MOLECULAR MARKERS AND GEOGRAPHIC INFORMATION SYSTEMS AS A TOOL
TO STUDY NATIVE PLANT SPECIES IN THE BRAZILIAN SAVANNAS
Fábio Gelape Faleiro1, Ana Maria Costa1, Cláudio Takao Karia1, Ronaldo Pereira de Andrade2,
Nilton Tadeu Vilela Junqueira1, Ailton Vítor Pereira2, Elainy Botelho C. Pereira3, Sueli Matiko
Sano1 (1Embrapa Cerrados, BR 020, Km 18, Caixa Postal 08223, 73010-970 Planaltina, DF. email: ffaleiro@cpac.embrapa.br; 2Embrapa Transferência de Tecnologia; 3Agência Rural)
Index terms: GIS; molecular markers, genetic diversity, native species
Introduction
The Brazilian Cerrado, a region with a savanna-like vegetation, is located in the country’s
Central Plateau and it is the second largest Brazilian biome, only surpassed by the Amazon Region
(Ribeiro e Walter, 1998). The flora of the Cerrado region shows great botanical and genetic
variability and many of the native plant species have actual or potential economic use. At the
Embrapa Cerrados Research Center, plant species with potential for forrage, fruits, medicinals or oil
production are the main targets in the studies regarding conservation, characterization and
utilization of native genetic resources.
Unsustainable land use, predatory extractivism and climate change are real threatens for the
Cerrado native plant species variability. The loss of genes or genetic combinations existing in plants
which have actual and potential agricultural use can be irreversible, and actions are needed to
prevent or minimise it. Establishment of germplasm banks is one of the effective actions and, along
this, germplasm characterization is essential for a sustainable use of the native plant resources.
Knowledge about the intro and inter genetic variability of these native Cerrado plants will
support effective conservation strategies and sustainable use of natural resources in the region. The
utilization of DNA based molecular markers and Geographical Information Systems (GIS) have
proved to be important tools to complement characterization data of accessions from different
species (Faleiro, 2007).
This study shows how molecular markers and Geographic Information System (GIS) have
been used, at Embrapa Cerrados, for characterization and monitoring of genetic and spatial
variability in economically important native plant species from the Brazilian Cerrados region.
Material and Methods
Germplasm: A range of Cerrado native species are object of studies at the Embrapa Cerrados using
DNA analisys or/and GIS. Among them, there are pasture legumes [Stylosanthes guianensis, S.
macrocephala, Arachis pintoi], fruit plants [Dipteryx alata Vog., Caryocar brasiliense Camb.,
Hancornia speciosa Gomes, Eugenia dysenterica Mart. ex DC. and Annona crassiflora Mart.,
Passiflora spp.], oil production species [Acrocomia aculeata (Jacq.) Lodd] and medicinal species
[Dimorphandra mollis Benth. and Stryphnodendron barbadetimam (Vell.) Forrero].
DNA extraction: DNA extraction native species was developed from a protocol previously
established by Faleiro et al. (2003). DNA samples were extracted from foliar tissue from a range of
native species with present or potential economical importance. Foliar tissue showed differences
regarding hair and nervure densities, polysaccharide presence, and latex production, among others.
For each species, extraction was done in leaves at an intermediate maturation stage. Leaves were
collected in the field and transferred to the laboratory in paper bags. Extraction happened from an
foliar tissue sample of approximately 500 mg, which was macerated in a porcelain recipient in
contact with liquid N2.
