MICROSATELLITE MARKERS FOR COFFEE TREE
Raphael José Nascif RUFINO1, Eveline Teixeira CAIXETA1,2, Eunize Maciel ZAMBOLIM1,3, Guilherme Ferreira PENA1,
Robson Ferreira de ALMEIDA1; Samuel Mazzinghy ALVARENGA1; Laércio ZAMBOLIM3 e Ney Susssumu SAKIYAMA1,4
Universidade Federal de Viçosa (UFV)/BIOAGRO, Laboratório de Biotecnologia do Cafeeiro (BioCafé), Viçosa – MG. E –
mail: biocafe@ufv.br; 2Embrapa Café; 3UFV/Depto de Fitopatologia; 4UFV/Depto de Fitotecnia.
1
Microsatellite markers developed for Coffea arabica were tested for different species of coffee. Eighteen out of 24 previously
reported pairs of coffee microsatellites primers were adapted for routine use. A high level of polymorphism was observed
among 60 genotypes, including C. arabica, C. canephora, C. eugenioides and Híbrido de Timor (HT). The number of alleles
per marker ranged from 2 to 9. Fifty percent of the tested primers generated polymorphic markers for C. canephora, which
allowed the discrimination among all the genotypes of this species studied here. The level of polymorphism for HT was 33.3%,
and its band patterns were, in general, similar to the C. arabica. Six primers allowed the discrimination of 13 accessions of HT,
which were not differentiated in previous attempt with RAPD markers. The results demonstrate the potential of microsatellite
markers to distinguish coffee genotypes. Ongoing research in our lab using the Coffee Genome ESTs will produce new
microsatellite markers for coffee tree, in the near future.
Key words: SSR, molecular markers, coffee breeding, Coffee sp
INTRODUCTION
In plant breeding programs, molecular markers have been used to increase the efficiency of the genetic factor transference.
Among the molecular markers currently available, the microsatellite or SSRs (Simple Sequence Repeats) has been the most
used due to its technical simplicity, speed, resolution, and polymorphism levels. The high polymorphism allows a precise
discrimination, even among the individuals genetically related. The microsatellites also show advantages over the other markers
based on PCR, because they are co-dominant and easily reproducible, besides being abundant and uniformly dispersed in the
genome. These characteristics make these molecular markers efficient tools for the genetic mapping, linkage studies, genotype
identification, variety protection, seed purity evaluation, use and conservation of germplasm, genetic analyses, pedigree
analyses, marker assisted selection, and analysis of DNA library for gene cloning. In spite of the proved efficiency of these
markers for the coffee, the SSRs are not broadly used due to the limited numbers of primers developed and available for this
plant. In the present work we tested and adapted the microsatellites markers to be routinely incorporated in our genetic and
breeding program.
MATERIALS AND METHODS
Twenty four pairs of coffee microsatellites primers published in the literature (ROVELLI et al., 2000; COMBES et al., 2000)
were synthesized. To adapt these primers, we adjusted the amplification conditions and PCR components concentrations, in two
stages.
1st Stage: preliminary tests
The 24 pairs of primers were tested in six genotypes, being two C. arabica, two C. canephora and two Híbrido de Timor (HT),
which are natural hybrids from the C. arabica and C. canephora species. Different concentrations and commercial brands of
the PCR components were tested including the taq DNA polymerase, MgCl 2, dNTPs and primers.
The PCR was carried out in a thermo cycler JM Research PTC-200. The resulting products were separated in an agarose gel
1.5%, stained with ethidium bromide and visualized with UV. In this phase, the conditions for the functioning of most of the
primers were obtained.
2nd Phase: complementary tests
The reaction and amplification conditions defined in the first phase were re-adapted, aiming to eliminate smears and bad
amplification. The PCR reaction resulting products were separated by electrophoresis in a denaturant polyacrylamide gel 6%
and died with silver stain.
After establishing the PCR conditions, the polymorphism of the primers was tested. The use of these primers in different coffee
species was also evaluated. Sixty coffee genotypes were analyzed, including six accessions of C. arabica, five of C. canephora,
one C. eugenioides and 48 of HT. Young leaves from the coffee trees were collected, frozen and lyophilized. The DNA was
extracted using a method previously adapted in our laboratory for the coffee tree. The DNA quality was analyzed in agarose gel
and its quantification in a Smart Spec BioRad spectrophotometer. The purified DNA was amplified in thermo cycler separated
and visualized in a 6% denaturant polyacrylamide gel stained with silver.
