Electronic Journal of Biotechnology ISSN: 0717-3458
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile
Vol.10 No.3, Issue of July 15, 2007
Received October 11, 2006 / Accepted March 5, 2007
RESEARCH ARTICLE
DOI: 10.2225/vol10-issue3-fulltext-2
Extent and structure of genetic diversity in a collection of the tropical
multipurpose shrub legume Cratylia argentea (Desv.) O. Kuntze as
revealed by RAPD markers
Meike S. Andersson*
Biodiversity International
c/o CIAT, A.A. 6713
Cali, Colombia
Tel: 57 2 4450048. Ext. 113
Fax: 57 2 4450069
E-mail: m.andersson@cgiar.org
Rainer Schultze-Kraft
Institute of Plant Production and Agroecology
in the Tropics and Subtropics
University of Hohenheim
D-70593 Stuttgart, Germany
Tel: 49 711 459 23538
Fax: 49 711 459 24207
E-mail: rsk@uni-hohenheim.de
Michael Peters
International Center for Tropical Agriculture
A.A. 6713
Cali, Colombia
Tel: 57 2 4450000. Ext. 3267
Fax: 57 2 445 0073
E-mail: m.peters-ciat@cgiar.org
Myriam C. Duque
International Center for Tropical Agriculture
A.A. 6713
Cali, Colombia
Tel: 57 2 4450000. Ext. 3487
Fax: 57 2 445 0073
E-mail: mc.duque@cgiar.org
Gerardo Gallego
International Center for Tropical Agriculture
A.A. 6713
Cali, Colombia
Tel: 57 2 4450000. Ext. 3265
Fax: 57 2 445 0073
E-mail: g.gallego@cgiar.org
Websites: http://www.uni-hohenheim.de
http://www.ciat.cgiar.org
Keywords: agroforestry, molecular markers, MPT (multipurpose shrubs and trees), tropics.
Abbreviations: cv.: cultivated variety
MCA: multiple correspondence analysis
RAPD: random amplified polymorphic DNA
The tropical multipurpose shrub legume Cratylia
argentea is well adapted to acid soils of low to medium
fertility and has excellent drought-tolerance. Due to its
high nutritive value it is particularly suited as forage for
dry-season supplementation. A collection of 47 C.
argentea accessions in a collection, derived from seed
*Corresponding author
This paper is available on line at http://www.ejbiotechnology.info/content/vol10/issue3/full/2/
Andersson, M.S. et al.
replicating of original accessions with differing
geographic origin and morphological and agronomic
characteristics was investigated using molecular
markers (RAPD (random amplified polymorphic
DNA)). Genetic diversity (HT = 0.145) in the collection
was low, with 30% of differentiation among groups and
high genetic similarity among accessions (GS = 0.805).
Within-accession variability was high. One taxonomic
mismatch and five possible duplicate accessions were
identified. Our results suggest that the genetic diversity
in the C. argentea accessions studied is relatively
homogeneously distributed, indicating the likelihood of
extensive outcrossing. The genetic diversity of original
accessions should be assessed to determine if
outcrossing has occurred during or before ex situ
storage. This might also support any decision on
whether accessions should be bulked rather than
maintaining them individually.
Cratylia argentea (Desv.) O. Kuntze (syn. C. floribunda
Benth., Dioclea argentea Desv.) is a drought-tolerant
tropical multipurpose shrub legume with high potential for
animal
nutrition,
particularly
for
dry
season
supplementation and silage, due to its high nutritive value
(Argel and Lascano, 1998). The leafy shrub usually reaches
a height of 1.5 to 3 m and has the potential to remain green
and productive during dry seasons up to seven months. It is
well adapted to acid soils of low to medium fertility and to
elevations up to 1200 m.a.s.l.
Cratylia argentea contains only traces of tannins and its
nutritive value is higher than that of most other shrub
legumes adapted to acid soils (Schultze-Kraft, 1996). The
species shows excellent regrowth after cutting and can be
used as forage, soil cover, mulch and green manure (Argel
and Lascano, 1998; Lascano et al. 2002). It is a valuable
protein source in livestock production systems, particularly
during the dry season, both in cut-and-carry (fresh fodder
and silage) and in grazing systems (Argel and Lascano,
1998; Lascano et al. 2002). C. argentea can be easily
propagated by seed, whereas vegetative propagation was
unsuccessful (Pizarro et al. 1996).
The genus Cratylia is native to South America where it
occurs south of the Amazon river, from western Peru to the
state of Ceará in Brazil. The taxonomic delimitations within
the genus are not yet defined. According to Queiroz and
Coradin (1996) there are five species distinguished based
on morphological characteristics and geographical
distribution. Four of them, C. mollis Mart. ex Benth., C.
hypargyraea Mart. ex Benth., C. bahiensis L.P. Queiroz
and C. intermedia (Hassl.) L.P. Queiroz and R. Monteiro,
are restricted to Brazil. The distribution range of C.
argentea extends from western Peru to Bolivia and Brazil.
The species occurs in a broad range of biogeographical
regions such as the Cerrado, Caatinga, Yungas and Amazon
provinces.
