: Universal linker and ligation procedures for construction of genomic DNA
libraries enriched for microsatellites. Biotechniques 27, 500–506.
Jarne, P.; Lagoda, P. J. L., 1996: Microsatellites from molecules to population and back. Trends Ecol. Evol. 11, 424–429.
Katip, J. Y., 1994: Prospecci on y estudio de la moniliasis (Moniliophthora roreri (Cif. y Par.) Evans et al.) del cacao en la cuenca del rıo
Mara~ n. Thesis Ing. Agr. UNAS, Tingo Marıa, Per
no u.
Krauss, U.; Hoopen, T. M.; Hidalgo, E.; Martınez, A.; Arroyo, C.; Garcıa, J.; Portuguez, A.; Sanchez, V., 2003: Manejo integrado de la moniliasis
(Moniliophthora roreri) del cacao (Theobroma cacao) em Talamanca, Costa Rica. Agroforesterıa en la Americas 10, 52–58.
Liu, Y. C.; Double, M. L.; MacDonald, W. L.; Cortesi, P.; Milgroom, M. G., 1996: Diversity and multilocus genetic structure in populations of
Cryphonectria parasitica. Phytopathology 86, 1344–1351.
Phillips-Mora, W. 2003: Origin, biogeography, genetic diversity and taxonomic affinities of the cacao fungus Moniliophthora roreri as deter-
mined using molecular, phytopathological and morpho-physiological evidence [Doctoral dissertation]. University of Reading, Reading,
UK, 349 p.
Phillips-Mora, W.; Aime, M. C.; Wilkinson, M. J., 2007: Biodiversity and biogeography of the cacao (Theobroma cacao L.) pathogen Monilioph-
thora roreri (Cif.) Evans et al.. Plant. Pathol. 56, 911–922.
Rehner, S. A.; Buckley, E. P., 2003: Isolation and characterization of microsatellite loci from the entomopathogenic fungus Beauveria bassi-
ana (Ascomycota: Hypocreales). Mol. Ecol. Notes 3, 409–411.
324 B. L. B. Melo, J. T. de Souza, R. M. F. Santos et al.

Reusch, T. B. H., 2001: New markers-old questions: population genetics of seagrasses Mar. Ecol. Prog. Ser. 211, 261–274.
Silva, J. R. Q.; Figueira, A.; Pereira, G. A. G.; Albuquerque, P., 2008: Development of novel microsatellites from Moniliophthora perniciosa,
causal agent of the witches broom disease of Theobroma cacao. Mol. Ecol. Resour. 8, 783–785.
Taylor, J. W.; Jacobson, D. J.; Fisher, M. C., 1999: The evolution of asexual fungi. Annu. Rev. Phytopathol. 37, 197–246.
Van Oosterhout, C.; Hutchinson, W. F.; Wills, D. P. M.; Shipley, P., 2004: Micro-checker: software for identifying and correcting genotyping
errors in microsatellite data. Mol. Ecol. Notes 4, 535–538.
Zolan, M. E.; Pukkila, P. J., 1986: A rapid high yield miniprep meted for isolation of total genomic DNA. Mol. Cell. Biol. 6, 195–200.

You might also like