RESULTS AND DISCUSSION
The first tested condition for the 24 primers amplification was exactly the one published by ROVELLI et al. (2000). Some of
these primers did not amplify the genotypes’ DNA (no bands visualized in the polyacrylamide gel), while others resulted in
amplification of multiple bands (Fig. 1). Based on this result, different concentrations of the PCR components and amplification
conditions were tested. The amplification and reaction conditions optimized resulted in the use of 18 primers which amplified
well defined bands (Fig. 2 and 3). The other six primers, even under changes in the PCR conditions, did not amplify any bands
or amplified multiple bands, being, therefore, discarded.
SSR 1
a
b
c
d
SSR 5
e
f
a
b
c
d
SSR 6
e
f
a
b
c
d
SSR 7
e
f
a
b
c
d
SSR 8
e
f
a
b
c
d
e
f
Fig 1. Electrophoretic patterns, in agarose gel, of the products amplified with some microsatellite primers. SSR 1, 5, 6, 7, and 8
correspond to the different primers tested. The letters a and b correspond to two genotypes of C. arabica; c and d, to two
genotypes of C. canephora; e and f, to two genotypes of Híbrido de Timor.
The 24 pairs of microsatellites primers available and developed to amplify the C. arabica specie, were tested in genotypes of C.
arabica, C. canephora, C. eugenioides and HT (natural hybrid from C. arabica and C. canephora). This test was carried out to
verify the usefulness of these markers as heterologous primers to be used in the two species cultivated in Brazil and in
interspecific hybrids important for the breeding programs. From the 18 microsatellites validated, two amplified no bands and
one multiple bands in the C. canephora trees. In C. eugenioides, only one primer was not amplified successfully. This result
showed that the majority of the microsatellites primers can be efficiently used for different coffee species analyzed. The
heterologous primers usefulness associated to these DNA markers unique-locus-natured guarantees their validity as connectors
to a future consolidation of genetic and physical maps. In particular, it provides the opportunity of using microsatellites markers
to investigate the high genetic diversity range existing in related wild species, important for the coffee trees breeding programs.
The efficiency and polymorphism of the 18 primers were tested. The amplification products of these markers showed
monomorphic bands inside the six genotypes of C. arabica, confirming the narrow genetic base of this specie. However, they
presented a high polymorphism among the five accessions of C. canephora; nine primers amplified polymorphic bands
corresponded to 50% of the total tested primers. A polymorphism example found in the C. canephora is shown in Figure 2.
Analyzing the Híbrido de Timor (TH) accessions it was observed that six primers showed polymorphic bands, which
corresponds to 33 % of the total primers. Figure 3 shows an electrophoresis gel containing the amplified products by one of
these primers. The smallest polymorphism found among the Híbrido de Timor when compared to the C. canephora genotypes,
can be explained by its possible origin. The designation of Híbrido de Timor is given to the progenies of a coffee tree, with has
phenotype similar to the C. arabica species, found in a Típica cultivar field in the Timor Island. The best hypothesis is that this
plant was originated from a spontaneous interspecific cross between C. arabica and C. canephora. The progenies of this plant,
as the HT accessions which were used in the present study, are probably originated from the natural backcrossing for the C.
arabica specie. Thus, these genotypes show a greater C. arabica genome proportion, and therefore, carry the narrow genetic
base of this specie. The HT intermediary polymorphism level, found with the microsatellites tested, supports this hypothesis.
SSR 8


C. arabica
C. canephora
E
Híbridos de Timor
Fig. 2. Electrophoretic pattern of the amplified products with the SSR 8 primer. The six first genotypes are C. arabica follow
by five C. canephora, one C. eugenioides (E) and 27 Híbridos de Timor. The gel shows an example of polymorphism
among C. canephora genotypes.