Collections of C. argentea are held in Brazil by Embrapa
Recursos Genéticos e Biotecnologia, Brasília and Embrapa
Cerrados, Planaltina (45 and 48 accessions, respectively),
and by the International Center for Tropical Agriculture
(CIAT), Cali, Colombia (57 accessions). However, a
reconciled total collection probably comprises not more
than 50 different accessions. The germplasm was mostly
collected during general wild-legume collecting missions,
i.e., where the target was a wide range of species.
Depending on topography and soil variability, not only the
distance between general legume sampling sites in these
missions was variable but, subsequently, also the distance
between individual C. argentea accessions. The accessions
were collected in a diversity of environments and broadly
represent the natural distribution of the species.
Agronomic and morphological evaluations across a range
of sites in tropical America concentrated on a collection of
11 accessions (Schultze-Kraft, 1996) and as a result,
acommercial variety consisting of a mixture of accessions
CIAT 18516 and CIAT 18668 was released in Costa Rica
as cv. (cultivated variety) Veraniega and in Colombia as cv.
Veranera (Lascano et al. 2002). Recently, the agronomic
and morphological diversity in an expanded collection of
38 accessions was described by Andersson et al. (2006a).
The aim of the present study was to complement the latter
variability assessment by a molecular marker approach. The
specific objectives were to assess and describe the genetic
diversity (a) within a collection of 47 accessions, and (b)
within accessions, and (c) identify possible duplicates and
taxonomic mismatches. When choosing a molecular marker
technique, one should not only consider the particular
objective(s) of the study, but also the technical
requirements and the cost and time investments implied. In
the present case, random amplified polymorphic DNA
(RAPD) markers were chosen since they are an effective
and relatively inexpensive technique and do not require any
prior sequence information (Jones et al. 1997; Chenuil,
2006). These dominant neutral markers sample the genome
randomly generating multiple numbers of amplifiable
polymorphic fragments, and can be applied to a wide range
of plant and animal taxa. They are especially useful for
distinguishing individuals, cultivars or accessions, and
allow the quantification of similarities or differences (Jones
et al. 1997; Parker et al. 1998).
MATERIALS AND METHODS
Plant material and DNA extraction
The 47 Cratylia argentea accessions used in this study
were collected mainly in the Brazilian states of Mato
Grosso and Goiás in three collection missions in 1984,
1993 and 1995 (Figure 1, Table 1). Accessions are bulk
samples of three plants per accession, representing
populations at their respective collection sites. Two
accessions (CIAT 18672 and CIAT 18957) were collected
at the northern limit of the natural distribution range of the
387
RAPD genetic diversity in Cratylia argentea
species in the Brazilian states of Pará and Tocantins, south
of the Amazon river (03º 45’ S and 06º 30’ S, respectively).
The only non-erect but prostrate and strongly climbing
accession (CIAT 22397 'Yapacaní') was collected in Santa
Cruz, Bolivia. Cratylia mollis CIAT 7940 was included as
additional reference to validate the genetic assessment of C.
argentea, expecting the genetic similarity among C.
argentea accessions to be greater than between accessions
of C. argentea and C. mollis, another drought tolerant
legume shrub native to the dry Caatinga of north-eastern
Brazil (Queiroz and Coradin, 1996).
At CIAT, the entire C. argentea collection is replicated
every 7 to 10 years. Accordingly, the plant material used in
this study has undergone one (accessions collected between
1993 and 1998) or two (accessions collected in 1984)
multiplication cycles, respectively (A. Ciprián, CIAT GRU,
personal communication 2006). Seedlings were raised in
the greenhouse and transplanted to the field at the CIATQuilichao Experimental Station (Cauca department,
Colombia; 03º 06' N, 76º 31' W; 990 m.a.s.l). Young leaves
were harvested in the field just before reaching fully
developed size. For the determination of genetic variability
among accessions, bulked samples were taken from three
individual plants per accession. For the assessment of
within-accession genetic variability, leaves were harvested
separately from ten individual plants of each of five
accessions (CIAT 18516, CIAT 18668, CIAT 18674, CIAT
22408 and CIAT 22409). Samples were macerated in liquid
nitrogen and total genomic DNA extracted from 50 mg
tissue using a small-scale DNA extraction method (Qiagen
DNeasy® Plant Mini Extraction Kit, Qiagen Inc., Valencia,
CA, USA) with minor modifications: 600 instead of 400 μl
Buffer AP1, 2 instead of 4 μl RNAse, and 150 instead of
130 μl Buffer AP2. DNA was quantified by means of a
DyNA QuantTM 200 fluorometer (Hoefer Scientific
Instruments, San Francisco, CA, USA) and diluted to a
final concentration of 5 ng DNA/μl.
RAPD markers
The protocol for RAPD analysis was as described in
Andersson et al. (2006b), with the volume of the final
reaction (25 μl) being composed of 1X PCR buffer (50 mM
KCl, 10 mM Tris-HCl pH 8.8, 0.1% Triton x-100), 2.5 mM
MgCl2, 0.2 mM of each dNTPs, 0.2 μM primer (Series
OPD, OPG, OPI and OPJ from Operon Technologies,
Alameda, CA, USA), 1 U Taq DNA polymerase (Promega
Corp., Madison, WI, USA) and 25 ng of template DNA. A
negative control without template DNA was included in
each round of reactions. Amplifications were performed in
a thermocycler (PTC-100TM, MJ Research Inc., Watertown,
MA, USA) with an initial denaturing step of 5 min at 94ºC
followed by 40 cycles of 30 sec at 94ºC, 30 sec at 38ºC and
1 min at 72ºC and a final extension step of 5 min at 72ºC.