Molecular markers: The molecular markers have been obtained in the Molecular Biology and
Genetics Laboratory at Embrapa Cerrados Research Center. DNA samples, from each germplasm,
are amplified using the RAPD (Random Amplified Polymorphic DNA) technique. Amplification
reactions are done in 13 uL total volume, containing Tris-HCl 10 mM (pH 8,3), KCl 50 mM,
MgCl2 3 mM, 100 uM of each deoxyribonucleotide (dATP, dTTP, dGTP e dCTP), 0,4 uM of a
primer (Operon Technologies Inc., Alameda, CA, EUA), one unit of the Taq polimerase enzyme
and, approximatelly , 15 ng of DNA. The amplifications happen in a termociclator programmed for
40 cycles, each one with the following sequence: 15 seconds at 94 ºC, 30 seconds at 35 ºC and 90
seconds at 72 ºC. After the 40 cycles, there is a final extension phase, which lasts for 6 minutes at
72 ºC, and finally, temperature is reduced to 4 ºC. After the amplification, each sample receives the
adition of 3 ul of a mixture containing bromophenol blue (0,25%) and glicerol (60%) in water.
These samples are applied in agarose gel (1,2%), stained with ethidium bromide, submerged in
tamponing TBE (Tris-Borato 90 mM, EDTA 1 mM). Electrophoretic separation last for four hours,
approximatelly, at 90 volts.
Following this electroforesis, the gels are photographed under
ultraviolet light.
Statistical analysis: The RAPD markers are converted in a binary data matrix to estimate the genetic
distances among the accessions. The genetic distance, based on the Nei & Li similarity coefficient
complement, is estimated using the Genes Program. The genetic distances matrix is used for
dendrogram grouping analysis and UPGMA ("Unweighted pair-group arithmetic average") is the
grouping criteria. Graphical dispersion is based in multidimensional scales using the principal
coordinates method. SAS and Statistic are the statical packages used in the analysis. Beside distance
analysis, DNA fingerprinting is used for intra-specific comparisons.
Geographical Information System: ArcView GIS (www.esri.com) was used to plot the collection
site co-ordinates for each accession. Ecological descriptors for each of these sites were obtained by
superimposing SIG maps containing rainfall, vegetation and soil information, state, municipalities
and hydrographic basins borders (scale 1:1.000.000)
Results and Discussion
DNA extraction methodology was developed and successfully tested for a number of
Cerrado native species. Estimations about the extracted DNA amount by spectrophotometry at 260
nm (A260), and the A260 / A280 purity analysis showed that it was possible to extract over 37 g of
good quality DNA from each sample. Electrophoresis in 0,8% agarose gel showed the DNA
integrity in samples from all species in the study (Figure 1).
1 2
3 4 5 6 7 8
9 10
Figure 1. Genomic DNA from 10 Cerrado native species: 1. Stylosanthes guianensis; 2. S.
macrocephala; 3. Dypterix alata; 4. Caryocar brasiliense; 5. Hancornia speciosa; 6.
Eugenia dysenterica; 7. Annona crassiflora; 8. Acrocomia aculeata; 9. Dimorphrandra
mollis; 10. Stryphnodendron barbadetimam.
DNA analysis using molecular markers have several applications at Embrapa Cerrados:
Identification of duplicated or redundant germplasm: The study using RAPD markers in Arachis
pintoi showed that cv. Belmonte has high genetic similarity (respectivelly, 0,961 e 0947) with the
accessions Ap 61 e Ap 65. Based on this high similarity and considering the collection site of the
three materials, (Itabuna, Simões Filho e Jussari), all municipalities of Bahia State, it was verified
that these accessions had the same genetic origin (Figure 2).
Figure 2. Cluster analysis of 10 Arachis
pintoi accessions, based on a genetic
distances matrix, and adopting the UPGMA
(Unweighted pair-group arithmetic average)
method.
Seed genetic purity analysis: Using RAPD markers, studies about genetic variability in progenies
from accessions of Stylosanthes macrocephala (Figure 3) has allowed genetic purity analysis in
each accession and the identification of seed mixtures and of plants from casual cross pollination.
Figure 3. Amplification products from 11
plants of S. macrocephala CPAC 1192
generated by OPE-01 primer. Arrow shows
polymorphism in plant 3.
1 2 3 4 5 6 7 8 9 10 11
B
Development of core collections: The characterization of Cerrado native plants, using molecular
markers, allowed the choice of accessions to compose core collections, representing the variability
existing in base collections, which, normally, have a high number of accessions. Figure 4 shows the
procedures for the selection of 20 Stylosanthes macrocephala accessions, corresponding to 15% of
base collection with 135 accessions.
