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Analysis of random and specific sequences of nuclear and cytoplasmic DNA in diploid and tetraploid American wild rice species (Oryza spp.)

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A sample of American wild rice and other accessions of the genus Oryza were studied at polymorphic regions of nuclear, mitochondrial, and chloroplastic genomes. First, flow cytometry, genome-specific RAPD markers, and chromosome counting were utilized to verify the original ploidy and classification of 230 accessions studied. Based on these methods, 8% of the accessions were considered to be misclassified either taxonomically or as a result of contamination. Second, a fine resolution analysis was conducted at genomic regions sampled at random by RAPD markers and at specific sites of the chloroplast and mitochondrial DNA by cleaved amplified polymorphic sequence (CAPS) analysis. Phylogenetic trees resulting from phenetic and cladistic analyses of RAPD, cpDNA, and mtDNA polymorphisms were obtained. The results indicated that the American diploid species O. glumaepatula should be considered an individual species, distinct from O. rufipogon, and confirmed that the American tetraploid species (O. alta, O. grandiglumis, and O. latifolia) belong to the O. officinalis complex. The data indicate that these species should still be treated as a group rather than as three distinct species and that their closest relative is a CC-genome species. It was estimated that the diploid and tetraploid American species diverged from O. sativa - O. nivara (AA genome) and CC- and BBCC-genome species, respectively, 20 million years ago.
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Analysis of random and specific sequences of
nuclear and cytoplasmic DNA in diploid and
tetraploid American wild rice species (Oryza spp.)
Glaucia Salles Cortopassi Buso, Paulo Hideo Nakano Rangel,
and Márcio Elias Ferreira
Abstract: A sample of American wild rice and other accessions of the genus Oryza were studied at polymorphic re-
gions of nuclear, mitochondrial, and chloroplastic genomes. First, flow cytometry, genome-specific RAPD markers, and
chromosome counting were utilized to verify the original ploidy and classification of 230 accessions studied. Based on
these methods, 8% of the accessions were considered to be misclassified either taxonomically or as a result of contami-
nation. Second, a fine resolution analysis was conducted at genomic regions sampled at random by RAPD markers and
at specific sites of the chloroplast and mitochondrial DNA by cleaved amplified polymorphic sequence (CAPS) analy-
sis. Phylogenetic trees resulting from phenetic and cladistic analyses of RAPD, cpDNA, and mtDNA polymorphisms
were obtained. The results indicated that the American diploid species O. glumaepatula should be considered an indi-
vidual species, distinct from O. rufipogon, and confirmed that the American tetraploid species (O. alta,
O. grandiglumis, and O. latifolia) belong to the O. officinalis complex. The data indicate that these species should still
be treated as a group rather than as three distinct species and that their closest relative is a CC-genome species. It was
estimated that the diploid and tetraploid American species diverged from O. sativa – O. nivara (AA genome) and CC-
and BBCC-genome species, respectively, 20 million years ago.
Key words: RAPD, cleaved amplified polymorphic sequences, flow cytometry, Oryza glumaepatula, rice evolution.
Résumé : Des échantillons de riz sauvage américain et d’autres accessions du genre Oryza ont été étudiés au niveau de
régions d’ADN polymorphes au sein des génomes nucléaire, mitochondrial et chloroplastique. D’abord, de la cyto-
métrie en flux, des marqueurs RAPD spécifiques d’un génome et des décomptes chromosomiques ont été utilisés pour
vérifier le niveau de ploïdie original et la classification des 230 accessions étudiées. Sur la base de ces travaux, 8 %
des accessions se sont avérées incorrectement classifiées sur le plan taxonomique ou encore souffraient de contamina-
tions. Ensuite, une analyse à résolution fine a été réalisée sur des régions génomiques échantillonnées au hasard à
l’aide de marqueurs RAPD de même que sur des régions spécifiques de l’ADN chloroplastique ou mitochondrial grâce
à des marqueurs CAPS (« cleaved amplified polymorphic sequence »). Des arbres phylogénétiques, résultant d’analyses
phénétiques et cladistiques sur les polymorphismes RAPD, ADNcp et ADNmt, ont été obtenus. Les résultats indiquent
que l’espèce diploïde américaine O. glumaepatula devrait être considérée comme une espèce à part, distincte de l’O.
rufipogon. Il a été confirmé que les espèces américaines tétraploïdes (O. alta,O. grandiglumis et O. latifolia) appar-
tiennent au complexe O. officinalis. Les données suggèrent que ces trois espèces devraient être traitées comme un
groupe plutôt qu’en tant qu’espèces distinctes et que leur plus proche parent est une espèce à génome CC. Il a été es-
timé que les espèces américaines diploïdes et tétraploïdes ont divergé de l’O.sativa O. nivara et des espèces à gé-
nome CC/BBCC ilya20millions d’années.
Mots clés : RAPD, CAPS, cytométrie en flux, Oryza glumaepatula, évolution du riz.
[Traduit par la Rédaction] Buso et al. 494
Introduction
Rice and its wild relatives (Oryza spp.) have a wide geo-
graphical distribution and are found on all continents. Ge-
netic information has been accumulated for a number of rice
species, particularly for cultivated rice (Oryza sativa L.),
which has been a model for plant genetic analysis since the
beginning of the 20th century. The evolutionary history of
Genome 44: 476–494 (2001) © 2001 NRC Canada
476
DOI: 10.1139/gen-44-3-476
Received July 6, 2000. Accepted February 13, 2001. Published on the NRC Research Press Web site May 23, 2001.
Corresponding Editor: G. Jenkins.
G.S.C. Buso1and M.E. Ferreira. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Recursos Genéticos e Biotecnologia,
Laboratório de Genética, C.P. 02372, 77770-900 Brasília DF, Brasil.
P.H.N. Rangel. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Arroz e Feijão, C.P. 179, 75375-000 Santo Antônio de
Goiás, Goiânia, Brasil.
1Corresponding author (e-mail: buso@cenargen.embrapa.br).
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the genus Oryza has been studied by several means, includ-
ing cytogenetic analysis (Morinaga 1964; Nayar 1973),
morphometry (Morishima and Oka 1960), isozyme variation
(Shahi et al. 1969; Second 1985), and polymorphism of nu-
clear DNA (nDNA), chloroplast DNA (cpDNA), and mito-
chondrial DNA (mtDNA) sequences (Dally and Second
1990; Sano and Sano 1990; Second and Wang 1992; Wang
et al. 1992; Ishii et al. 1993, 1996; Provan et al. 1997). All
these analyses have contributed in complementary ways to
the current understanding of the genus Oryza. However,
knowledge of the wild rice species found on the American
continent is limited and based on few samples. Little is
known about the genetic relationships and diversity within
and among the recognized American wild species, which
comprise an unexploited gene pool that needs to be better
known and used by breeding programs.
The rice genome has been classified into five diploid (AA,
BB, CC, EE, and FF) and two tetraploid (BBCC and CCDD)
genomes, but the genome constitution of some diploid and
tetraploid species is still unknown (Vaughan 1994). This
classification was originally proposed during the Symposium
on Rice Genetics and Cytogenetics, held at the International
Rice Research Institute (IRRI) in 1963 (Katayama 1997).
Among approximately 20 recognized Oryza spp., only two
are cultivated: O. sativa, originally from Asia, and
O. glaberrima, cultivated in North Africa, both of which
have the AA genome. All diploid species described so far on
the American continent also have the AA genome (e.g.,
O. glumaepatula, sometimes described as O. rufipogon
(Vaughan 1994)), while the allotetraploid species (e.g.,
O. alta,O. latifolia, and O. grandiglumis) have the CCDD
genome (Tateoka 1962a, 1962b; Vaughan 1994; Watanabe
1997). Of particular interest is the DD genome, which has
not yet been described in a diploid species and could even
be extinct (Nayar 1973; Jena and Kochert 1991). The num-
ber and classification of the American diploid and tetraploid
species is debatable (Morishima 1984; Vaughan 1989;
Vaughan 1994; Kiefer-Meyer et al.1995; Martin et al. 1997).
Hence, the origin of rice on the American continent and how
the South and Central American AA-genome species are re-
lated to AA species from other continents is not clear. Simi-
larly, the origin of the CCDD allotetraploid, given that
diploid species with the CC and DD genomes have not been
described on the American continent, demands investigation.
Recently, a broad prospection of wild rice populations
was carried out in the Amazon Forest and Western Brazil.
As a result, a large collection of American wild rice was
gathered, comprising a valuable resource that has already
provided information about the population structure and
mating system of American diploid species (Buso et al.
1998).
The molecular evaluation of genetic diversity is a useful
way to study the amount and partitioning of genetic variabil-
ity in cultivated species and their wild relatives. It also clari-
fies phylogenetic relationships and may provide a rationale
for choosing strategies for breeding, germplasm collection,
conservation, and use of genetic resources. The main objec-
tive of the present study was to analyze a sample of culti-
vated and wild rice accessions from all continents and
compare the data with a large sample of diploid and
tetraploid accessions of American wild rice. First, the ploidy
of the accessions was checked by flow cytometry,
chromosome counting, and genome-specific RAPD (ran-
domly amplified polymorphic DNA) markers. Then, DNA
polymorphism was studied at genomic regions sampled at
random by RAPD markers (Williams et al. 1990) and at spe-
cific sites of cpDNA and mtDNA by cleaved amplified poly-
morphic sequence (CAPS) analysis (Konieczny and Ausubel
1993). Therefore, DNA regions with different mutation rates
would be studied to provide an assessment of wild rice and
cultivated rice genetic diversity and phylogeny.
Materials and methods
Genetic material
A total of 230 cultivated and wild Oryza accessions from all
continents were used in this study, including 123 new accessions of
wild rice recently collected from riverine habitats in the Amazon
forest and the western river basins of Brazil (Table 1). Some of the
accessions were morphologically analyzed in the field and tenta-
tively classified at species and genome levels (Morishima and Mar-
tins 1994). Additionally, two species were used as outgroups in
this study: (a)Rhynchoryza subulata, a monotypic genus from
South America that belongs to the same tribe as Oryza (Oryzeae),
and (b)Bambusa tuldoides, from the same subfamily as rice
(Bambusoideae). Seeds from each accession were germinated di-
rectly in the soil and kept in the screen house for leaf-sampling for
DNA extraction.
Chromosome counting
Chromosome numbers were determined in meristematic cells of
root tips taken from plants maintained in the screen house. Roots
were pretreated in a saturated solution of α-bromonaphthalene for
2 h and fixed in alcohol – glacial acetic acid 3:1 for a minimum of
3 h. The root tips were then submitted to hydrolysis in1MHCl
for 10 min at 60°C. Staining followed the Feulgen technique. Root
tips were squashed on a slide in a drop of 2% acetocarmine. Chro-
mosomes in prometaphase or metaphase cells were examined,
counted, and photographed. At least 10 cells were evaluated for
each accession examined, to determine the chromosome number.
The accessions submitted to chromosome counting are indicated in
Table 1.
Flow cytometry
Total-DNA quantitation by flow cytometry was carried out for
all samples (Table 1). Young leaves were excised, kept under re-
frigeration, and chopped with a razor blade in a glass petri dish
containing a “chopping” buffer (pH 7.0) of the following composi-
tion: 45 mM MgCl2, 30 mM sodium citrate, 20 mM 4-
morpholinepropane sulfonate, and Triton X-100 (1 mg/mL)
(Galbraith et al. 1983), with bis-benzimide as the fluorochrome.
The homogenate was passed through a 50-µm nylon filter. After
that, the filtrate was kept for 4 min at room temperature, and the
stained nuclei were analyzed by microfluorometry with a Partec
CAII flow cytometer, using an O. sativa accession (accession 90)
as the internal standard (Arumuganathan and Earle 1991). Several-
hundred nuclei were assayed for each measurement of DNA con-
tent. The ploidy estimation was based on the fluorescence histo-
gram by comparing the mean peak position of the plant nuclei of
each sample with the mean peak position of the standard. The coef-
ficient of variation (CV) of DNA-content estimates was calculated
for each accession analyzed.
Genome-specific RAPD markers
DNA pooling can be an effective strategy for detecting genetic-
marker differences among groups of individuals with similar geno-
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types or phenotypes (Michelmore et al. 1991). Only abundant
highly frequent bands are usually detected using a DNA-pooling
strategy. Bulks were made with DNA samples of six accessions of
each Oryza genome. A total of 127 random decamer primers were
screened in RAPD assays, to identify highly polymorphic well-
resolved genome-specific bands. The polymerase chain reaction
(PCR) conditions were as described in Ferreira and Grattapaglia
(1995). Each 10-µL reaction contained: 10 mM Tris-HCl (pH 8.3),
1.5 mM MgCl2, 1.0 µg/µL BSA, 0.2 mM each dNTP, 0.4 µM
primer, 7.5 ng genomic DNA, and 1 U Taq polymerase, overlayed
with 50 µL of mineral oil to prevent evaporation. RAPD reactions
were performed in a Perkin Elmer 9600 thermal cycler pro-
grammed for 40 cycles of 1 min at 92°C, 1 min at 35°C, 2 min at
72°C. Reaction products were separated by electrophoresis in 1.5%
agarose gels using a Tris-borate–EDTA (TBE) buffer system. Per-
manent records were obtained by photographing ethidium bro-
mide-stained gels under UV light. Band sizes were determined by
comparison with a 1-kb ladder in each gel.