The PCR products were run on a 1.4% agarose gel at 20
V/cm during 45 min. The amplified DNA fragments were
visualised by ethidium bromide staining (0.5 μg/ml
bromide in gel and buffer) under UV-light, and
photographed with a Kodak digital camera DC 120
(Software Kodak Digital ScienceTM 1997).
Data analysis
Amplified DNA fragments were manually scored as present
(1) or absent (0) for each primer, and variations in band
presence were recorded as polymorphisms.
Data were converted into a similarity matrix using the Nei
and Li similarity coefficient based on the proportion of
shared fragments. The Nei and Li similarity coefficient
(Nei and Li, 1979) calculates the genetic similarity (GS)
between two samples, i and j, with the formula GS(i,j) =
2Nij / (2Nij + Ni + Nj), where Nij is the number of bands
present in both i and j, Ni is the number of bands present in
i and absent in j, and Nj is the number of bands present in j
and absent in i.
Then, a similarity tree was produced by clustering the
similarity matrix based on the Average Linkage UPGMA
(unweighted pair group method with arithmetic averages)
algorithm. The dendrogram was cut considering a
cophenetic value of 0.97 (R2), which resulted in 7 groups
and a level of 77% of genetic similarity. The calculation of
genetic similarity and cluster analysis were performed
using the NTSYS package version 2.1. In addition, multiple
correspondence analysis (MCA) was performed on the
original matrix to provide an additional representation of
genetic similarity and to visualize the dispersion of
individuals in relation to their molecular profile, based on
the first three principal axes of variation. Means of genetic
similarity and genetic diversity within and among groups
were determined for both methods (cluster analysis and
MCA) to analyse the coherence of the resulting
classifications. MCA and means of genetic diversity were
calculated using SAS version 8.2.
The efficiency of the RAPD primers to differentiate among
accessions was evaluated by calculating the discriminatory
power DL = 1 - ∑ pi2, with pi being the proportion of
accessions carrying the ith banding pattern at the jth primer
and calculating pi for each pattern generated by the primer
(Tessier et al. 1999). DL is an extension of the
polymorphism information content (PIC) and provides an
estimate of the probability that two randomly chosen
individuals show different banding patterns for the same
primer, and thus are distinguishable from one another.
RESULTS
RAPD profiles
Out of the 47 oligonucleotide decamer primers initially
screened with three C. argentea accessions, 17 gave
smeared or faint bands and seven gave no amplification
products. Fifteen primers identified high levels of
polymorphism and were repeated to test for reproducibility.
Out of these, six primers detected distinct, clearly resolved
388
Andersson, M.S. et al.
and consistently reproducible amplification products and
were, therefore, selected for the amplification of RAPD
sequences. They generated a total of 72 scorable fragments
across the 47 C. argentea accessions and the C. mollis
reference accession (Table 2). On average 11 scorable
fragments were obtained per primer, ranging from six
(primer OPJ 12) to 16 (primers OPG 12 and OPI 07). The
band size ranged from 320 to 2900 bp. A RAPD profile
generated by primer OPD 15 is shown in Figure 2.
Out of the 72 fragments detected across all analysed
accessions, 68 (94%) were polymorphic. Among C.
argentea accessions, 61 fragments were detected, 56 (92%)
being polymorphic. Eleven markers generated by primers
OPD 15, OPG 12, OPI 07 and OPJ 12 were unique to C.
mollis, and five markers (all generated by primer OPI 07)
were unique to C. argentea accessions. Of these, three were
unique to the only non-erect C. argentea accession CIAT
22397 ('Yapacaní'), and the other two were found in CIAT
22399 and CIAT 22403, respectively. In addition, three
markers (generated by primers OPJ 06 and OPJ 12) were
common to C. mollis and 'Yapacaní', but were absent in the
other C. argentea accessions. Although the two accessions
CIAT 18672 and CIAT 18957 were collected at the
northern limit of the natural distribution range of the
species and were geographically as distant from the rest of
the collection as was the Bolivian accession 'Yapacaní'
(Figure 1), no fragments unique to these accessions were
detected.
The discriminatory power (DL) of the selected primers
ranged from 0.615 to 0.942, with four primers having >
85% probability of discriminating between two accessions
(Table 2). As reported by Tessier et al. (1999) our data
showed that the discriminatory power of a primer does not
solely depend on the number of bands and banding patterns
it generates. For example, primer OPD 15, which generated
nine polymorphic bands and 18 different banding patterns,
had a discriminatory power of 90%, whereas primer OPI 07
with more polymorphic bands (16) and a similar number of
banding patterns (16) had a much lower discriminatory
power (75%).
Cluster and multiple correspondence analyses
The dendrogram based on Nei and Li genetic similarity,
provides seven groups at the 77% level of genetic similarity
(Figure 3). First, the C. mollis reference accession is
separated, followed by the only non-erect accession
'Yapacaní' (CIAT 22397). Subsequently, two groups each
containing only one accession are split: CIAT 18668 and
CIAT 22389. Next, a 4-member group comprising
accessions CIAT 18674, CIAT 22384, CIAT 22386 and
CIAT 22401, and a single accession (CIAT 22408) are
separated. The remaining 39 accessions (81%) fall into one
main cluster (Figure 3).