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Figura 4. Graphical dispersion of 135
Stylosanthes macrocephala accessions based
on a genetic distances matrix from 180
RAPD markers. In the figure, asterisks
marked accessions (*) are the accessions
included in the core collection.
*
Parental selection in breeding programs: Molecular markers have been used in genetical diversity
analysis for parental selection in hybridisation, a basic and fundamental phase for a breeding
program success. Parental selection based on genetic diversity maximises the chances for gene
combinations for the desired character, increasing the breeding program success. As an example,
Figure 5 shows the selection of 10 parentals from a work collection containing 35 Stylosanthes
guianensis accessions.
Confirmation of hybridization: Hybridization can be confirmed from easily visualized dominant
characters which are contrasting among species or accessions (genetic markers). DNA markers are
useful when these characters are not available, when they are not visually detected or when they are
expressed only in specific plant growth phases. In a passion flower breeding program, seeking
multiple disease resistance, DNA markers have been used to confirm hybridization between
commercial varieties and wild expontaneous species existing in the Cerrados flora. For
hybridization
studies, DNA markers and morphological characters follow the same principle
(Figure 6).
Figure 5. Grouping analysis for 35 S.
guianensis accessions based on genetic
distances generated by 159 RAPD
markers. UPGMA method was used as a
grouping criteria. Parentals selected on a
genetic diversity base are shown by
arrows.
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6
7
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11
Relative Genetic Distance
Relative Genetic Distance
Figura 6. Use of molecular markers and morphological
characters to confirm artificial hybridization.
Fingerprinting studies: Among other applications, fingerprinting is useful for Stylosanthes
guianensis botanical classification, identification of seed contamination and as a complement in
agronomic characterization. Figure 7A and 7B show, respectively, fingerprinting analysis in
mangaba (Hancornia speciosa) and pequi (Caryocar brasiliense) selected stock plants. These
results increase the possibilities for selecting the best stock plants and allow the expansion of the
genetic basis of stock plants which are both adapted and recommended for distinct agro-ecosystems.
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Figure 7. Grouping analysis of 8 mangaba (Hancornia speciosa) (A) and 15 pequi (Caryocar
brasiliense) selected stock plants based on the genetic distances matrix generated by
RAPD markers. UPGMA method was used as a grouping criteria.
Applications in ecological studies: Integrated use of genetic diversity obtained by molecular
markers and GIS data makes possible the identification of areas (hot spots) showing great diversity
for a species. This information is important for orientation of collection and “in situ” conservation.
Ecological information about collecting sites is also important for establishment of core and subset
collections, and for the identification of accessions with desirable agronomic characteristics, such as
high productivity, drought or illness resistance, and low soil fertility tolerance. Figure 8 shows the
sites in Bahia e Minas Gerais states where S. macrocephala acessions showing high variability were
collected. Identification of these sites was based on a integrated use of molecular markers and GIS
data (Costa, 2003).
Figure 8. Collecting sites of S. macrocephala showing high
genetic diversity.
Conclusions
The association of molecular markers and GIS data is an important information source about
the whole conservation process and for an efficient assessment and use of genetic resources.
However, the molecular markers can not substitute genetic markers such as those based on
morphological and agronomic characters.
References
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FALEIRO, F.G. Marcadores genético-moleculares aplicados aos programas de conservação e
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FALEIRO, F.G.; FALEIRO, A.S.G.; CORDEIRO, M.C.R., KARIA, C.T. Metodologia para
operacionalizar a extração de DNA de espécies nativas do cerrado. Planaltina: Embrapa
Cerrados, 2003. (Comunicado Técnico No.92) 6p.
COSTA, A.M. Uso de marcadores moleculares e do Sistema de Informações Geográficas no
estudo da diversidade genética de Stylosanthes macrocephala. Brasília: UCB, 2003. 79p. (M.Sc.
thesis). Universidade Católica de Brasília, 2003.