DNA-polymorphism analysis using RAPD markers
Total genomic DNA was isolated from young leaves according
to the procedures described by Doyle and Doyle (1987). RAPD
analysis was performed on all 232 accessions, following the proce-
dures described above. RAPD-primer screening was based on 127
random decamers, including 40 primers previously selected for
O. sativa germplasm analysis. Eight of the most informative prim-
ers were used to evaluate all 232 accessions. The experiment was
repeated with all primers, to ensure the repeatability of the bands,
using 95 accessions representing all the species and genomes used.
Then, the selection of bands for inclusion in the data set was based
on band reliability, clarity, signal strength, and resolution. Another
precaution was taken with respect to reliability: when including
DNA independently extracted from different plants from the same
accession (CICA 8), different code numbers (159 and 212) were
used in the analysis.
CAPS for the analysis of DNA polymorphisms
Following examination of the results of RAPD fingerprinting,
flow cytometry, and chromosome counting, a sample of 65 acces-
sions was selected that represented all known Oryza genomes and
a large number of American wild rice accessions (Table 1). The se-
lection of the accessions was based on genome- and (or) species-
typical amplification patterns. Chloroplast and mitochondrial uni-
versal primers were used to amplify specific regions of these
genomes (Table 2). These primers were designed to amplify
noncoding regions separating two conserved coding sequences of
the cpDNA and mtDNA (Demesure et al. 1995). Such noncoding
sequences were then digested with several endonucleases, to detect
informative polymorphisms. The chloroplast primers anchored
preferably within the highly conserved tRNA genes, while mito-
chondrial primers anchored within the nad1 and nad4 genes, which
are interrupted by introns. The template for PCR amplification con-
sisted of 15 ng of genomic DNA. The reaction mixtures (25 µL)
contained: 10 mM Tris-HCl (ph 8.3), 1.5 mM MgCl2, 1.0 µg/µL
BSA, 0.2 mM each of the four dNTPs, 0.2 µM each primer, and
0.2 U Taq polymerase. Amplifications were carried out using one
cycle of 4 min at 94°C and 30 cycles of 45 s at 92°C, 45 s at 56°C,
3 min at 72°C. The reactions were performed in a Hot Bonnet
PTC-100 thermocycler (MJ Research Inc., Watertown, Mass.). The
PCR products were visualized by UV fluorescence after electro-
phoresis in 1.5% agarose gels and staining with ethidium bromide.
Twenty-two restriction endonucleases (Sau3A, HaeIII, MseI,
HindIII, TaqI, HinfI, HpaI, AluI, PalI, MspI, EcoI, SalI, CfoI, XbaI,
XhoI, BamHI, NciI, Sau96I, ClaI, PstI, DdeI, and RsaI) were tested
and five (HaeIII, HinfI, MspI, RsaI, and TaqI) were chosen for
their ability to restrict and identify polymorphic fragments. The re-
striction products were visualized in ultrapure – low melting (2:1)
agarose and stained with ethidium bromide. Photographs were
taken at different times during electrophoresis, to certify that all
fragments resulting from endonuclease cleavage were detected.
The fragment sizes were estimated using SEQAID II version 3
software (Rhoads and Roufa 1989).
Phenetic analysis
The total DNA, cpDNA, and mtDNA polymorphic data were en-
tered into the respective binary data matrices as discrete variables
(“1” for presence and “0” for absence). To generate similarity ma-
trices, data were subjected to the Dice algorithm (1945), which is
equivalent to the coefficient of Nei and Li (1979). Both algorithms
calculate coefficients of similarities based on shared presence of at-
tributes, and exclude shared absence as a criterion of similarity.
The resulting similarity matrices were utilized to group genotypes
via the unweighted pair-group method (UPGMA; Sneath and Sokal
1973). Dendrograms were produced using the NTSYS-pc 2.0 pack-
age for numerical taxonomy (Rohlf 1992).
Cladistic analysis
The character state was the fragment that was either present
(coded as “1”) or absent (coded as “0”) in an accession. Therefore,
the final data set consisted of a binary data matrix of the presence
and absence of fragments. Phylogenetic analysis was performed
using PAUP 3.1.1 (phylogenetic analysis using parsimony;
Swofford 1993), by unweighted maximum parsimony, considering
each character as equally weighted and unordered. In view of the
large number of accessions under consideration, the most efficient
method of searching for the minimal tree was the heuristic search
method in PAUP. For tree searches, tree bisection–reconnection
(TBR) branch swapping was selected, with the ACCTRAN (accel-
erated transformation) optimization options. PAUP was also used
to compute consensus trees (strict, semi-strict, and 50%-majority
rule), consensus indices (to assess the degree of congruence sup-
porting each branch), and character statistics, such as the consis-
tency index (CI; Kluge and Farris 1969) and the retention index
(RI; Farris 1989).
Results
Flow cytometry and chromosome counting
All 47 accessions that had their ploidy level assessed by
chromosome counting were also analyzed by flow
cytometry. The root-tip cells observed consistently showed
2n=2x= 24 for diploid species and 2n=4x=48for
tetraploid species. For all analyzed accessions, the results
from the two methodologies were in agreement. Conse-
quently, flow cytometry alone was used to determine the
ploidy levels of the remaining accessions. However, when
these results were compared with classifications based on
morphological descriptors made during the prospection trip
(Morishima and Martins 1994; P.H.N. Rangel, unpublished
data), there was some disagreement. Accession 87 was clas-
sified as tetraploid by morphological classification but was
classified as diploid by flow cytometry. The reverse occurred
for other accessions, such as 1, 4, 6, 12, 26, and others,
which were classified as diploids using morphological
descriptors but classified as tetraploids by flow cytometry
and (or) chromosome counting (Table 1).
Flow-cytometric analysis of cultivated and wild rice ac-
cessions indicated a correlation between ploidy number and
DNA content, as has generally been observed in other spe-
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cies and genera (Ozias-Akins and Jarret 1994; Jarret et al.
1995). Accessions from the same Oryza species showed
similar DNA content. In general, the DNA content (pg/2C)
of diploid and tetraploid rice species was lower than 1.5 and
higher than 2.0, respectively, with the exception of
O. australiensis (EE genome), which, although considered a
diploid (Vaughan 1989), has a DNA content of around 2.0
pg/2C (Table 1). DNA content varied from 0.50 pg/2C for
O. brachyantha (FF genome) to 2.52 pg/2C for the
tetraploid O. ridleyi (unknown genome). The average CV
was 6.65, with only 15% of the accessions having a CV
above 10.00 (Table 1).
Genome identification using RAPD markers
Eight primers (OPAB-01, OPAB-07, OPAB-10, OPK-11,
OPL-20, OPN-09, OPY-01, and OPZ-15) that amplified re-
peatable Oryza genome specific DNA markers were se-
lected. The observation and comparison of the amplified-
fragment patterns made it possible to identify the specific
genomes of each of the samples analyzed. Some intrinsic
patterns of intragenomic variation could also be identified.
For example, accessions of the BBCC and CC genomes
could be easily classified into their respective genome
groups according to their typical RAPD-band patterns
(Fig. 1). The combination of flow-cytometric analysis and
genome-specific RAPD markers enabled classification of
most accessions to the species level.
The genomic classification of Oryza accessions based on
genome-specific RAPD markers agreed completely with the
results of flow cytometry and chromosome counting (Ta-
ble 1). However, when compared with the classification
based on morphological descriptors, there was some dis-
agreement (Table 1). Accession 154 (OR 39), for example,
was originally classified as O. rufipogon (AA), but the anal-
ysis of genome-specific RAPD markers indicated that it was
actually an O. punctata (BBCC) accession. This was later
confirmed by the analysis of nDNA and cytoplasmic DNA
polymorphisms. Three accessions (166 (OA 27), 174 (OA
4), and 175 (OA 36)) previously classified as
O. australiensis (EE genome) (Second and Wang 1992;
Wang et al. 1992) were reclassified as BBCC-genome acces-
sions. Some disagreement between the original morphologi-
cal classification and the classification resulting from
molecular analysis was also indicated in other instances, for
example, accession 171, which was originally classified as
O. officinalis (CC) and reclassified as O. punctata (BBCC)
(Wang et al. 1992).
Phenetic and cladistic analyses with genome-wide
RAPD
The 230 accessions of wild and cultivated rice represent-
ing the different genomes of the genus Oryza and two
outgroup species (R. subulata and B. tuldoides) were ana-
lyzed with 309 RAPD markers. The repetition of RAPD re-
actions with a sample of 95 accessions representing all
species and genomes ensured the identification of the most
reliable markers. The relatively high number of polymorphic
products per primer was due to the high level of differentia-
tion among the accessions studied, including the outgroups.
Parsimony analysis resulted in 7356 maximally
parsimonious trees with equal lengths of 1204 steps (CI =
0.434; RI = 0.897). The clades of each genome and (or) spe-
cies were well defined and supported, as indicated by the
goodness-of-fit statistics. The 50% majority rule tree was
compared with the phenogram derived from the UPGMA
analysis. Both phenetic and cladistic analyses of genetic di-
versity were, in general, compatible with current known
Oryza phylogeny. In both analyses, the accessions were di-
vided into four main groups. For example, the phenogram
(Fig. 2) separated the accessions into (a) an AA-genome
group (the O. sativa complex); (b) a group composed of the
CCDD, BB, BBCC and CC, and EE genomes (the
O. officinalis complex); (c) a third group composed of Oryza
longiglumis and O. brachyantha (the O. ridleyi complex);
and (d) a group composed of the outgroup species
(R. subulata and B. tuldoides) and one accession of un-
known genome (O. granulata).
The O. sativa complex was divided into four large sub-
groups that predominantly included accessions of (subgroup
1) O. glumaepatula; (2) O. sativa,O. nivara,O. rufipogon,
and O. glaberrima; (3) O. longistaminata; and
(4) O. breviligulata and O. barthii. Two accessions were ex-
cluded from these four subgroups: O. meridionalis (acces-
sion 258) and one unknown-genome accession from Asia
(accession 176). In the first AA-genome subgroup,
O. glumaepatula accessions were clearly separated and
formed a cluster with 45% similarity with another cluster
containing O. sativa,O. nivara,O. rufipogon, and
O. glaberrima. These last species were grouped with a
greater similarity (56%). Within the O. glumaepatula sub-
group, it was possible to observe a small cluster containing
accessions from Western Brazil (accessions 87, 98, 99, 100,
89, 94, and 93), collected in the Paraguay River Basin, that
were separated from the Amazon River Basin accessions.
Six accessions (87, 119, 120, 153, 160, and 176) that origi-
nally were of unknown classification or classified as other
species were reclassified as AA-genome species. The
phenetic and cladistic analyses indicated that accession 153
is O. longistaminata. Two accessions of O. glaberrima (252
and 253) grouped with the Asian species, while a third ac-
cession (127) grouped with an African species
(O. longistaminata). As expected, accessions 159 and 212
(CICA 8), which were used as internal controls, grouped as
identical accessions.
The O. officinalis complex group was divided into four
main clusters composed of: (cluster 1) CCDD-genome ac-
cessions, (2) BB- and BBCC-genome accessions, (3) CC-
genome accessions, and (4) EE-genome accessions. In the
first cluster, the CCDD accessions were grouped with 67%
similarity and there was no clear evidence of clusters corre-
sponding to the three American allotetraploid species
(O. alta,O. grandiglumis, and O. latifolia). The CCDD
group clustered with other groups with only 12% similarity,
indicating that American allotetraploids are very dissimilar
from other O. officinalis complex species. In the second
cluster, the accessions of O. punctata (BBCC) are closer to
the accessions of the so-called diploid O. punctata (BB) than
to O. minuta (BBCC) accessions. In the third cluster, acces-
sions of O. eichingeri,O. rhizomatis, and O. officinalis were
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480 Genome Vol. 44, 2001
Sample Results
No. Accession Donor
instituteaOriginal species
classificationbOrigin Code Original genome
classificationbChromosome
counting (2n)Flow cytometry
(pg/2C) CV(%) Final genome
classificationc
1dRB1 1 O. glumaepatula Amazon, Brazil AA glu AA 48 2.27 3.57 CCDD
4 RB4 1 O. glumaepatula Amazon, Brazil AA glu AA 2.29 3.91 CCDD
12dRN1 1 O. glumaepatula Amazon, Brazil AA glu AA 2.18 3.33 CCDD
17 RN6 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 22.22 AA glu
25 RBJ1 1 O. glumaepatula Amazon, Brazil AA glu AA 24 AA glu
26 RBJ2 1 O. glumaepatula Amazon, Brazil AA glu AA 48 2.19 4.02 CCDD
28dRJA2 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.19 2.86 CCDD
29dRJA3 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.25 2.29 CCDD
30dRJA4 1 O. glumaepatula Amazon, Brazil AA glu AA CCDD
31dRP1 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.21 5.36 CCDD
32 RP2 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.15 3.60 CCDD
33 RP3 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 8.51 AA glu
35 RPA1 1 O. glumaepatula Western Brazil AA glu AA 48 2.26 4.95 CCDD
36 RS1 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 10.78 AA glu
37 RS2 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.03 3.45 CCDD
38 RS3 1 O. alta Amazon, Brazil CCDD CCDD 48 1.93 3.45 CCDD
39 RS4 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 9.57 AA glu
40dRS5 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.08 3.07 CCDD
41 RS6 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 8.33 AA glu
42 RS7 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.14 3.42 CCDD
43 RS8 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.19 4.09 CCDD
44 RS9 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.11 3.18 CCDD
45 RS10 1 O. alta Amazon, Brazil CCDD CCDD 2.15 3.60 CCDD
46 RS11 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 16.67 AA glu
47 RS12 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.16 2.75 CCDD
49 RS14 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.25 3.30 CCDD
50 RS15 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 9.78 AA glu
51 RS16 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 11.96 AA glu
52 RS17 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 13.04 AA glu
53 RS18 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.08 2.70 CCDD
55 RS20 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 10.64 AA glu
56 RS20A 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.28 4.25 CCDD
57 RS21 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.15 3.27 CCDD
59 RS23 1 O. alta Amazon, Brazil CCDD CCDD 48 2.06 4.46 CCDD
60 RS24 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 7.61 AA glu
61 RS25 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.17 4.02 CCDD
62 RS26 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 5.56 AA glu
63 RS27 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.17 3.60 CCDD
64 RS28 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.35 5.91 CCDD
65 RS29 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.27 2.27 CCDD
66 RS30 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.18 3.98 CCDD
67 RS31 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 12.24 AA glu
68 RS32 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 10.00 AA glu
69dRS33 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.06 10.64 CCDD
Table 1. Accessions used in the analysis of genomic regions sampled at random by RAPD markers and at specific sites of the cpDNA and mtDNA by cleaved amplified
ploymorphic sequences (CAPS).