MCA confirms the separation of the C. mollis reference and
the non-erect C. argentea accession 'Yapacaní' from the
remaining accessions (first and second dimension,
accounting for 27% and 14% of the total variation,
respectively). The multiple correspondence coordinates of
each of the three groups (C. mollis, 'Yapacaní' and the
remaining C. argentea accessions) form the extreme points
of an almost equally-sided triangle, indicating that the nonerect C. argentea accession 'Yapacaní' is genetically almost
as distant from all other C. argentea accessions as is C.
mollis. The third dimension discriminates the remaining 46
C. argentea accessions into two main and three smaller
groups (Figure 4). Accession CIAT 18668 is grouped
together with CIAT 22389 and CIAT 22403 (group 5 in
Figure 4), and CIAT 22386 with CIAT 22387 (group 4).
Accession CIAT 18674 makes up a group of its own (group
3). The remaining 40 accessions (83%) fall into two main
clusters, containing 28 and 12 accessions, respectively.
Geographic origin (i.e. distance) had only limited influence
on the composition of the groups. In several instances,
genetically similar accessions were detected from
geographically very distant areas (e.g. CIAT 22373 and
CIAT 22409), whereas geographically close accessions
often appeared to be genetically very distant in both cluster
analysis and MCA (e.g. CIAT 18675 and CIAT 22408).
Here again, it was not possible to distinguish the two
accessions CIAT 18672 and CIAT 18957, which were the
most distant from the rest of the collection in terms of
geographic distance (Figure 1). Both accessions clustered
tightly together with other accessions distributed widely
throughout Central Brazil, in cluster as well as in MCA.
Genetic diversity and genetic similarity among
and within groups
Total genetic diversity in the sample studied here was low
(HT = 0.169), with only 30% of differentiation among
groups (GST = 0.302) (Table 3). The mean genetic similarity
was high (GS = 0.776) and ranged from 0.388 to 0.814
among groups (Table 4) and from 0.316 to 0.947 among
accessions (data not shown). When excluding the two most
genetically distant accessions, 'Yapacaní' and C. mollis
(groups 6 and 7, respectively), total diversity decreased to
HT = 0.145, and the coefficient of gene differentiation was
reduced by half to 15.2% (Table 3). Mean GS increased to
0.805, ranging from 0.720 to 0.814 among groups (Table 4)
and from 0.604 to 0.947 among accessions (data not
shown). The genetic similarity between groups (Table 4,
above diagonal) was as high as or even higher than within
groups (Table 4, diagonal).
Genetic diversity and genetic similarity within
accessions
To compare mean genetic diversity within and among C.
argentea accessions, ten individual plants of each of five
accessions were chosen. Total genetic diversity HT among
these accessions was 0.180, while mean genetic diversity
within accessions (HS) was 0.152, representing 84% of total
genetic variation. Mean genetic similarity GS was 0.720,
389
RAPD genetic diversity in Cratylia argentea
ranging from 0.730 to 0.800 within the five accessions
studied (Table 5).
due to extensive outcrossing and geneflow occurring over
distances of several kilometres (Schierenbeck et al. 1997).
Possible duplicates in the collection
Mating system
If considering accessions as genetically identical when their
genetic similarity GS was equal to or greater than 0.95, then
all accessions were uniquely identified, indicating that the
collection does not contain genetic duplicates. However, a
group of possible duplicate candidates was identified
(CIAT 22373, CIAT 22378, CIAT 22380, CIAT 22381 and
CIAT 22411) with pairwise genetic similarities very close
to this value (GS > 0.94).
Among various factors that determine the genetic
composition of plant populations, mating system is the
most influential. High polymorphism levels of the
magnitude observed in the present study for C. argentea
(92%) are reported for outcrossing species, whereas
predominantly selfing and/or clonal species generally show
much higher proportions (45-80%) of monomorphic loci
(Zhivotovsky, 1999). Similarly, the relatively low
differentiation among groups and the high genetic diversity
within groups, are characteristic for predominantly
outcrossing species, whereas for inbreeding species this
relationship tends to be inverted (Nybom, 2004).
DISCUSSION AND CONCLUDING REMARKS
In this first study of genetic diversity in Cratylia with
RAPD molecular technique, the level of DNA
polymorphism detected with six decamer random primers
was very high (> 90%) and allowed the distinction of all
accessions analysed. The high discriminatory power of the
primers used (83% on average) indicates that the RAPD
technique provides an effective tool for germplasm analysis
in Cratylia.
Genetic structure within the collection
Overall genetic diversity in the collection was low, with
high genetic similarity among accessions. In other words,
the frequencies/abundances of polymorphic RAPD
fragments in the analysed collection were relatively low,
and the distribution pattern of these few polymorphic
fragments was very similar for the majority of the
accessions, leading to high genetic similarity values.
Cluster and multiple correspondence analyses showed the
clear separation of C. mollis and the non-erect accession
'Yapacaní'. The grouping of the remaining 46 C. argentea
accessions differed slightly when based on UPGMA
clustering or MCA, indicating that there was no clear
pattern within this bulk of accessions. The low genetic
differentiation and high genetic similarity among groups
show that the low genetic diversity is fairly homogeneously
distributed in the collection, without any particular pattern.