Analyzing all the 60 genotypes, a high polymorphism level of the microsatellite markers was observed, and all the 18 primers
pairs amplified polymorphic bands. The number of alleles per plant, is probably related to homozygosis or heterozygosis, that
is, the plants which show an allele are homozygotes, while the ones which show two alleles are heterozigotes for the locus
amplified by the microsatellite marker. However, the genotypes of the arabica and the HT species are tetraploids, and the fact
that they have two alleles can be related to the ploidy and not to the heterozygose. Some microsatellites primers amplified 3 and
4 alleles per plant in the arabica and HT genotypes (Table 1) confirming the tetraploid nature of these coffee trees.
Using a group of adapted microsatellite markers, it was possible to discriminate all the C. canephora accessions. The HT, in a
general, showed band patterns similar to those of C. arabica (Fig. 2), however, six primers showed polymorphism (Fig.3),
allowing the discrimination of 13 accessions. The RAPD markers used in these same genotypes, in earlier studies, did not
permit any of these accessions discrimination. The results obtained in the present work showed the potential of microsatellite
markers in discriminating coffee accessions genetically related.
SSR 18
C. arabica
C. canephora
E
Híbridos de Timor
Híbridos de Timor
Fig. 3. Electrophoretic pattern of the amplified products with the SSR 18 primer. The six first genotypes are C. Arabica follow
by five C. canephora, one C. eugenioides (E) and 27 Híbridos de Timor. The gel shows an example of polymorphism
among Híbrido de Timor accessions.
Table 1. Tested microsatellites and number of amplified alleles.
Identificação
Referência
Número de
Número de alelos/planta
alelos
Aa
Cb
Ec
SSR 1
Rovelli et al., 2000
5
3
2
2
SSR 2
Rovelli et al., 2000
2
1
1
SSR 3
Rovelli et al., 2000
SSR 4
Rovelli et al., 2000
SSR 5
Rovelli et al., 2000
5
1
1e2
2
SSR 6
Rovelli et al., 2000
2
2
2
SSR 7
Rovelli et al., 2000
5
2
2
1
SSR 8
Rovelli et al., 2000
6
2
2
1
SSR 9
Rovelli et al., 2000
4
2
SSR 10
Rovelli et al., 2000
SSR 11
Rovelli et al., 2000
5
3
1
1
SSR 12
Rovelli et al., 2000
SSR 13
Rovelli et al., 2000
6
2
2
2
SSR 14
Combes et al., 2000
SSR 15
Combes et al., 2000
2
1
1
1
SSR 16
Combes et al., 2000
3
1
1
1
SSR 17
Combes et al., 2000
8
2
1e2
2
SSR 18
Combes et al., 2000
8
1
1e2
1
SSR 19
Combes et al., 2000
4
4
2
2
SSR 20
Combes et al., 2000
4
2
1
2
SSR 21
Combes et al., 2000
6
2
2
2
SSR 22
Combes et al., 2000
SSR 23
Combes et al., 2000
9
2
1e2
2
SSR 24
Combes et al., 2000
8
2
1e2
1
a
A correspond to genotypes of C. arabica;
b
C correspond to genotypes of C. canephora;
c
E correspond to genotypes of C. eugenioides;
d
HT correspond to genotypes of Híbrido de Timor.
Observação
HTd
3
1
1
2
2
2
2
3
2
1
1
2e4
1e2
4
2
2
2
2e3
Descartado
Descartado
Descartado
Descartado
Descartado
Descartado
CONCLUSIONS
The adaptation of the reaction and amplification conditions for the 24 microsatellite primers permitted the use of 18, which
showed sharp bands. These markers will be incorporated in our genetics and breeding program. Ongoing research in our lab
using the Coffee Genome ESTs will produce new microsatellite markers for coffee tree, in the near future.
BIBLIOGRAPHIC REFERENCES
COMBES, M.C., ANDRZEJEWSKI, S., ANTHONY, F., BERTRAND, B., ROVELL, P., GRAZIOSI, G.SASHERMES, P.
Characterization of microsatelites loci in Coffea arabica and related coffee species. Molecular Ecology, v.9, p.1171-1193.
2000.
ROVELI, P., METTULIO, R., ANTHONY, F., ANZUETO, F., LASSHERMES, P., GRAZIOSI, G. Microsatellites in Coffea
arábica L. Coffee Biotechnology and Quality, Kluwer, Dordrecht, p. 123-133. 2000.