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70 RS34 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 12.77 AA glu
71 RS35 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.21 4.76 CCDD
72 RS36 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.03 2.73 CCDD
73 RS37 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 12.50 AA glu
74 RS38 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 10.23 AA glu
75 RS39 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.13 5.09 CCDD
77 RS41 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 10.20 AA glu
78dRS42 1 O. glumaepatula Amazon, Brazil AA glu AA 24 0.96 20.91 AA glu
79 RS43 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.30 4.05 CCDD
80 RS44 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.20 3.60 CCDD
81 RS45 1 O. grandiglumis Amazon, Brazil CCDD CCDD 48 2.18 2.78 CCDD
82 RS46 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.30 2.83 CCDD
83 RS47 1 O. glumaepatula Amazon, Brazil AA glu AA 0.96 7.00 AA glu
84 RS48 1 O. grandiglumis Amazon, Brazil CCDD CCDD 2.24 3.18 CCDD
85 RS49 1 O. alta Amazon, Brazil CCDD CCDD 2.01 4.13 CCDD
87 EC1 1 O. latifolia Western Brazil CCDD CCDD 0.96 17.05 AA glu
88dRNE1 1 O. latifolia Western Brazil CCDD CCDD 48 2.22 2.78 CCDD
89 RNE2 1 O. glumaepatula Western Brazil AA glu AA 0.96 16.67 AA glu
90dRPA1 1 O. glumaepatula Western Brazil AA glu AA 24 0.96 4.95 AA glu
91 RPA2 1 O. latifolia Western Brazil CCDD CCDD 2.09 2.75 CCDD
92 RPA3 1 O. latifolia Western Brazil CCDD CCDD 48 2.20 3.67 CCDD
93 RPA4 1 O. glumaepatula Western Brazil AA glu AA 24 0.96 14.13 AA glu
94 RPA5 1 O. glumaepatula Western Brazil AA glu AA 0.96 11.46 AA glu
95 RPA6 1 O. glumaepatula Western Brazil AA glu AA 0.96 8.33 AA glu
96 RPA7 1 O. latifolia Western Brazil CCDD CCDD 2.08 4.02 CCDD
97 RPA8 1 O. latifolia Western Brazil CCDD CCDD 2.06 2.73 CCDD
98 RPA9 1 O. glumaepatula Western Brazil AA glu AA 0.96 8.16 AA glu
99 RTA1 1 O. glumaepatula Western Brazil AA glu AA 0.96 8.89 AA glu
100 RTA2 1 O. glumaepatula Western Brazil AA glu AA 0.96 11.22 AA glu
101 RTA3 1 O. glumaepatula Western Brazil AA glu AA 48 2.08 3.98 CCDD
102 IRRI 105661 2 O. glumaepatula Amapá, Brazil AA glu AA AA glu
103 IRRI 105662 2 O. glumaepatula Amapá–Brazil AA glu AA 0.96 8.00 AA glu
104 IRRI 105663 2 O. glumaepatula Pará, Brazil AA glu AA 0.96 13.00 AA glu
105 IRRI 105665 2 O. glumaepatula Amazon, Brazil AA glu AA 0.96 6.25 AA glu
106 IRRI 105666 2 O. glumaepatula Amazon, Brazil AA glu AA 0.96 14.58 AA glu
107 IRRI 105667 2 O. glumaepatula Amazon, Brazil AA glu AA 0.96 13.27 AA glu
108 IRRI 105667 2 O. glumaepatula Amazon, Brazil AA glu AA 0.96 8.33 AA glu
109 IRRI 105670 2 O. glumaepatula Amazon, Brazil AA glu AA 1.02 7.41 AA glu
110 IRRI 105672 2 O. glumaepatula Amazon, Brazil AA glu AA 0.96 8.51 AA glu
111 IRRI 105686 2 O. glumaepatula Pará, Brazil AA glu AA AA glu
112 IRRI 105687 2 O. glumaepatula Pará, Brazil AA glu AA 0.96 7.78 AA glu
113 IRRI 105688 2 O. glumaepatula Amazon, Brazil AA glu AA 0.96 10.87 AA glu
114 IRRI 105689 2 O. glumaepatula Amazon, Brazil AA glu AA AA glu
115 IRRI 105692 2 O. glumaepatula Amapa, Brazil AA glu AA AA glu
116dIR36 1 O. sativa IRRI AA sat AA 0.96 6.25 AA sat
117 IR34583 1 O. sativa IRRI AA sat AA 0.96 7.45 AA sat
118dBulu Dalam 1 O. sativa AA sat AA 0.96 6.52 AA sat
119 1 LL 116 3 AA 0.81 7.00 AA lon
120dWL 02 3 AA 0.77 6.12 AA lon
121 IRRI 103848 2 O. rufipogon IRRI AA ruf AA 0.96 4.35 AA ruf
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482 Genome Vol. 44, 2001
Donor
institutea
Sample Results
No. Accession Original species
classificationbOrigin Code Original genome
classificationbChromosome
counting (2n)Flow cytometry
(pg/2C) CV(%) Final genome
classificationc
127dIRRI 103350 2 O. glaberrima Africa AA gla AA 0.96 8.00 AA gla
128 BL25 3 O. longistaminata Bissau, Guinea AA lgt AA 0.76 7.14 AA lgt
129 CL2 3 O. longistaminata Senegal AA lgt AA 0.73 6.25 AA lgt
131dEB22 3 O. breviligulata Tanzania AA lgt AA AA bre
132dEL01 3 O. longistaminata Tanzania AA lgt AA 0.79 6.73 AA lgt
133 EL16 3 O. longistaminata Tanzania AA lgt AA 0.86 7.41 AA lgt
134 EL34 3 O. longistaminata Tanzania AA lgt AA 0.79 4.00 AA lgt
135 EL51 3 O. longistaminata Tanzania AA lgt AA 0.74 4.81 AA lgt
136dEL89 3 O. longistaminata Tanzania AA lgt AA 0.78 5.56 AA lgt
139 SL305 3 O. longistaminata Senegal AA lgt AA 0.77 5.77 AA lgt
140 TL31 3 O. longistaminata Tchad AA lgt AA 0.73 7.00 AA lgt
142 TL70 3 O. longistaminata Tchad AA lgt AA 0.82 6.73 AA lgt
143 UL62 3 O. longistaminata Cameroon AA lgt AA 0.74 10.19 AA lgt
144 UL48 3 O. longistaminata Cameroon AA lgt AA 0.75 7.41 AA lg
146dW1228 3 O. longiglumis lgg — lgg
147 ZL01 3 O. longistaminata Zambia AA lgt AA 0.65 7.27 AA lgt
148 ZL03 3 O. longistaminata Zambia AA lgt AA 0.65 11.11 AA lgt
149 ZL13 3 O. longistaminata Zambia AA lgt AA 0.73 21.59 AA lgt
150 ZL14 3 O. longistaminata Zambia AA lgt AA 0.73 6.86 AA lgt
151 ZL22 3 O. longistaminata Zambia AA lgt AA 0.79 10.20 AA lgt
152dZB12 3 O. breviligulata Zambia AA lgt AA AA bre
153dOR54 3 O. rufipogon Australia AA ruf AA 0.75 5.45 AA
154dOR39 3 O. rufipogon Australia AA ruf AA 2.33 2.73 BBCC pun
157 BG90-2 1 O. sativa Brazil AA sat AA 0.96 11.70 AA sat
158 IRGA 409 1 O. sativa Brazil AA sat AA AA sat
159dCICA 8 1 O. sativa Brazil AA sat AA AA sat
160d001 GO 1 O. rufipogon Central Brazil AA ruf AA AA glu
162dW1144 3 O. alta America CCDD CCDD 48 2.17 3.15 CCDD
163dW1168 3 O. latifolia America CCDD CCDD 48 2.19 3.18 CCDD
164 W0017 3 O. alta America CCDD CCDD 48 2.13 3.13 CCDD
165 W1483 3 O. grandiglumis America CCDD CCDD 2.26 4.59 CCDD
166 OA27 3 O. australiensis Australia EE aus EE 2.40 2.52 BBCC min
167dOR07 3 O. rufipogon Australia AA ruf AA BBCC min
168 OR10 3 O. rufipogon Australia AA ruf AA 0.96 10.20 AA ruf
169dW510 3 O. subulata sub 0.40 8.00 sub
170 OR09370 3 2.21 3.21 BBCC pun
171 IRRI 100180 2 O. officinalis Malaysia CC off CC 2.12 2.68 BBCC pun
172 OR09374 3 1.09 5.08 BB
173 OR09435 3 2.33 2.19 CCDD
174 OA04 3 O. australiensis Australia EE aus EE 2.30 3.42 BBCC min
175 OA36 3 O. australiensis Australia EE aus EE 2.23 19.00 BBCC pun
176dGSCB7 O. australiensis EE aus EE 0.70 19.57 AA
177dIRRI 101150 2 O. officinalis Asia CC off CC 1.33 15.56 CC off
178dIP7 3 O. eichingeri Ivory Coast CC eic CC 2.23 2.29 BBCC pun
179dGSCB8 Brazil 2.26 5.66 CCDD
180d1IP2 3 O. punctata Africa BBCC pun BB/BBCC 2.17 3.77 BBCC pun
Table 1 (continued).