Also, geographic origin (i.e. distance) apparently did not
affect the composition of these groups, since in several
instances genetically similar accessions were detected from
geographically very distant areas, whereas geographically
close accessions often appeared to be genetically very
distant in both cluster analysis and MCA. In addition, the
genetic variability within accessions was high, with genetic
similarities within accessions being only slightly greater
than mean similarity among accessions. These findings
suggest the existence of a single, widely shared genepool
with limited genetic separation among accessions, and
indicate the likelihood of extensive outcrossing. These
results agree with studies of tropical tree population
structures. Tropical tree species appear to have large
genepools, with high diversity and little genetic structure,
Generally, tropical legumes have been considered as
predominantly self-pollinated, but there is increasing
evidence of cross-pollination in many species, suggesting
that some outcrossing may occur in most legume species
(Maass and Torres, 1998). Very little is known about the
genetics of C. argentea and it is unclear whether C.
argentea is allogamous or autogamous. While Queiroz et
al. (1997) reported from Planaltina, Brazil, the occurrence
of outcrossing and suggested a mixed mating system,
Bystricky et al. (2006) found C. argentea to be selfincompatible under the conditions of CIAT-Quilichao. Our
findings provide further evidence for this. Furthermore,
field observations suggest that tripping is involved in
fertilization (Andersson et al. 2006a). Pollinators such as
the exotic honeybee Apis mellifera, and other bees and
bumblebees belonging to the genera Bombus, Centris and
Xylocopa (Hymenoptera: Apoidea) have been observed to
visit C. argentea flowers loaded with pollen (Queiroz,
1996). Some of these pollinators have been reported to be
capable of flight distances of several kilometers thus
effecting pollen dispersal between plants at great distances,
e.g. the honeybee A. mellifera (Ricketts, 2004) and the
carpenter bee Xylocopa fimbriata Fabr. (Gonzalez and
Engel, 2004). At CIAT, C. argentea accessions are
multiplied in plots that are at least 300 m distant and hence,
isolated from each other to a certain extent (A. Ciprián,
CIAT GRU, personal communication 2006). The
accessions studied here have not undergone more than two
multiplication cycles since seed increase has started. It thus
appears that cross-pollination may have occurred also
among and within original populations in the wild.
However, we suggest precautions to be taken by genebanks
during the regeneration of C. argentea accessions to avoid
cross-pollination, and the re-consideration of multiplication
protocols in order to maintain the genetic integrity of
accessions. More detailed studies (e.g. with co-dominant
molecular markers) are required to identify the prevailing
reproductive strategy of C. argentea and to assess the rate
and impact of outcrossing in this species.
390
Andersson, M.S. et al.
Possible duplicates and taxonomic mismatches in
the collection
supported by the German Academic Exchange Service
(DAAD) and the Eiselen-Stiftung Ulm, Germany.
Although genetically all accessions were significantly
different (P < 0.05) from each other, a group of five
possible duplicate accessions (CIAT 22373, CIAT 22378,
CIAT 22380, CIAT 22381, CIAT 22411) was identified,
which shared more than 94% of their banding pattern,
differing in three bands only. Re-analysing these accessions
with an additional primer or with additional marker types
such as AFLP (amplified fragment length polymorphisms)
would likely lead to a clearer molecular differentiation
between pairs.
References
Ordination was particularly useful in visualising that the
non-erect accession 'Yapacaní', the only germplasm from
Bolivia, was genetically almost as distinct from all other C.
argentea accessions as was C. mollis. This, together with its
unusual growth habit (climbing vine), suggests that there
may be a misclassification of the 'Yapacaní' accession - in
spite of the fact that according to the botanical literature
(Queiroz and Coradin, 1996), the only Cratylia speciesthat
occurs in Bolivia is C. argentea. Botanical specimens from
Bolivia and Peru should be re-examined for an eventual
taxonomic revision of the genus Cratylia. Also, further
germplasm from longitudes higher than 58º W (Figure 1)
should be collected and its genetic diversity analyzed in
support of taxonomic re-considerations.
With respect to accessions CIAT 18516 and CIAT 18668
which form the commercial cultivar, it was striking that
CIAT 18668 was genetically very different from CIAT
18516 as well as from most other C. argentea accessions
(Figure 3). In agronomic evaluations, however, these two
accessions were not only very similar regarding
morphological characteristics and forage quality
(Andersson et al. 2006a), but also regarding consistently
high DM production in different environments. The fact
that in terms of RAPD variation these accessions appear to
be very distinct might be an indicator of the neutral (i.e.
non-functional) nature of the polymorphisms detected by
this molecular technique. Furthermore, it is unknown to
which extent the agronomic traits above are influenced by
the environment. RAPD markers therefore are not an
adequate tool for predicting or explaining agronomic
characteristics. These contradictions probably could be
solved either using morphological characters that are less
sensitive to environmental variation, or performing more
thorough examinations with more sophisticated techniques
(e.g. ISSR-PCR).
Acknowledgments
We express our gratitude to two anonymous referees
designated by CIAT for helpful comments on an earlier
version of this draft. Thanks are also due to J. Tohme from
the CIAT Biotechnology Unit for facilitating laboratory
infrastructure. The study is part of a project financially
ANDERSSON, Meike S.; PETERS, Michael; SCHULTZEKRAFT, Rainer; FRANCO, Luis-Horácio and LASCANO,
Carlos E. Phenological, agronomic and forage quality
diversity among germplasm accessions of the tropical
legume shrub Cratylia argentea. Journal of Agricultural
Science, June 2006a, vol. 144, no. 3, p. 237-248.
ANDERSSON, Meike S.; PETERS, Michael; SCHULTZEKRAFT, Rainer; GALLEGO, Gerardo and DUQUE,
Myriam C. Molecular characterization of a collection of the
tropical
multipurpose
shrub
legume
Flemingia
macrophylla. Agroforestry Systems, November 2006b, vol.
68, no. 3, p. 231-245.
ARGEL, Pedro J. and LASCANO, Carlos E. Cratylia
argentea (Desvaux) O. Kuntze: una nueva leguminosa
arbustiva para suelos ácidos en zonas subhúmedas
tropicales. Pasturas Tropicales, April 1998, vol. 20, no. 1,
p. 37-43.
BYSTRICKY, Maria; PETERS, Michael; ESCOBAR,
Germán; SCHULTZE-KRAFT, Rainer and FRANCO, Luis
Horacio. Floral biology of Cratylia argentea - First results
of a study in Colombia. In: Deutscher Tropentag 2006:
Prosperity and poverty in a globalized world - challenges
for agricultural research (11th - 13th October, 2006,
University of Bonn, Germany). Book of abstracts. p. 349.
CHENUIL, Anne. Choosing the right molecular genetic
markers for studying biodiversity: from molecular
evolution to practical aspects. Genetica, May 2006, vol.
127, no. 1-3, p. 101-120.
GONZÁLEZ, Victor H. and ENGEL, Michael S. The
tropical Andean bee fauna (Insecta: Hymenoptera:
Apoidea), with examples from Colombia. Entomologische
Abhandlungen, June 2004, vol. 62, no. 1, p. 65-75.
JONES, Neil; OUGHAM, Helen and THOMAS, Howard.
Markers and mapping: we are all geneticists now. New
Phytologist, September 1997, vol. 137, no. 1, p. 165-177.
LASCANO, Carlos E.; RINCÓN, Álvaro; PLAZAS,
Camilo; ÁVILA, Patricia; BUENO, Guillermo and
ARGEL, Pedro J. Cultivar Veranera (Cratylia argentea
(Desvaux) O. Kuntze): leguminosa arbustiva de usos
múltiples para zonas con períodos prolongados de sequía
en Colombia. Cali, Colombia, Corporación Colombiana de
Investigación Agropecuaria (CORPOICA), International
Center for Tropical Agriculture (CIAT), 2002. 28 p.
Available
from
Internet:
http://www.ciat.cgiar.org/forrajes/pdf/cratyllia_argentea_cv
_veranera.pdf.
391
RAPD genetic diversity in Cratylia argentea
MAASS, Brigitte L. and TORRES, Alba M. Off-types
indicate natural outcrossing in five tropical forage legumes
in Colombia. Tropical Grasslands, June 1998, vol. 32, no.
2, p. 124-130.
NEI, Masatoshi and LI, Wen-Hsiung. Mathematical model
for studying genetic variation in terms of restriction
endonucleases. Proceedings of the National Academy of
Sciences of the United States of America, October 1979,
vol. 76, no. 10, p. 5269-5273.
RICKETTS, Taylor H. Tropical forest fragments enhance
pollinator activity in nearby coffee crops. Conservation
Biology, October 2004, vol. 18, no. 5, p. 1262-1271.
SCHIERENBECK, Kristina A.; SKUPSKI, Marian P.;
LIEBERMAN, Diana and LIEBERMAN, Milton.
Population structure and genetic diversity in four tropical
tree species in Costa Rica. Molecular Ecology, February
1997, vol. 6, no. 2, p. 137-144.
NYBOM, Hilde. Comparison of different nuclear DNA
markers for estimating intraspecific genetic diversity in
plants. Molecular Ecology, May 2004, vol. 13, no. 5, p.
1143-1155.
SCHULTZE-KRAFT, Rainer. Leguminous forage shrubs
for acid soils in the tropics. In: ELGERSMA, Anjo;
STRUIK, Paul C. and MAESEN, Jos L.G. van der eds.
Grassland
Science
in
Perspective.
Wageningen
Agricultural University Papers 96-4, 1996, p. 67-81.
PARKER, Patricia G.; SNOW, Allison A.; SCHUG,
Malcolm D.; BOOTON, Gregory C. and FUERST, Paul A.
What molecules can tell us about populations: choosing and
using a molecular marker. Ecology, March 1998, vol. 79,
no. 2, p. 361-382.
TESSIER, Cécile; DAVID, Jaques L.; THIS, Patrice;
BOURSIQUOT, Jean-Michel and CHARRIER, André.
Optimization of the choice of molecular markers for
varietal identification in Vitis vinifera L. Theoretical and
Applied Genetics, January 1999, vol. 98, no. 1, p. 171-177.