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Buso et al. 483
181dW0051 3 O. minuta BBCC min BBCC 2.33 3.57 BBCC min
182 IRRI 105671 2 O. grandiglumis Brazil CCDD CCDD 2.27 3.37 CCDD
183dAmL3 3 2.30 2.56 CCDD
184 AmL5 3 2.06 1.82 CCDD
185 W1539 3 O. latifolia CCDD CCDD 2.08 2.78 CCDD
186dIRRI 100963 2 O. latifolia Guatemala CCDD CCDD 2.47 3.04 CCDD
187 IRRI 105685 2 O. alta CCDD CCDD 2.18 2.38 CCDD
188 IRRI 105664 2 O. grandiglumis CCDD CCDD 2.15 3.30 CCDD
189 IRRI 105669 2 O. grandiglumis CCDD CCDD 2.11 2.83 CCDD
190 W0613 3 O. grandiglumis CCDD CCDD 2.40 2.16 CCDD
192dW0047 3 O. latifolia CCDD CCDD 2.44 2.68 CCDD
193 W0046 3 O. latifolia CCDD CCDD 2.37 2.16 CCDD
194 AmL1 3 CCDD 2.37 3.42 CCDD
195 W0048 3 O. latifolia CCDD CCDD 2.40 2.17 CCDD
199dGSCB9 2.48 2.19 CCDD
209 W811 3 O. latifolia CCDD CCDD 2.10 2.73 CCDD
211 GSCB10 O. sativa Brazil AA sat AA AA sat
212 CICA 8 1 O. sativa Brazil AA sat AA AA sat
213 CNASS8S × O. rufipogon O. sativa Brazil AA sat AA 0.96 6.52 AA sat
215 W504 3 2.08 1.40 CCDD
217 IRRI 101408 2 O. punctata Ghana BBCC pun BB/BBCC 2.26 3.24 BBCC pun
218 IRRI 101417 2 O. punctata Kenya BB pun BB/BBCC 0.96 4.63 BB
220dIRRI 103889 2 O. punctata Tanzania BB pun BB/BBCC 0.96 9.09 BB
221 IRRI 103903 2 O. punctata Tanzania BB pun BB/BBCC 0.96 5.81 BB
222dIRRI 103917 2 O. punctata Tanzania BB pun BB/BBCC 0.96 10.00 BB
223 IRRI 105137 2 O. punctata Zaire BBCC pun BB/BBCC 2.25 3.70 BBCC pun
224 IRRI 100881 2 O. eichingeri Sri Lanka CC eic CC 2.19 2.75 BBCC pun
225 IRRI 101426 2 O. eichingeri Uganda CC eic CC 1.30 3.73 CC eic
226 IRRI 105161 2 O. eichingeri Uganda CC eic CC 1.29 4.55 CC eic
227 IRRI 105162 2 O. eichingeri Uganda CC eic CC 1.34 5.30 CC eic
228 IRRI 105181 2 O. eichingeri Uganda CC eic CC 2.24 2.75 BBCC pun
229 IRRI 105407 2 O. eichingeri Sri Lanka CC eic CC 1.25 5.38 CC eic
230dIRRI 105414 2 O. eichingeri Sri Lanka CC eic CC 1.26 5.30 CC eic
231 IRRI 105412 2 O. eichingeri Sri Lanka CC eic CC 1.40 4.73 CC eic
232dIRRI 105415 2 O. eichingeri Sri Lanka CC eic CC 1.44 2.86 CC eic
233dIRRI 105961 2 O. officinalis Indonesia CC off CC 1.57 3.33 CC off
234 IRRI 105962 2 O. officinalis Indonesia CC off CC 1.45 8.22 CC off
235 IRRI 105964 2 O. officinalis Indonesia CC off CC 1.56 3.42 CC off
237dIRRI 106246 2 O. officinalis Indonesia CC off CC 1.52 4.73 CC off
238dIRRI 103410 2 O. rhizomatis Sri Lanka CC rhi CC 1.51 4.79 CC rhi
239dIRRI 103414 2 O. rhizomatis Sri Lanka CC rhi CC 0.96 7.78 BB
240 IRRI 105440 2 O. rhizomatis Sri Lanka CC rhi CC CC rhiz
241dIRRI 105443 2 O. rhizomatis Sri Lanka CC rhi CC 1.56 5.48 CC rhiz
242 IRRI 105449 2 O. rhizomatis Sri Lanka CC rhi CC 1.61 7.78 CC rhiz
243 IRRI 105450 2 O. rhizomatis Sri Lanka CC rhi CC 1.59 6.94 CC rhiz
244 IRRI 101083 2 O. minuta Phillipines BBCC min BBCC 2.28 4.42 BBCC min
245 IRRI 101101 2 O. minuta Philippines BBCC min BBCC 2.26 5.22 BBCC min
246 IRRI 101122 2 O. minuta Philippines BBCC min BBCC 1.36 7.04 CC eic
247dIRRI 103874 2 O. minuta Philippines BBCC min BBCC 2.34 4.78 BBCC min
248 IRRI 105132 2 O. minuta Philippines BBCC min BBCC 2.27 5.17 BBCC min
249 IRRI 100161 2 O. alta Brazil CCDD CCDD 2.10 6.60 CCDD
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© 2001 NRC Canada
484 Genome Vol. 44, 2001
Donor
institute Original species
classificationa
Sample
Original genome
classificationb
Results
No. Accession Origin Code Chromosome
counting (2n)Flow cytometry
(pg/2C) CV(%) Final genome
classficationc
250dIRRI 100223 2 O. barthii Guinea AA bar AA AA bar
251dIRRI 100122 2 O. barthii Gambia AA bar AA 0.83 3.77 AA bar
252dIRRI 102202 2 O. glaberrima Liberia AA gla AA 0.81 6.73 AA gla
253 IRRI 102254 2 O. glaberrima Liberia AA gla AA AA gla
254dIRRI 100901 2 O. nivara – O. sativa India AA sat AA 0.86 8.65 AA
255dIRRI 103831 2 O. nivara – O. sativa Bangladesh AA sat AA 0.96 11.96 AA
256dIRRI 101524 2 O. nivara India AA niv AA 0.96 12.50 AA
257 IRRI 101508 2 O. nivara India AA niv AA 0.96 7.14 AA
258dIRRI 101147 2 O. meridionalis Australia AA mer AA 0.96 11.00 AA mer
259dIRRI 101411 2 O. meridionalis Australia AA mer AA 0.96 7.14 AA ruf
260 IRRI 101202 2 O. longistaminata Niger AA lgt AA 0.75 9.00 AA lgt
261 IRRI 100211 2 O. rufipogon India AA ruf AA 0.96 8.16 AA ruf
262 IRRI 104075 2 O. longistaminata Niger AA lgt AA 0.76 7.41 AA lgt
263 IRRI 100970 2 O. glumaepatula Brazil AA glu AA 0.96 8.33 AA glu
264dIRRI 100894 2 O. glumaepatula AA glu AA 0.83 6.48 AA glu
265 IRRI 100924 2 O. glumaepatula Brazil AA glu AA 0.96 8.16 AA glu
266 IRRI 100968 2 O. glumaepatula Suriname AA glu AA 0.96 10.00 AA glu
267 IRRI 103810 2 O. glumaepatula Venezuela AA glu AA 0.96 6.38 AA glu
268 IRRI 105412 2 O. eichingeri Sri Lanka CC eic CC 1.31 4.41 CC eich
269 IRRI 105161 2 O. eichingeri Uganda CC eic CC 1.37 6.25 CC eich
270 IRRI 101144 2 O. australiensis Australia EE aus EE 1.97 4.25 EE aus
271 IRRI 103318 2 O. australiensis Australia EE aus EE 2.04 4.76 EE aus
272dIRRI 100115 2 O. brachyanta Guinea FF bra FF 0.50 9.38 FF bra
274 IRRI 103878 2 O. minuta Phillippines BBCC min BBCC 2.32 2.97 BBCC min
275 IRRI 105307 2 O. minuta Philippines BBCC min BBCC 2.40 2.52 BBCC min
276 IRRI 100161 2 O. alta Brazil CCDD CCDD 2.13 2.75 CCDD
277 IRRI 105143 2 O. alta Guyana CCDD CCDD 2.10 2.73 CCDD
278 IRRI 101405 2 O. grandiglumis Brazil CCDD CCDD 2.26 2.38 CCDD
279 IRRI 105156 2 O. grandiglumis Brazil CCDD CCDD 2.08 2.29 CCDD
280 IRRI 100914 2 O. latifolia Mexico CCDD CCDD 2.43 4.27 CCDD
281 IRRI 105133 2 O. latifolia Paraguay CCDD CCDD 2.26 3.64 CCDD
283dIRRI 105146 2 O. longiglumis Indonesia lgg 2.22 1.83 lgg
284dIRRI 100879 2 O. granulata Sri Lanka gra 1.89 2.69 gra
286dIRRI 100886 2 O. punctata India BBCC pun BB/BBCC 2.21 3.10 BBCC pun
287 IRRI 101417 2 O. punctata Kenya BB pun BB/BBCC 0.96 7.14 BB
290 Formoso1 O. glumaepatula Central Brazil AA glu AA 0.96 6.38 AA glu
291 Formoso2 Central Brazil CCDD 2.18 2.83 CCDD
292 IRRI 105080 2 O. officinalis Vietnam CC off CC 1.48 4.61 CC off
293 IRRI 105095 2 O. officinalis Brunei CC off CC 1.51 4.05 CC off
303dGSCB 3 48 2.22 4.95 BBCC pun
306dGSCB 6 2.11 3.88 BBCC pun
aDonor institutes: 1, Centro Nacional de Pesquisa de Arroz e Feijão (CNPAF), Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Goiânia, Brasil (Dr. Paulo Hideo N. Rangel); 2, International Rice
Research Institute (IRRI), Manila, Philippines (Dr. Michael Jackson); 3, Institut Français de Recherché Scientifique pour le Développement en Coopération (ORSTOM), Montpellier, France (Dr. Gerard Second).
bClassified by donor.
cBased on chromosome counting, flow cytometry, and total-DNA, cpDNA, and mtDNA analyses.
dAnalyzed by CAPSs.
Table 1 (concluded).
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separated into three subgroups. Two accessions of
O. australiensis (270 and 271) formed a distinct cluster.
Phenetic and cladistic analyses with CAPS of cpDNA
and mtDNA regions
Polymorphisms at five cpDNA and two mtDNA regions
were studied in a subsample of 65 of the 230 Oryza acces-
sions analyzed (Table 1). The accessions selected included
materials that were found difficult to classify in previous
studies. The predominant fragment sizes of the different re-
gions showed no variation among genomes and (or) Oryza
spp. upon amplification, except for the cp3 (trnC–trnD)
fragment, which was larger for the FF-genome accession
(O. brachyantha) in comparison with accessions of the other
genomes (Fig. 3a). Genetic diversity among accessions was
investigated after digesting the amplified fragments with se-
lected restriction endonucleases (Fig. 3b). A total of 274
chloroplast and 108 mitochondrial polymorphic fragments
were detected (Table 3). The cp3 fragment was the most
variable, with a total of 86 polymorphic cpDNA fragments,
while the mt2 (nad4exon1–nad4 exon 2) was the most
variable one for mtDNA. The HinfI restriction site was the
most frequent among the cpDNA fragments analyzed, while
the RsaI restriction site was the most frequent among the
mtDNA fragments analyzed.
The parsimony analysis based on CAPS-cpDNA frag-
ments resulted in 26 112 maximally parsimonious trees with
equal lengths of 521 steps (CI = 0.645; RI = 0.847). The
50% majority rule tree was compared with a UPGMA-based
phenogram. The major clades were well supported, as indi-
cated by the goodness-of fit statistics. UPGMA and parsi-
mony analyses both produced clusters that, in general, were
in agreement with the known taxonomic classification. There
was little polymorphism within genomes. The cpDNA frag-
ments examined showed great proximity among AA-genome
accessions, which formed a monophyletic group in the
cladistic analysis (Fig. 4). The O. sativa indica accessions IR
36 (116) and CICA 8 (159) were closely related when com-
pared with Bulu Dalam (118), a japonica cultivated rice.
In general, the cpDNA analysis differed from the total-
DNA analysis in some clusters. Accessions of the diploid
O. punctata (BB) and the allotetraploid O. minuta (BBCC)
formed a cluster more closely related to AA-genome acces-
sions than to other species of the O. officinalis complex.
Two main clusters of CCDD species could be observed, one
containing accessions 30 and 69 and another of several ac-
cessions. Accessions 30 and 69 differed from the other
CCDD accessions in the cladistic (Fig. 4) and phenetic anal-
yses of the cpDNA (data not shown). The accessions of
O. minuta (BBCC) and O. punctata (BB) grouped together,
separated from the other species of the O. officinalis com-
plex. Accession 179 grouped with O. longiglumis,
O. brachyantha, and O. granulata accessions in a separate
cluster. The clustering of CCDD-genome, CC-genome, and
BBCC-genome (O. punctata) accessions confirmed results
obtained with total-DNA analysis. The CCDD-genome ac-
cessions grouped with the CC- and BBCC-genome acces-
sions with 96% similarity.
The amplified fragments of two mitochondrial regions of
the 65 Oryza accessions were digested with the same restric-
tion enzymes used for cpDNA analysis, producing 102 poly-
morphic products. These products showed less intra- and
inter-genomic polymorphism than the products of the
cpDNA digestion. The mtDNA regions analyzed represent a
small segment of this genome. The a priori expectation of
low polymorphism was also confirmed.
The parsimony analysis of the 102 mitochondrial poly-
morphic fragments produced two maximally parsimonious
trees with 96 steps (CI = 0.792; RI = 0.939) that distributed
the 65 selected accessions into four main clades. Both trees
have, in general, the same topology, differing only in the
placement of the O. punctata (BB genome) accessions (data
not shown). The strict- and 50% majority rule consensus
trees were identical and all clades were well supported by
the goodness-of-fit statistics. The first clade contained AA-
genome accessions. The mtDNA analysis indicated that
O. glumaepatula accessions were closer to Oryza
breviligulata accessions than to other AA-genome acces-
sions. This agrees with the results from the phenetic analysis
of mtDNA data (data not shown). The mtDNA of the mt1
and mt2 fragments did not show any polymorphism among
accessions of the same species. It was enough, however, to
© 2001 NRC Canada
Buso et al. 485
Primer Amplified region Primers Expected fragment size (bp)
Chloroplast
cp1 trnH [tRNA-His (GUG)] to 5-ACGGGAATTGAACCCGCGCA-31690
trnK [tRNA-Lys (UUU) exon 1] 5-CCGACTAGTTCCGGGTTCGA-3
cp2 trnK [tRNA-Lys (UUU) exon 1] to 5-GGGTTGCCCGGGACTCGAAC-32580
trnK [tRNA-Lys (UUU) exon 2] 5-CAACGGTAGAGTACTCGGCTTTTA-3
cp3 trnC [tRNA-Cys (GCA)] to 5-CCAGTTCAAATCTGGGTGTG-33000
trnD [tRNA-Asp (GUC)] 5-GGGATTGTAGTTCAATTGGT-3
cp5 psbC [psII 44 kd protein] to 5-GGTCGTGACCAAGAAACCAC-31680
trnS [tRNA-Ser (UGA)] 5-GGTTCGAATCCCTCTCTCTC-3
cp6 trnS [tRNA-Ser (UGA)] to 5-GAGAGAGAGGGATTCGAACC-31700
trnM [tRNA-fMet (CAU)] 5-CATAACCTTGAGGTCACGGG-3
Mytochondria
mt1 nad1 exon B 5-GCATTACGATCTGCAGCTCA-31500
nad1 exon C 5-GGAGCTCGATTAGTTTCTGC-3
mt2 nad4 exon 1 5-CAGTGGGTTGGTCTGGTATG-31700
nad4 exon 2 5-TCATATGGGCTACTGAGGAG-3
Table 2. Sequence of cpDNA and mtDNA universal primers used in this study and their expected amplified frag-
ment sizes.