PIZARRO, Esteban A.; CARVALHO, Marcelo A. and
RAMOS, Allan K.B. Introducción y evaluación de
leguminosas forrajeras arbustivas en el Cerrado brasileño.
In: PIZARRO, Esteban A. and CORADIN, Lídio eds.
Potencial del género Cratylia como leguminosa forrajera.
Memorias del taller de trabajo realizado el 19 y 20 de julio
1995, Brasilia, DF, Brasil. Documento de Trabajo No. 158,
Cali, Colombia, International Center for Tropical
Agriculture (CIAT), 1996, p. 40-49. Available from
Internet:
http://www.ciat.cgiar.org/forrajes/pdf/Cratylia_02(783).pdf.
ZHIVOTOVSKY, Lev A. Estimating population structure
in diploids with multilocus dominant DNA markers.
Molecular Ecology, June 1999, vol. 8, no. 6, p. 907-913.
QUEIROZ, Luciano P. de. Pollination ecology studies in
Cratylia Mart. ex Benth. (Leguminosae: Papilionoideae)
and its taxonomic and evolutionary implications. Sitientibus
(UEFS), 1996, vol. 15, p. 119-131.
QUEIROZ, Luciano P. de and CORADIN, Lídio.
Biogeografia de Cratylia e areas prioritárias para coleta. In:
PIZARRO, Esteban A. and CORADIN, Lídio eds.
Potencial del género Cratylia como leguminosa forrajera.
Memorias del taller de trabajo realizado el 19 y 20 de julio
1995, Brasilia, DF, Brasil. Documento de Trabajo No. 158,
Cali, Colombia, International Center for Tropical
Agriculture (CIAT), 1996, p. 1-28. Available from Internet:
http://www.ciat.cgiar.org/forrajes/pdf/Cratylia_02(783).pdf
QUEIROZ, Luciano P. de; SILVA, Mario M. da; RAMOS,
Allan K.B. and PIZARRO, Esteban A. Estudos
reprodutivos em Cratylia argentea (Desv.) O. Kuntze e
Cratylia mollis Mart. ex Benth. (LeguminosaePapilionoideae). Pasturas Tropicales, December 1997, vol.
19, no. 3, p. 20-23.
Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication.
Supported by UNESCO / MIRCEN network.
392
Andersson, M.S. et al.
APPENDIX
Figure 1. Geographical distribution of the 47 Cratylia argentea accessions used in this study. The accessions
were originally collected in the states Mato Grosso, Goiás, Tocantins and Pará in Brazil, and in Bolivia.
393
RAPD genetic diversity in Cratylia argentea
Figure 2. RAPD profile obtained using primer OPD15 for 47 Cratylia argentea accessions, C. mollis, one DNAfree control, and five samples of different individuals of accession CIAT 18516 for the assessment of withinaccession variability. Size markers (λ-DNA/PstI, Invitrogen, USA) for assessing base pair lengths are shown in the first
and last lanes.
394
Andersson, M.S. et al.
Figure 3. Grouping of 47 Cratylia argentea accessions and a C. mollis reference accession, using UPGMA. Genetic
distances are according to Nei and Li based on RAPD markers. Clusters were analysed at the 0.77 similarity level (dotted
line).
395
RAPD genetic diversity in Cratylia argentea
Figure 4. Three-dimensional representation of five groups derived from MCA of RAPDs among 46 Cratylia
argentea accessions (without 'Yapacaní').
396
Andersson, M.S. et al.
Table 1. Origin of the Cratylia argentea accessions used in the present study (Queiroz and Coradin, 1996; CIAT Tropical
Forages Database and G.P. da Silva, Embrapa Recursos Genéticos e Biotecnologia [personal communication]).
CIAT
No.
18516
18667
18668
18671
18672
18674
18675
18676
18957
22373
22374
22375
22376
22377
22378
22379
22380
22381
22382
22383
22384
22386
22387
22388
22389
22390
22391
22392
22393
22394
22395
22396
22397
22399
22400
22401
22402
22403
22404
22405
22406
22407
22408
22409
22410
22411
22412
EMBRAPA
No.
000167
000027
000035
000060
000086
000116
000124
000132
000175
000591
000612
000639
000655
000663
000671
000680
000698
000701
000710
000728
000736
000752
000761
000787
000795
000809
000817
000825
000833
000868
000892
000906
‘Yapacaní’
000604
000621
000841
000876
000884
000191
000213
000221
000514
000540
000566
000574
000647
000779
State
Goiás
Mato Grosso
Mato Grosso
Mato Grosso
Pará
Mato Grosso
Mato Grosso
Goiás
Tocantins
Goiás
Goiás
Goiás
Goiás
Goiás
Goiás
Goiás
Goiás
Goiás
Goiás
Goiás
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Mato Grosso
Goiás
Goiás
Bolivia, Santa Cruz
Goiás
Goiás
Mato Grosso
Mato Grosso
Mato Grosso
Goiás
Goiás
Goiás
Goiás
Mato Grosso
Goiás
Goiás
Goiás
Mato Grosso
Latitude
Longitude
Altitude
(masl)
Year of
collection
13º 22’ S
15º 43’ S
15º 22’ S
14º 46’ S
03º 45’ S
14º 38’ S
14º 54’ S
16º 21’ S
06º 30’ S
14º 05’ S
13º 16’ S
13º 01’ S
14º 23’ S
13º 54’ S
13º 37’ S
13º 21’ S
13º 14’ S
13º 14’ S
13º 17’ S
13º 51’ S
14º 14’ S
14º 34’ S
15º 50’ S
16º 23’ S
16º 26’ S
16º 01’ S
15º 49’ S
15º 58’ S
15º 42’ S
15º 52’ S
16º 25’ S
16º 32’ S
17º 25’ S
14º 30’ S
13º 10’ S
15º 51’ S
16º 30’ S
16º 34’ S
13º 01’ S
13º 15’ S
14º 15’ S
14º 54’ S
14º 06’ S
13º 27’ S
13º 17’ S
15º 12’ S
15º 42’ S
46º 25’ W
55º 43’ W
56º 13’ W
57º 05’ W
55º 14’ W
52º 22’ W
52º 17’ W
51º 20’ W
48º 37’ W
46º 23’ W
46º 25’ W
46º 36’ W
49º 09’ W
49º 03’ W
49º 02’ W
47º 07’ W
49º 28’ W
50º 40’ W
50º 12’ W
50º 20’ W
52º 10’ W
52º 21’ W
52º 25’ W
54º 01’ W
54º 19’ W
54º 55’ W
55º 30’ W
55º 00’ W
56º 42’ W
57º 49’ W
51º 35’ W
51º 03’ W
63º 56’ W
46º 24’ W
46º 40’ W
56º 49’ W
54º 36’ W
51º 45’ W
56º 37’ W
46º 28’ W
46º 30’ W
46º 56’ W
46º 25’ W
46º 22’ W
46º 25’ W
46º 47’ W
52º 43’ W
800
455
175
230
140
320
380
450
350
780
660
620
550
510
390
380
340
360
330
300
360
320
370
330
400
300
400
240
210
200
460
510
350
650
660
360
270
600
650
700
780
500
810
540
660
550
400
pre-1980
1984
1984
1984
1984
1984
1984
1984
pre-1980
1995
1995
1995
1995
1995
1995
1995
1995
1995
1995
1998
1995
1995
1995
1995
1995
1995
1995
1995
1995
1995
1995
1995
1998
1995
1995
1995
1995
1995
1993
1993
1993
1995
1995
1995
1995
1995
1995
397
RAPD genetic diversity in Cratylia argentea
Table 2. Oligonucleotide primers employed in RAPD analysis of Cratylia argentea, their sequence, number of polymorphic
(P) and monomorphic (M) bands obtained, percentage of polymorphic bands (% PB), and discriminatory power (DL).
Primer
code
OPD 15
OPG 12
OPI 07
OPJ 06
OPJ 07
OPJ 12
Sum
Total
PB (%)
Sequence
(5’ to 3’)
CATCCGTGCT
CAGCTCACGA
CAGCGACAAG
TCGTTCCGCA
CCTCTCGACA
GTCCCGTGGT
Number of bands
(including C. mollis)
(only C. argentea)
P
M
P
M
9
1
7
1
16
1
11
1
16
1
12
2
13
0
13
0
8
0
8
0
6
1
5
1
68
4
56
5
72
61
94.4
91.8
DL
0.903
0.942
0.755
0.898
0.862
0.615
Table 3. Nei estimates of genetic diversity (heterogeneity) among groups of Cratylia argentea.
Group
n
1
2
3
4
5
6 ('Yapacaní')
7 (C. mollis)
HS
HT
GST
28
12
1
2
3
1
1
Hi(1-7)
0.128
0.126
0.0
0.101
0.120
0.0
0.0
0.118
0.169
0.302
Hi(1-5)
0.128
0.126
0.0
0.101
0.120
0.123
0.145
0.152
n: number of accessions.
Hi: genetic diversity within each group.
HS: average genetic diversity within groups.
HT: total genetic diversity.
GST: coefficient of genetic differentiation (proportion of total genetic
diversity found among groups).
398
Andersson, M.S. et al.
Table 4. Average genetic similarity (GS) values between (above diagonal) and within (diagonal) groups of Cratylia
argentea germplasm, the non-erect accession 'Yapacaní' and the C. mollis reference accession, based on all pairwise
similarities between accessions according to molecular marker information (RAPDs).
Group
1
2
3
4
5
C. argentea
'Yapacaní'
C. mollis
Total
n
28
12
1
2
3
46
1
1
48
1
0.825
2
0.814
0.839
Cratylia argentea
3
4
5
0.769 0.774 0.759
0.720 0.764 0.721
1.000 0.757 0.754
0.717 0.748
0.757
Total
'Yapacaní'
C. mollis
0.487
0.515
0.426
0.479
0.457
0.413
0.404
0.433
0.400
0.388
1.000
0.444
1.000
0.776
0.805
n: number of accessions.
Table 5. Genetic similarity among (above diagonal) and within (diagonal) five Cratylia argentea accessions,
based on all pairwise similarities between individuals according to molecular marker information (RAPDs).
CIAT No.
18516
18668
18674
22408
22409
Total
n
18516
18668
18674
22408
22409
10
10
10
10
10
50
0.730
0.704
0.763
0.724
0.727
0.782
0.669
0.667
0.692
0.737
0.735
0.719
0.753
0.710
0.800
Total
0.720
n: number of individuals per accession.
399