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discriminate the O. glumaepatula accessions from the other
AA-genome species. The mtDNA analysis also indicated
O. granulata,R. subulata, and B. tuldoides as outgroups.
O. granulata was invariably the most diverse of all Oryza
accessions studied.
Discussion
This study surveyed the largest sample of wild rice spe-
cies collected so far on the American continent. The sample
was composed of 52 accessions of O. glumaepatula (AA ge-
nome) and 71 CCDD-genome accessions. Nuclear- and cyto-
plasmic-DNA polymorphisms were compared among
accessions of Oryza spp. from Asia, Africa, and Oceania.
Flow cytometry was used to identify the ploidy level of
the accessions and proved to be simpler than and as precise
as chromosome counting and quite adequate for a fast and
reliable analysis of large samples from collection expedi-
tions. DNA-content values measured by flow cytometry
were very similar among accessions of the same species.
The majority of the AA-genome accessions, for example,
had similar DNA-content values (approximately 0.96
pg/2C), except for the O. longistaminata accessions, which
had lower values (0.65–0.82 pg/2C). These results are in
agreement with previous analyses of AA-genome species
(Arumuganathan and Earle 1991). The flow cytometric data
were very useful for a first classification of the accessions
into the O. sativa or O. officinalis complex. All accessions
with the AA genome had a DNA content of between 0.65
and 1.02 pg/2C. The DNA content of O. officinalis complex
accessions ranged from 1.25 to 2.47 pg/2C. The DNA con-
tent of the outgroup species and O. brachyantha was below
0.5 pg/2C. The exceptions were the diploid BB-genome ac-
cessions from Africa, generally classified as O. punctata,
that had DNA-content values of around 0.96 pg/2C. As ex-
pected, the DNA content of tetraploid species accessions
was above 2.0 pg/2C. The exceptions were the accessions of
O. granulata (284) and O. australiensis (270 and 271) that
are diploids and had DNA-content values of 1.89 and 1.97–
2.04 pg/2C, respectively. Some authors have also found a
high DNA content for O. australiensis (1.99 pg/2C) (Marti-
nez et al. 1998), probably because its chromosomes are
larger than those of the other species (Katayama 1997).
Several samples originally classified in the field by mor-
phological criteria were actually misclassified. The ploidy
number was checked by flow cytometry and chromosome
counting and a total of 29 samples (8%) were reclassified.
Fourteen other accessions of unknown genome were first
classified by flow cytometry and later studied in detail at the
total and cytoplasmic DNA levels. When large numbers of
wild rice samples are to be analyzed, flow cytometry can
provide a first and accurate classification into the major
Oryza complexes, with a finer-scale analysis then being pur-
sued using random and specific DNA markers.
The PCR pattern obtained for each genome represented a
consensus of RAPD markers for various accessions of the
same genome. Therefore, each marker used was clearly
polymorphic among genomes and not among accessions
within the same genome. The results of this analysis corrob-
orated those obtained with flow cytometry and chromosome
counting and were more detailed, as the samples could also
be separated at the species level within the same genome.
The use of genome-specific RAPD markers may be an effi-
cient and fast way to check accession identification in large
collections. Some of the misclassifications identified in the
present study were also identified in other studies with other
types of markers, such as RFLPs (restriction fragment length
polymorphisms), as in the case of accession 171, originally
classified as O. officinalis and reclassified as O. punctata
(BBCC) by Wang et al. (1992). The same authors suggested
© 2001 NRC Canada
486 Genome Vol. 44, 2001
Fig. 1. Genome-specific RAPD patterns using primer OP-L20 that allow species and genome discrimination among accessions with BBCC
and CC genomes. The arrows indicate the different patterns of misclassified accessions. The first and last lanes are a 1-kb ladder.
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that an accession classified as O. australiensis (166) was
tetraploid and not diploid, owing to the large number of
RFLP bands observed in their experiment. In the present
study, accession 166 was actually reclassified as O. minuta
(BBCC) (Table 1). Recently, Martin et al. (1997) described
five accessions originally classified as O. meridionalis that
were reclassified as O. sativa and O. rufipogon; four acces-
sions originally classified as O. glumaepatula that were re-
classified as O. rufipogon and O. nivara; and six O. nivara
and one O. rufipogon accessions that were found to be
misclassified, when RAPD patterns were compared. Using a
combination of techniques, such as flow cytometry and ge-
nome-specific RAPD markers, proved to be very efficient
for determining the germplasm organization of wild rice ac-
cessions.
Genome-wide RAPD analysis
Some authors contend that RAPD markers should not be
recommended for phylogenetic analysis (Dowling et al.
1996) at the species level, unless patterns that are constant
within the species are identified. This could be achieved by,
for example, the utilization of genome-specific markers.
Bulking of accessions of the same genome was used in this
study to select genome-specific RAPD markers for wild rice
species. The use of RAPD markers for phylogenetic analysis
potentially leads to homoplasy (Furman et al. 1997). If this
occurs, it would result in tree topologies without statistical
support. However, the data analyzed in the present study
produced highly supported trees.
Phylogenetic algorithms may be classified into distance-
based versus character-state approaches (Avise 1994), and it
is generally advisable to attempt multiple methods of data
analysis, particularly if these entail philosophically distinct
approaches. Both UPGMA-clustering (a distance-based
method) and parsimony (a character-state approach) methods
can be reasonably applied to many molecular data sets and
the results compared (Avise 1994).
The phylogenetic trees produced by phenetic and cladistic
analyses of the wild rice samples showed four major clades
that correspond to the four complexes proposed by Vaughan
(1994): the O. sativa complex with AA-genome species; the
O. officinalis complex, containing CCDD-, CC-, BB-, and
BBCC-genome species; the O. ridleyi complex with the spe-
cies O. longiglumis and O. brachyantha; and the
O. meyeriana complex, containing O. granulata species.
The O. sativa complex
The classification of the Brazilian diploid species
O. glumaepatula is controversial. Diploid American wild
rice has been classified variously as O. glumaepatula,
O. rufipogon,O. perennis,O. paraguayensis, and
O. cubensis (Morishima et al. 1962; Tateoka 1962bVaughan
1989, 1994). Vaughan (1989) suggested that the lack of
extravaginal ramification and the semi-erect habit could be
used to distinguish O. glumaepatula from O. rufipogon but,
recently, it was contended that there are no good key charac-
ters that distinguish the two species (Vaughan 1994).
O. glumaepatula and O. rufipogon have lower reproductive
affinities and produce highly sterile F1hybrids when
crossed. Fertility is higher, for example, in crosses between
O. rufipogon and O. sativa (Morishima 1969). Martin et al.
(1997) suggested that O. glumaepatula should be reclassi-
fied as separate from O. rufipogon or O. perennis.Inthe
present study, a large collection of Brazilian diploid acces-
sions clustered together with O. sativa – O. nivara –
O. rufipogon with 45% similarity. This similarity value is
smaller than any within-group similarity. This study includes
the greatest sample of American accessions studied so far.
Phenetic and cladistic analyses of the cpDNA and mtDNA
of a subsample of O. glumaepatula accessions also separated
them from O. rufipogon and other AA-genome species.
Actually, at the chloroplast and mitochondrial levels,
O. glumaepatula accessions were more similar to African
O. breviligulata than to O. rufipogon accessions. All this in-
formation suggests that O. glumaepatula should be reclassi-
fied as a species separate from O. rufipogon or O. perennis,
in accordance with Martin et al. (1997). Furthermore,
O. glumaepatula accessions formed subgroups according to
their geographical origin: Amazon or Paraguay river basins.
This corroborates our previous studies, in which the popula-
tion differences between these two regions were evident
(Buso et al. 1998).
The species O. sativa and O. nivara were not separated
into different clusters. These species, in general, are consid-
ered to be very close and the original classification is based
on annual or perennial habit. Some authors refer to
O. nivara,O. rufipogon, and O. sativa as a complex
(Vaughan 1989). Others, however, do not consider O. nivara
to be a distinct species (Chang 1976). The present results
confirm the proximity of these species.
Some differences between the phenetic and cladistic anal-
yses were observed in comparisons of African AA-genome
species. O. breviligulata (also known as O. barthii) clustered
with O. longistaminata in the phenetic analysis of RAPD
markers. In the cladistic analysis, however, O. breviligulata
clustered with an O. glaberrima accession, the placement
that is generally accepted (Morishima et al. 1962). In addi-
tion, in both analyses, some O. glaberrima accessions (252
and 253) clustered with O. sativa – O. nivara – O. rufipogon.
Field observations have suggested the existence of spontane-
ous hybrids between O. glaberrima and O. sativa (Second
1985), which could explain the clustering of O. glaberrima
with other AA-genome species. The O. meridionalis acces-
sion is the most dissimilar AA-genome species in the
O. sativa complex, and this agrees with what is known about
this diploid species from Australia (Vaughan 1994; Martin et
al. 1997).
The O. officinalis complex
The O. officinalis complex is the largest complex of the
genus and includes related species groups from Asia, Africa,
and Latin America. The CCDD amphidiploid species
(O. alta, O. grandiglumis, and O. latifolia) are found only in
America. O. latifolia is widely distributed, growing in Cen-
tral and South America as well as the Caribbean islands.
O. alta and O. grandiglumis are usually found only in South
America (Oliveira 1994). Chromosome pairing has identified
the CC genome as one of the likely genome components of
this species (Katayama 1997). However, little has been dis-
covered about the DD genome of this allotetraploid, since it
was first proposed (Morinaga 1939 cited by Katayama
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1997). So far, all attempts to describe the donor ancestor of
the DD genome have failed.
In both types of analysis of RAPDs, phenetic and
cladistic, the CCDD-genome accessions clustered together,
separated from the other species of the complex. They have
a closer affinity to each other than to any known diploid spe-
cies. This raises the possibility that all CCDD species were
derived from a single polyploidization event. The main mor-
phological difference among the three species is in spikelet
size. The spikelets of O. latifolia are less than 7 mm long,
while those of O. alta and O. grandiglumis are longer
(Vaughan 1989). Another basic difference is that, in
O. grandiglumis, the structure and size of the sterile and fer-
tile lemmas are usually the same. With only these differ-
ences, Chevalier (1932) suggested that the three taxa should
be treated as varieties of the same species. Analysis of the
samples studied here show great variation in seed size and in
the size of the sterile lemmas of the three so-called species.
Also, 24 bivalents were observed in studies of F1hybrids be-
tween these species (Katayama 1997). In the present study,
clear groups corresponding to the three species with the
CCDD genome (O. latifolia,O. alta, and O. grandiglumis)
were not observed. This is probably an indication that these
species really form a species group rather than being three
distinct species or, that perhaps they are all just one species.
This CCDD-genome data is consistent with other reports
based on morphology, crossability, isozymes, and
chloroplasts (Sampath 1962; Gopalakrishnan and Sampath
1966; Second 1985; Jena and Kochert 1991; Wang et al.
1992; Kiefer-Meyer et al. 1995; Jena and Kush 1998).
Phenetic and cladistic analyses confirmed the classification
of the CCDD-genome accessions in the O. officinalis com-
plex. The data indicated that CC-genome accessions are the
closest relatives of the CCDD-genome accessions. This
means that the DD-genome species could be either extinct
(Jena and Kochert 1991), not yet recognized for what it is,
or yet to be collected. Although this study attempted to ana-
lyze the greatest sample of American accessions of wild
© 2001 NRC Canada
Buso et al. 489
Fig. 3. (a) Amplification of the cpDNA trnC [tRNA-Cys (GCA)] to trnD [tRNA-Asp (GUC)] region. Polymorphism could be detected
between the FF genome (O. brachyantha) (lane 1) and the CCDD (O. latifolia) (lane 2), BB (O. punctata (lane 3), BBCC (O. minuta)
(lane 4), CC (O. officinalis) (lane 5), CCDD (O. grandiglumis) (lane 6), and AA (O. sativa) (lane 7) genomes. (b) Restriction fragment
patterns of cpDNA trnC [tRNA-Cys (GCA)] to trnD [tRNA-Asp (GUC)] digested with MspI. Lanes 1 and 2 are 1-kb and 123-bp lad-
ders, respectively. The arrows indicate misclassified accessions.
Fig. 2. Analysis of 230 accessions of wild and cultivated rice (Oryza spp.) based on 309 polymorphic RAPD markers. The phenogram
was constructed following pairwise estimates of coefficients of similarity and UPGMA analysis. The species Rhynchoryza subulata and
Bambusa tuldoides were used as outgroups.
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Oryza spp., it was restricted to accessions of the Amazon
and Paraguay river basins. Therefore, a broader prospection
should be made to collect more samples of diploid and
tetraploid American wild rice.
The BBCC-, BB-, and CC-genome species are in the sec-
ond subgroup of the O. officinalis complex. The so-called
O. punctata species with BB and BBCC genomes are en-
demic to Africa. The diploid and tetraploid species occupy
different habitats, although they cannot be easily separated
by morphological descriptors (Vaughan 1989). In the present
study, these species clustered separately based on nDNA,
cpDNA, and mtDNA data, indicating that they are actually
two distinct species. This suggests the need for a taxonomic
revision of these two species. It is interesting to note the per-
sistence of the same species name for diploid and tetraploid
specimens.
The O. ridleyi and O. meyeriana complexes
The O. ridleyi and O. meyeriana complexes are less
known, with few samples in collections and herbaria. The
genome group of the species belonging to these complexes
is still unknown. Based on genomic DNA hybridization,
Aggarwal et al. (1996) recently suggested that the
O. meyeriana complex genomes be designated GG and the
allotetraploid O. ridleyi complex genomes be designated
HHJJ.
The diploid species O. brachyantha grows on the African
continent. It is the species of the genus Oryza that is most
closely related to the genus Leersia (Vaughan 1989). The ge-
nome of this diploid species, designated FF, is different from
all other species of the genus (Vaughan 1989). The analysis
of embryo structure and other morphological characteristics
has placed O. brachyantha in the O. ridleyi complex
(Morishima and Oka 1960; Tateoka 1964). RFLP analysis,
however, placed it in the O. sativa complex (Wang et al.
1992). The present results support the placement of
O. brachyantha in the O. ridleyi complex, since in the
phenetic and cladistic analyses, O. brachyantha clustered
with O. longiglumis from the O. ridleyi complex.
O. longiglumis is related to O. ridleyi, but each has mor-
phological peculiarities that result in its classification as a
separate species (Vaughan 1989). Cytological observation of
the F1hybrid derived from crosses of O. ridleyi and
O. longiglumis confirmed that both species have the same
genome constitution (Katayama 1997; Sitch et al. 1998).
Aggarwal et al. (1996) designated the genome of both spe-
cies HHJJ.
The O. meyeriana complex consists of scarcely studied
species. The most common and widely spread species of the
complex are O. granulata and O. meyeriana. This complex
highlights a typical problem of plant phylogeny. When a
taxon or related taxa are poorly represented in herbaria or
germplasm collections, it is difficult to be sure what status
they should be given. In the present study, O. granulata
seems to be the most distant species in the genus. In both
analyses, phenetic and cladistic, the O. granulata accession
studied clustered with one of the outgroup species,
R. subulata. Their DNA-content values, however, are very
different: R. subulata seems to be a diploid, whereas the
DNA-content value for O. granulata lies in the range of the
tetraploid species.
cpDNA and mtDNA analysis
Polymorphism analysis of cpDNA
Significant levels of polymorphism were detected among
genomes and at lower levels among the wild rice species, but
few intraspecific polymorphisms were observed. In general,
phenetic and cladistic analyses agreed, and there was low
resolution at the intragenomic level.
The O. sativa complex species clustered together, and the
O. glumaepatula accessions formed a group with 96% simi-
larity with the O. sativa – O. nivara group. Oryza
glumaepatula is endemic to Latin America and is considered
by some authors to be derived from ancestors that predate
the division of the continent of Gondwanaland (Chang
1985). The pantropical and subtropical distribution of the
wild relatives of the two cultivated species in Africa, South
and Southeast Asia, Oceania and Australia, and Central and
South America strongly suggests a common progenitor that
existed in the humid zone of Gondwanaland before its
breakup and drift (Chang 1976). Chatterjee (1951), on the
other hand, suggested that the three species of Oryza found
in Latin America might have arrived from Africa during the
early Tertiary period. This period extended from 66.4 to 1.6
million years ago and, during this period, the continents pro-
gressively assumed their present configuration. Second
(1985), however, suggested that O. glumaepatula is a recent
introduction to the American continent, brought by coloniz-
ers 500 years ago. The synonymous rate for cpDNA evolu-
tion among grasses is estimated to be1×10
–9 nucleotide
substitutions per year (Zurawski et al. 1984). If the
chloroplast genome of Oryza evolves at a similar rate, then
the estimated time of divergence between O. glumaepatula
© 2001 NRC Canada
490 Genome Vol. 44, 2001
Enzyme
Region HaeIII TaqIHinfI MspIRsaI Total no. of fragments
cp1 7 2 12 8 3 32
cp2 3 19 11 17 6 56
cp3 10 14 32 21 9 86
cp5 6 5 5 4 15 35
cp6 11 18 12 11 13 65
Total no. of fragments 37 58 72 61 46 274
mt1 9 12 7 6 14 48
mt2 12 12 12 13 11 60
Total no. of fragments 21 24 19 19 25 108
Table 3. Number of polymorphic fragments produced by the digestion of five cpDNA and two mtDNA re-
gions with five enzymes.
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and O. sativa O. nivara is about 20 million years ago. This
estimate agrees with the hypothesis of Chang (1976) or
Chatterjee (1951).
The O. officinalis complex showed more than one group
of related species. Two species from this complex clustered
with the O. sativa complex species, O. punctata (BB ge-
nome) and O. minuta (BBCC), with similar chloroplast se-
quences. The other BBCC-genome species (O. punctata)
clustered with the CC-genome species. Many studies de-
tected the same relationship between these species (Dally
and Second 1990; Second and Wang 1992; Nakazono et al.
1995; Provan et al. 1997). The current hypothesis is that, in
forming the tetraploid O. punctata (BBCC genome), the cy-
toplasm came from a CC-genome ancestor species, whereas
in forming O. minuta (BBCC genome), the cytoplasm came
from a BB-genome ancestor species.
The cpDNA-polymorphism analysis grouped the CCDD-
and CC-genome accessions. An ancestral CC-genome spe-
cies seems to be the cytoplasm donor of the CCDD genome.
This corroborates the conclusions of other studies (Second
1985; Dally and Second 1990). The estimated divergence
time of CCDD and CC chloroplast genomes is also approxi-
mately 20 million years ago. Based on isozyme electropho-
retic calibration, Second (1985) estimated a time of
divergence of roughly 15 million years between the BB, CC,
DD, and EE genomes.
Some authors indicated a genetic proximity between the
CCDD- and EE-genome species based on molecular data
(Second and Wang 1992; Wang et al. 1992; Nakazono et al.
1995). Some EE-genome accessions utilized in the present
study are the same ones used by the just-cited authors (ac-
cessions 166, 174, and 175). These accessions are tetraploids
and were reclassified in the present study as BBCC-genome
accessions. Therefore, the reason for the detected proximity
between EE- and CCDD-genome species could be the
misclassification of BBCC-genome accessions as EE-
genome accessions. A repetitive DNA sequence from
O. sativa that is present in the AA, BB, CC, and DD
genomes but not in the EE genome suggests that there is a
distant relationship between the EE and other genomes
(Zhao et al. 1989; Zhao and Kochert 1993). The chromo-
somes of O. australiensis (EE) are larger than those of the
other species, and studies of F1hybrids between
O. australienses and O. alta (CCDD) showed that, owing to
size differences and degrees of staining between paired chro-
mosomes, the majority of bivalents resulted from
autosyndesis between the C and D genomes of O. alta
(Katayama 1997). Two accessions classified as
O. australiensis (270 and 271) have a DNA content in the
range of the tetraploids, which would be expected, since
chromosomes of this species are larger.
Polymorphism analysis of mtDNA
The large size, complexity, and rearrangements of the
plant mitochondrial genomes create highly variable restric-
tion patterns that limit interpretation of experimental data.
These limitations can be circumvented by analyzing
polymorphisms of specific mtDNA sequences using cleaved
polymorphic sequences.
The DNA sequences of plants evolve at different rates, de-
pending on whether they are located in the nuclear, mito-
chondrial, or chloroplast genome. In angiosperms, the silent
substitution rate in mtDNA is less than one-third of that ob-
served in cpDNA, which in turn evolves only half as fast as
© 2001 NRC Canada
Buso et al. 491
Fig. 4. Analysis of 65 accessions of wild and cultivated rice
(Oryza spp.) based on 246 polymorphic cpDNA markers. The
cladogram represents the 50% majority rule consensus tree of
26 112 maximally parsimonious trees generated by a heuristic
search. Numbers above the branches refer to the number of
times that the particular clade was obtained in trees generated by
PAUP. The species Rhynchoryza subulata and Bambusa tuldoides
were used as outgroups.
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plant nDNA (Wolfe et al. 1987). In this study, mtDNA frag-
ments were, as expected, less polymorphic than cpDNA
fragments. The low polymorphism and limited genome cov-
erage influenced the phenetic analysis based only on
mtDNA, which did not completely reflect the taxonomic
classification. On the other hand, this did not influence the
cladistic analysis, which produced trees with high statistical
support reflecting the taxonomic classification currently ac-
cepted. High levels of conservation of angiosperm mtDNA
have also been found in Lycopersicon spp. (McCleen and
Hanson 1986), commercial lines of Zea mays (Levings and
Pring 1977), and Hordeum spp. (Holwerda et al. 1986; Rines
et al. 1988).
In the O. sativa complex, the O. glumaepatula accessions
formed a cluster distinct from the other AA-genome species
but closer to O. breviligulata accessions. Diploid accessions
of O. punctata (BB) and O. minuta (BBCC) clustered to-
gether, corroborating the cpDNA results. The mtDNA results
also indicated at least two different allotetraploidization
events, one leading to tetraploid O. punctata, with cytoplasm
from a CC-genome ancestral donor, and one leading to
O. minuta, with cytoplasm from an ancestral maternal BB
genome. Also, for species with CC, CCDD, and BBCC
(O. punctata) genomes, mtDNA results agreed with cpDNA
results, indicating maternal proximity among them. All
CCDD-genome accessions clustered together. For the re-
maining complexes, mtDNA corroborated cpDNA results,
indicating parallel evolution of cpDNA and mtDNA.
Conclusions
The molecular evaluation of genetic diversity is a useful
way to study the amount and partitioning of genetic variabil-
ity in cultivated species and their wild relatives. It can be
used to study their phylogenetic relationships and may pro-
vide a rationale for choosing strategies for breeding,
germplasm collection, conservation, and use of genetic re-
sources. This investigation examined a large sample of
American wild rice species at DNA regions evolving at dif-
ferent rates, comparing the polymorphism found in these re-
gions among accessions of wild and cultivated rice from
America, Africa, Asia, and Oceania. The results suggest: (i)
The combination of techniques such as flow cytometry, ge-
nome-specific markers, and random and specific analyses of
nDNA, mtDNA, and cpDNA is a powerful approach for
germplasm analysis of large-scale samples of genetic re-
sources. (ii) The three tetraploid American wild rice species
(O. alta, O. grandiglumis, and O. latifolia) could be treated
as the same species. (iii) The diploid American species
(O. glumaepatula) should be considered a species distinct
from O. rufipogon or other AA-genome species. Oryza
glumaepatula represents an important gene pool to be con-
served and is currently being used in breeding programs
aimed at introgressing new genes into cultivated rice
(Brondani et al. 1998). (iv) The estimated time of divergence
between the American diploid O. glumaepatula and
O. sativa O. nivara and between American tetraploid spe-
cies and CC- and BBCC-genome species was about 20 mil-
lion years ago. (v) The cpDNA and mtDNA analyses
confirmed the current hypothesis of two maternal ancestors
for the BBCC genome, one leading to the tetraploid
O. punctata, with CC-genome cytoplasm, and another lead-
ing to O. minuta, with BB-genome cytoplasm.
(vi)O. punctata species with BB and BBCC genomes clus-
tered separately, based on nDNA, cpDNA, and mtDNA data,
indicating that they are actually two distinct species. This
suggests a need for taxonomic revision of these two species.
It is interesting to note the persistence of the same species
name for diploid and tetraploid specimens. (vii)O. granulata
seems to be the most distant species in the genus. In
phenetic and cladistic analyses of RAPD and mtDNA mark-
ers, the O. granulata accession studied clustered with one of
the outgroup species, R. subulata.(viii) The results of the
present study support the placement of O. brachyantha in
the O. ridleyi complex.
Acknowledgements
We thank M.I.O. Penteado for flow cytometric analyses
and M.T. Pozzobon and A. del P.S. Peñaloza for assistance
with the chromosome-counting technique. This work was
supported, in part, by a grant provided by the Brazilian Na-
tional Program for Supporting Scientific and Technological
Development – National Council of Scientific and Techno-
logical Development (PADCT/CNPq) and Research Sup-
porting Foundation of Distrito Federal (FAP-DF).
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... Misclassification (misidentification) has been reported in several species, including Oryza glaberrima, O. sativa, and O. barthii (Orjuela et al. 2014), other wild rice species (Buso et al. 2001), Dioscorea spp. (Girma et al. 2012), andBrassica spp. ...
... All the 37 regenerated seed lots were misclassified, which might be caused by labeling errors made during seed regeneration/multiplication and seed processing/ handling. The proportion of misclassification observed in our study was much lower than the 5-21% misclassification reported in other studies (Buso et al. 2001;Girma et al. 2012;Orjuela et al. 2014;Mason et al. 2015). Orjuela et al. (2014) reported misclassification of 13 of 266 O. glaberrima accessions (4.9%), which were supposed to be O. sativa; however, the proportion of misclassification in their study seems over 20%, as there were several O. barthii accessions that were clustered with O. glaberrima in both neighbor-joining phylogenetic analysis and PCA. ...
... Orjuela et al. (2014) reported misclassification of 13 of 266 O. glaberrima accessions (4.9%), which were supposed to be O. sativa; however, the proportion of misclassification in their study seems over 20%, as there were several O. barthii accessions that were clustered with O. glaberrima in both neighbor-joining phylogenetic analysis and PCA. Buso et al. (2001) samples and 104 randomly selected lines from the germplasm collection. The authors found out that (i) 18% of the 180 lines were misclassified based on species; (ii) 30% of the 76 suspected samples were misclassified; (iii) 9% of the randomly selected samples were misclassified; (iv) several individuals were found to be the product of interspecific hybridization events; and (vi) SNP markers proved to be effective at confirming species identity. ...
Article
Full-text available
Species misclassification (misidentification) and handling errors have been frequently reported in various plant species conserved at diverse gene banks, which could restrict use of germplasm for correct purpose. The objectives of the present study were to (i) determine the extent of genotyping error (reproducibility) on DArTseq-based single-nucleotide polymorphisms (SNPs); (ii) determine the proportion of misclassified accessions across 3134 samples representing three African rice species complex (Oryza glaberrima, O. barthii, and O. longistaminata) and an Asian rice (O. sativa), which are conserved at the AfricaRice gene bank; and (iii) develop species- and sub-species (ecotype)-specific diagnostic SNP markers for rapid and low-cost quality control (QC) analysis. Genotyping error estimated from 15 accessions, each replicated from 2 to 16 times, varied from 0.2 to 3.1%, with an overall average of 0.8%. Using a total of 3134 accessions genotyped with 31,739 SNPs, the proportion of misclassified samples was 3.1% (97 of the 3134 accessions). Excluding the 97 misclassified accessions, we identified a total of 332 diagnostic SNPs that clearly discriminated the three indigenous African species complex from Asian rice (156 SNPs), O. longistaminata accessions from both O. barthii and O. glaberrima (131 SNPs), and O. sativa spp. indica from O. sativa spp. japonica (45 SNPs). Using chromosomal position, minor allele frequency, and polymorphic information content as selection criteria, we recommended a subset of 24 to 36 of the 332 diagnostic SNPs for routine QC genotyping, which would be highly useful in determining the genetic identity of each species and correct human errors during routine gene bank operations. Electronic supplementary material The online version of this article (10.1007/s11032-018-0885-z) contains supplementary material, which is available to authorized users.
... Oryza latifolia is widely distributed, occurring in Central and South America as well as the Caribbean islands, while O. alta and O. grandiglumis are found only in South America, primarily in the Amazon basin (Vaughan, 1989(Vaughan, , 1994. Due to their homogeneous genome type, similar morphological characteristics, as well as overlapping distribution, the delimitation and phylogenetic relationships of the three species have long been controversial (Tateoka 1962, Nayar 1973, Jena and Kochert 1991, Aggarwal et al. 1996, Fukui et al. 1997, Buso et al. 2001). In addition, because diploid species with the C and D genomes have not been reported on the American continent, the debates over possible origins of these American tetraploids with the CD genome have been continued for decades (Nayar 1973, Wang et al. 1992, Fukui et al. 1997, Li et al. 2001, Federici et al. 2002. ...
... By studying the meiotic pairing of hybrids between Oryza species, Morinaga (1939Morinaga ( , 1943 identified five different genomes with the A, B, C, D, and E genomes in diploids and BC and CD genomes in tetraploids. Although many previous studies indicated that the CD genomes originated from a single hybridization event (Jena and Kochert 1991, Wang et al. 1992, Aggarwal et al. 1996, Ge et al. 1999, Buso et al. 2001, the diploid donors of CD genome species have remained unclear. Besides supporting the single origin of the CD genome species, the present study was in good agreement with the previous recognition that the C genome served as the maternal parent of these tetraploids (Ge et al. 1999, Buso et al. 2001. ...
... Although many previous studies indicated that the CD genomes originated from a single hybridization event (Jena and Kochert 1991, Wang et al. 1992, Aggarwal et al. 1996, Ge et al. 1999, Buso et al. 2001, the diploid donors of CD genome species have remained unclear. Besides supporting the single origin of the CD genome species, the present study was in good agreement with the previous recognition that the C genome served as the maternal parent of these tetraploids (Ge et al. 1999, Buso et al. 2001. Wang et al. (1992) more closely related to the CD genome species (Fig. 2 and Fig. 4). ...
Article
Full-text available
Oryza officinalis complex includes five diploid species involving the B, C and E genomes, and is distributed in Asia, Africa and Oceania. These species are very important because O. australiensis is the only species with E genome, while O. punctata is the single species with B genome. Although there are three species with the C genome (O. eichingeri, O. officinalis and O. rhizomatis), these species might have played different roles in speciation of polylploids due to their disjunction in Asia and Africa. The phylogenetic relationships among these diploids are still unclear, and polyploidy speciation involving these diploids has long been in debate. In this study, we utilized the sequence data of multiple genes, including the chloroplast matK, internal transcribed spacer (ITS) fragment of nuclear ribosomal DNA, and two nuclear genes (Adh1 and Adh2), to infer the phylogenetic relationships among the diploids and the related genomes. The separate and combined analyses were implemented by both maximum parsimony and maximum likelihood methods. The results indicate that (1) the C genome showed a closer affinity to the B genome than to the E genome; (2) of the three species with the C genome, O. officinalis and O. rhizomatis showed a closer relationship to each other than to O. eichingeri; (3) notably, in O. eichingeri, there exists higher divergence between the samples from Africa and those from Sri Lanka although they formed one monophyletic clade with lower bootstrap support.
... 育关系上, 相对于具有A染色体组的栽培稻而言也 较远, 但是这些野生多倍体中孕育的大量遗传多样 性却是栽培稻分子育种不可多得的宝贵资源。目前 通过胚拯救等手段, 一些多倍体中的有益基因已经 被成功地转移到栽培稻中 (Amante-Bordeos et al., 1992;Brar et al., 1996;Multani et al., 2003)。但是这 些应用仅仅涉及多倍体本身大量遗传多样性中的 极少部分, 更多的有益基因还有待深入开发。因此 加强对这些多倍体的研究, 是合理利用这部分资源 的重要前提。正是基于此种原因, 众多学者在长达 两个半世纪里从整个稻属的角度, 对其中的多倍体 植 物 开 展 了 较 为 广 泛 的 研 究 (Roschevicz, 1931;Tateoka, 1963;Vaughan, 1989Vaughan, , 1994Vaughan, , 2003Morishima et al., 1992;Wang et al., 1992;Aggarwal et al., 1999;Ge et al., 1999;Buso et al., 2001;Li et al., 2001a, b;Thomas et al., 2001;Bao & Ge, 2004) (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003b卢宝荣等, 2001) Table 1 Genomic constitutions and geographical distributions of Oryza species (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003bLu et al., 2001 (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003aAggarwal et al., 1999;Buso et al., 2001) (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003 (Nayar, 1973;Vaughan, 1989Vaughan, , 1994Vaughan, , 2003a卢宝荣等, 2001 Wang et al., 1992;Ge et al., 1999;Buso et al., 2001;Federici et al., 2002;Bao & Ge, 2004 (Sarker et al., 1993;Jena, 1994;Brar et al., 1998) ...
... 育关系上, 相对于具有A染色体组的栽培稻而言也 较远, 但是这些野生多倍体中孕育的大量遗传多样 性却是栽培稻分子育种不可多得的宝贵资源。目前 通过胚拯救等手段, 一些多倍体中的有益基因已经 被成功地转移到栽培稻中 (Amante-Bordeos et al., 1992;Brar et al., 1996;Multani et al., 2003)。但是这 些应用仅仅涉及多倍体本身大量遗传多样性中的 极少部分, 更多的有益基因还有待深入开发。因此 加强对这些多倍体的研究, 是合理利用这部分资源 的重要前提。正是基于此种原因, 众多学者在长达 两个半世纪里从整个稻属的角度, 对其中的多倍体 植 物 开 展 了 较 为 广 泛 的 研 究 (Roschevicz, 1931;Tateoka, 1963;Vaughan, 1989Vaughan, , 1994Vaughan, , 2003Morishima et al., 1992;Wang et al., 1992;Aggarwal et al., 1999;Ge et al., 1999;Buso et al., 2001;Li et al., 2001a, b;Thomas et al., 2001;Bao & Ge, 2004) (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003b卢宝荣等, 2001) Table 1 Genomic constitutions and geographical distributions of Oryza species (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003bLu et al., 2001 (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003aAggarwal et al., 1999;Buso et al., 2001) (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003 (Nayar, 1973;Vaughan, 1989Vaughan, , 1994Vaughan, , 2003a卢宝荣等, 2001 Wang et al., 1992;Ge et al., 1999;Buso et al., 2001;Federici et al., 2002;Bao & Ge, 2004 (Sarker et al., 1993;Jena, 1994;Brar et al., 1998) ...
... 育关系上, 相对于具有A染色体组的栽培稻而言也 较远, 但是这些野生多倍体中孕育的大量遗传多样 性却是栽培稻分子育种不可多得的宝贵资源。目前 通过胚拯救等手段, 一些多倍体中的有益基因已经 被成功地转移到栽培稻中 (Amante-Bordeos et al., 1992;Brar et al., 1996;Multani et al., 2003)。但是这 些应用仅仅涉及多倍体本身大量遗传多样性中的 极少部分, 更多的有益基因还有待深入开发。因此 加强对这些多倍体的研究, 是合理利用这部分资源 的重要前提。正是基于此种原因, 众多学者在长达 两个半世纪里从整个稻属的角度, 对其中的多倍体 植 物 开 展 了 较 为 广 泛 的 研 究 (Roschevicz, 1931;Tateoka, 1963;Vaughan, 1989Vaughan, , 1994Vaughan, , 2003Morishima et al., 1992;Wang et al., 1992;Aggarwal et al., 1999;Ge et al., 1999;Buso et al., 2001;Li et al., 2001a, b;Thomas et al., 2001;Bao & Ge, 2004) (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003b卢宝荣等, 2001) Table 1 Genomic constitutions and geographical distributions of Oryza species (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003bLu et al., 2001 (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003aAggarwal et al., 1999;Buso et al., 2001) (Vaughan, 1989(Vaughan, , 1994(Vaughan, , 2003 (Nayar, 1973;Vaughan, 1989Vaughan, , 1994Vaughan, , 2003a卢宝荣等, 2001 Wang et al., 1992;Ge et al., 1999;Buso et al., 2001;Federici et al., 2002;Bao & Ge, 2004 (Sarker et al., 1993;Jena, 1994;Brar et al., 1998) ...
Article
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The genus Oryza belongs to the tribe Oryzeae, subfamily Oryzoideae of the grass family Poaceae, consisting of more than 20 species. Among the species, about half are polyploids. These polyploids include BC, CD, HJ and HK genomic constitutions and are distributed in Asia, Oceania, Africa and Latin America. Because of its economic and theoretic significance, Oryza has attracted enormous attentions from scientists worldwide. Correspondingly, many research data are accumulated for the polyploid members in the genus. In this paper, we have reviewed the history of Oryza, especially the study of the polyploids. Based on our recent molecular studies on the origin and phylogeny of Oryza species, we summarized the current status of the investigations on the classification and phylogeny of the polyploid species. In addition, we discussed the perplexity and future direction of the studies on the Oryza polyploids.
... Based on AFLP analysis, for example, Aggarwal et al. (1999) indicated that two accessions determined as diploid O. eichingeri (CC) were actually tetraploids with the BC genome, and one accession as diploid O. punctata (BB) was the tetraploid O. punctata (BBCC). In recent studies of American wild rice species, Buso et al. (2001) found that 8% of the 230 accessions studied was misidentified as a result of either taxonomic error or contamination. Of these materials, four accessions of O. punctata (BBCC) were originally misidentified as O. eichingeri (CC) and O. officinalis (CC), one accession of O. eichingeri (CC) was misidentified as O. minuta (BBCC), and one accession of O. punctata (BB) was misidentified as O. rhizomatis (CC). ...
... Similarly, Aggarwal et al. (1999) indicated that one Indian accession (105329) labeled as O. malampuzhanensis (BBCC) should be a species with the CD genomes. Many other studies also pointed out the misidentifications of Oryza species from genebank storage (Martin et al. 1997;Buso et al. 2001). Therefore, misidentification poses a considerable problem for the efficient utilization and management of the wild rice germplasm for breeding and research. ...
... Of the nine accessions determined as O. eichingeri, we found through our survey that five accessions (three from Sri Lanka and two from Uganda) were diploids with the C genome and other four (one from Sri Lanka and three from Uganda) were tetraploids (Table 1). It is noteworthy that out of the four tetraploid O. eichingeri accessions in this study, three were previously studied and considered to be misidentified (Aggarwal et al. 1999;Buso et al. 2001). Using AFLP markers, Aggarwal et al. (1999) indicated that the two Uganda accessions designed as the tetraploid O. eichingeri (Acc. ...
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The genus Oryza consists of approximately 24 species with 10 recognized genome types (A, B, C, BC, CD, E, F, G, HJ, HK). The species with the CD genome are endemic to the Central and South America and comprise three species, i.e. O. latifolia Desv., O. alta Swallen and O. grandiglumis (Doell) Prod. Although the three species can be easily distinguished from the species with the other genomes in Oryza by their morphological characteristics, it is very difficult to distinguish them from one another. Recent studies suggested that O. alta and O. grandiglumis should be the same species (O. grandiglumis), whereas O. latifolia retains its taxonomic position unchangeably. In this paper, a total of 77 clones of the nuclear ribosomal internal transcribed spacer (ITS) from 11 samples representing different geographical races were sequenced. Using DNA Strider 1.2 software, the restriction enzyme digestion sites of these clone sequences were analyzed. Based on restriction fragment length polymorphism (RFLP) of ITS sequences, a method to identify the species with CD genome in Oryza was proposed. The method is rapid and convenient and all experimental procedure includes only three steps: (1) to amplify the ITS fragment with the routine primers: (2) to digest PCR products with restriction enzymes Fok I and Dra III or both: (3) to run the digested product on 1% agarose gel and identify the sample based on the restriction profiles.
... Mislabeling, misclassification (misidentification), and mixing of samples www.nature.com/scientificreports/ are common problems in genebanks 15 and have been reported in several species, including multiple Oryza species 26,73,74 , Dioscorea spp. 75 , Brassica spp 76 . ...
... 77 . The percentage of mislabeled or misclassified samples reported in the literature is highly variable depending on sample size, the species, and the methods used for assessing the error rates, which varied from 3 to 21% 26,[73][74][75][76][77] . In one of our recent studies, we found that 3.1% of 3,134 of accessions from four rice species were either mislabeled or misclassified 26 , which can easily be checked using a subset of the diagnostic SNPs that we developed in the previous study. ...
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To minimize the cost of sample preparation and genotyping, most genebank genomics studies in self-pollinating species are conducted on a single individual to represent an accession, which may be heterogeneous with larger than expected intra-accession genetic variation. Here, we compared various population genetics parameters among six DNA (leaf) sampling methods on 90 accessions representing a wild species (O. barthii), cultivated and landraces (O. glaberrima, O. sativa), and improved varieties derived through interspecific hybridizations. A total of 1,527 DNA samples were genotyped with 46,818 polymorphic single nucleotide polymorphisms (SNPs) using DArTseq. Various statistical analyses were performed on eleven datasets corresponding to 5 plants per accession individually and in a bulk (two sets), 10 plants individually and in a bulk (two sets), all 15 plants individually (one set), and a randomly sampled individual repeated six times (six sets). Overall, we arrived at broadly similar conclusions across 11 datasets in terms of SNP polymorphism, heterozygosity/heterogeneity, diversity indices, concordance among genetic dissimilarity matrices, population structure, and genetic differentiation; there were, however, a few discrepancies between some pairs of datasets. Detailed results of each sampling method, the concordance in their outputs, and the technical and cost implications of each method were discussed.
... The use of molecular techniques for genomic analysis, associated with classic breeding methods, can greatly facilitate the utilization of this germplasm in the development of rice cultivars which are superior to those currently being used (Ferreira and Grattapaglia, 1995). Molecular research showed that O. glumaepatula is genetically related to O. sativa, which significantly facilitates gene introgression from the wild into the cultivated species (Buso, 1998;Buso et al., 2001). Using samples of O. glumaepatula populations harvested in Brazil, Embrapa is conducting a pre-breeding program with the goal to incorporate genes from this species into O. sativa elite lineage; this process is monitored based on molecular markers and genetic maps. ...
... Using molecular markers (microsatellites), were selected those of the obtained F1's which have wild species genes; following this selection, they underwent retrocrossings for 'BG 90-2'. Chromosomes genetic maps and an extended phenotypical analysis for yield components were used to locate the genomic chromosomal regions controlling quantitative features (QTLs) of interest -chiefly those related to the genetic control of yield (Brondani et al., 2002). Examination of genetic maps and, as a consequence, knowledge of the allele composition of segregating families allowed the selection of improved families, with wild species genes. ...
... Most taxonomic misclassification and mislabeling are due to human error during planting of material, characterization of accessions for phenotypic traits, and mislabeling or misreading of the germplasm names 18 during the various genebank operations. Such errors have been reported in various crop species and error rates vary from 3 to 28% 5, [18][19][20][21][22] . Recently, the impact of taxonomic misnaming was highlighted in a study of the genus Citrullus (watermelon) in two major databases: Genesys PGR (https:// www. ...
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Morphological identification of closely related rice species, particularly those in the Oryza AA genome group, presents major challenges and often results in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNP) markers specific for several rice species and subspecies based on DArTseq next-generation sequencing technology (“DArTseq”). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validation data for a subset of them; these can be used for low-cost routine genotyping quality control (QC) analysis. Of the 224 species/subspecies’ diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 70%. Two validation experiments were run with 87 of the 158 SNP markers to ensure that the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool to facilitate germplasm curation and management of these four Oryza AA genome species across multiple genebanks.
... The three allotetraploid (CCDD) species, O. alta, O. grandiglumis, and O. latifolia, which formed a monophelytic clade, were closely related to the three diploid CC genome species, that is, O. officinalis, O. rhizomatis, and O. eichingeri, with 100% bootstrap support values or 1.00 posterior probabilities. Complete chloroplast genome-based results convincingly demonstrate the hypothesis that the CCDD genome originated from a single hybridization event 12,14,19,[54][55][56] . Although it was well documented that the CC genome species (O. ...
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The course, tempo and mode of chloroplast genome evolution remain largely unknown, resulting in limited knowledge about how plant plastome gene and genome evolve during the process of recent plant speciation. Here, we report the complete plastomes of 22 closely related Oryza species in chronologically ordered stages and generate the first precise map of genomic structural variation, to our knowledge. The occurrence rapidity was estimated on average to be ~7 insertions and ~15 deletions per Myr. Relatively fewer deletions than insertions result in an increased repeat density that causes the observed growth of Oryza chloroplast genome sizes. Genome-wide scanning identified 14 positively selected genes that are relevant to photosynthesis system, eight of which were found independently in shade-tolerant or sun-loving rice species. psaA seemed positively selected in both shade-tolerant and sun-loving rice species. The results show that adaptive evolution of chloroplast genes makes rice species adapt to diverse ecological habitats related to sunlight preferences.
... The close relationship between O. barthii and many of the African rice accessions undoubtedly reflects the direct relationship between the two species, but it may also be caused by some misclassification of accessions. In previous studies, we found out that 3.1% of accessions across four rice species were misclassified/misidentified (Ndjiondjop et al. 2018b), which was smaller than the 4-21% misclassification reported in other studies (Buso et al. 2001;Girma et al. 2012;Orjuela et al. 2014;Mason et al. 2015). To minimize errors due to misclassification during germplasm collection, acquisitions and routine genebank operations, our group identified 332 species-and subspecies-specific diagnostic SNP markers in O. glaberrima/O. ...
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Key message The extent of molecular diversity parameters across three rice species was compared using large germplasm collection genotyped with genomewide SNPs and SNPs that fell within selective sweep regions. Abstract Previous studies conducted on limited number of accessions have reported very low genetic variation in African rice (Oryza glaberrima Steud.) as compared to its wild progenitor (O. barthii A. Chev.) and to Asian rice (O. sativa L.). Here, we characterized a large collection of African rice and compared its molecular diversity indices and population structure with the two other species using genomewide single nucleotide polymorphisms (SNPs) and SNPs that mapped within selective sweeps. A total of 3245 samples representing African rice (2358), Asian rice (772) and O. barthii (115) were genotyped with 26,073 physically mapped SNPs. Using all SNPs, the level of marker polymorphism, average genetic distance and nucleotide diversity in African rice accounted for 59.1%, 63.2% and 37.1% of that of O. barthii, respectively. SNP polymorphism and overall nucleotide diversity of the African rice accounted for 20.1–32.1 and 16.3–37.3% of that of the Asian rice, respectively. We identified 780 SNPs that fell within 37 candidate selective sweeps in African rice, which were distributed across all 12 rice chromosomes. Nucleotide diversity of the African rice estimated from the 780 SNPs was 8.3 × 10⁻⁴, which is not only 20-fold smaller than the value estimated from all genomewide SNPs (π = 1.6 × 10⁻²), but also accounted for just 4.1%, 0.9% and 2.1% of that of O. barthii, lowland Asian rice and upland Asian rice, respectively. The genotype data generated for a large collection of rice accessions conserved at the AfricaRice genebank will be highly useful for the global rice community and promote germplasm use.
Article
MITE-AFLP markers were successfully used to study the genetic variation and species relationship in Oryza species. Analysis of 53 accessions of Oryza species with seven MITE-AFLP primer combinations detected a total of 250 polymorphic fragments. High polymorphism was detected within and between Oryza species. Species relationships were analyzed by the pattern of presence or absence of homologous fragments, because nucleotide sequences of the detected MITE-AFLP fragments revealed that the same fragments in different species shared very high sequence homology. The genetic distances (GDs) between species were higher than those within species and the GDs in O. sativa complex were higher than those in O. officinalis complex. The phylogenetic tree recognized two major groups at 62% genetic similarity; group I consists of all AA genome species of the O. sativa complex, and group II consists of BB-, CC-, EE- and BBCC genome species of the O. officinalis complex. Therefore, this study demonstrated that the MITE-AFLP technique provide a tool for studying the genetic variation and species relationship in Oryza species.
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The genus Oryza to which cultivated rice belongs has 24 species (2n = 24 or 48), representing seven genomes (AA, BB, CC, EE, FF, BBCC and CCDD). The genomic constitution of five of these species is unknown. These five species have been grouped into two species complexes, the tetraploid ridleyi complex (O. ridleyi, O. longiglumis) and the diploid meyeriana complex (O. granulata, O. meyeriana, O. indandamanica). To evaluate the genomic structure of these species in terms of divergence at the molecular level vis-a-vis other known genomes of Oryza, we used the total genomic DNA hybridization approach. Total genomic DNA (after restriction digestion) of 79 accessions of 23 Oryza species, 6 related genera, 5 outgroup taxa (2 monocots, 3 dicots) and 6 F1s and BC1s derived from crosses of O. sativa with wild species were hybridized individually with 32P-labeled total genomic DNA from 12 Oryza species: O. ridleyi, O. longiglumis, O. granulata, O. meyeriana, O. brachyantha, O. punctata, O. officinalis, O. eichingeri, O. alta, O. latifolia, O. australiensis, and O. sativa. The labeled genomic DNAs representing the ridleyi and meyeriana complexes cross-hybridized best to all the accessions of their respective species, less to those representing other genomes of Oryza and related genera, and least to outgroup taxa. In general, the hybridization differential measured in terms of signal intensities was >50-fold under conditions that permit detection of 70-75% homologous sequences, both in the presence and in the absence of O. sativa DNA as competitor. In contrast, when total DNAs representing other Oryza genomes were used as probes, species of the O. ridleyi and O. meyeriana complexes did not show any significant cross-hybridization (<5%). These results demonstrate that the genome(s) of both of these complexes are highly diverged and distinct from all other known genomes of Oryza. We, therefore, propose new genomic designations for these two species complexes: GG for the diploid O. meyeriana complex and HHJJ for the allotetraploid O. ridleyi complex. The results also suggest that the uniqueness of these genomes is not restricted to species-specific highly repetitive DNA sequences, but also applies to dispersed sequences present in single or low to moderate copy numbers. Furthermore these appear to share relatively more genome-specific repeat sequences between themselves than with other genomes of rice. The study also demonstrates the potential of total genomic DNA hybridization as a simple but powerful tool, complementary to existing approaches, for ascertaining the genomic makeup of an organism.
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Spacer-length variation in ribosomal DNA (rDNA) was surveyed in two cultivated rice species and their wild relatives. Among 243 accessions observed, 18 different spacer-length variants were detected. Length heterogeneity was found within and among species as well as within individuals. Conventional genetic analysis revealed that two spacer-length variants were located at two unlinked loci. Restriction enzyme maps showed that length heterogeneity resulted from repetition of short repeated sequences in the intergenic spacer region in the Asian cultivar and its progenitor; however, the spacer region greatly differed from those of reproductively isolated taxa with respect to the length and the sequence. Furthermore, the Asian cultivated species and its progenitor were highly polymorphic for rDNA spacer-length variation and they were differentiated in frequencies of spacer-length variants as well as varietal groups within the cultivated species. Asian cultivars tended to carry homogeneous repeats of rDNA compared with their progenitor, suggesting different forms of homogenization occurring in Asian cultivars.Key words: ribosomal DNA, intergenic spacer, polymorphism, inheritance, Oryza.