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Following the track of “Híbrido de Timor” origin by cytogenetic and flow cytometry approaches

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The supposedly first plant of the coffee cultivar “Híbrido de Timor” (HT) was found in 1927, being denoted as HT CIFC 4106. According to different researchers, this plant originated from a natural interspecific hybridation between Coffea arabica (4x = 44) and Coffea canephora (2x = 22). From HT CIFC 4106, other HT accessions were obtained and employed to establish germplasm banks in some countries. As HT has been widely used in Coffea breeding programs, this study aimed to characterize different HT accessions with regard to ploidy, nuclear DNA content and base composition. Based on these data, the ploidy of HT CIFC 4106 was determined, suggesting that this accession is an allotriploid formed from reduced reproductive cell of C. canephora and of C. arabica. All HT CIFC 4106 plants exhibited the same 2C-value, AT% and chromosome number, showing that vegetative propagation has enabled the multiplication and germplasm conservation of this cytotype since 1927. Further five analyzed HT accessions showed distinct nuclear 2C-value and AT%. Since HT CIFC 4106 has been considered the first HT, it is suggested that aneuploid reproductive cells of this HT originated the other plants. Considering that HT accessions are used in the development of C. arabica cultivars, the findings of this study are important for the design of strategies to obtain new cultivars for breeding programs. Moreover, these data represent the first step to understand the origin and genome evolution of the HT.
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RESEARCH ARTICLE
Following the track of ‘‘Hı
´brido de Timor’’ origin
by cytogenetic and flow cytometry approaches
Wellington Ronildo Clarindo
Carlos Roberto Carvalho Eveline
Teixeira Caixeta Andre
´a Dias Koehler
Received: 12 December 2012 / Accepted: 18 March 2013
ÓSpringer Science+Business Media Dordrecht 2013
Abstract The supposedly first plant of the coffee
cultivar ‘‘Hı
´brido de Timor’’ (HT) was found in 1927,
being denoted as HT CIFC 4106. According to
different researchers, this plant originated from a
natural interspecific hybridation between Coffea arab-
ica (4x =44) and Coffea canephora (2x =22). From
HT CIFC 4106, other HT accessions were obtained
and employed to establish germplasm banks in some
countries. As HT has been widely used in Coffea
breeding programs, this study aimed to characterize
different HT accessions with regard to ploidy, nuclear
DNA content and base composition. Based on these
data, the ploidy of HT CIFC 4106 was determined,
suggesting that this accession is an allotriploid formed
from reduced reproductive cell of C. canephora and of
C. arabica. All HT CIFC 4106 plants exhibited the
same 2C-value, AT% and chromosome number,
showing that vegetative propagation has enabled the
multiplication and germplasm conservation of this
cytotype since 1927. Further five analyzed HT acces-
sions showed distinct nuclear 2C-value and AT%.
Since HT CIFC 4106 has been considered the first HT,
it is suggested that aneuploid reproductive cells of this
HT originated the other plants. Considering that HT
accessions are used in the development of C. arabica
cultivars, the findings of this study are important for
the design of strategies to obtain new cultivars for
breeding programs. Moreover, these data represent the
first step to understand the origin and genome
evolution of the HT.
Keywords Allotriploid Base composition
Coffea Cytotypes Karyotype Nuclear 2C-value
Introduction
‘‘ H ı
´brido de Timor’’ (HT) originated from a natural
interspecific cross between the allotetraploid Coffea
arabica L. (4x =44) and the diploid Coffea cane-
phora Pierre ex Froehner (2x =22) (Bettencourt
1973; Rodrigues et al. 1975,2004; Carvalho et al.
1989; Agwanda et al. 1997; Capucho et al. 2009). The
first HT plant was found in 1927, in a plantation of C.
arabica ‘Typica’ established around 1917/18, on the
Timor Island (Bettencourt 1973,1981).
W. R. Clarindo
Laborato
´rio de Citogene
´tica, Departamento de Biologia,
Centro de Cie
ˆncias Agra
´rias, Universidade Federal do
Espı
´rito Santo, Alegre, ES CEP 29500-000, Brazil
C. R. Carvalho (&)A. D. Koehler
Laborato
´rio de Citogene
´tica e Citometria, Departamento
de Biologia Geral, Centro de Cie
ˆncias Biolo
´gicas e da
Sau
´de, Universidade Federal de Vic¸osa, Vic¸osa, MG CEP
36570-000, Brazil
e-mail: ccarvalh@ufv.br
E. T. Caixeta
Embrapa Cafe
´, Empresa Brasileira de Pesquisa
Agropecua
´ria, Instituto de Biotecnologia Aplicada a
`
Agropecua
´ria (BIOAGRO), Laborato
´rio BioCafe
´,
Universidade Federal de Vic¸osa, Vic¸osa, MG CEP
36570-000, Brazil
123
Genet Resour Crop Evol
DOI 10.1007/s10722-013-9990-3
Seeds of this presumably first HT plant were
harvested by the ‘‘Empresa Sociedade Agrı
´cola Pa
´tria
e Trabalho’’ (SAPT), and started being cultivated in
Timor Island on the second half of the 1940s. Since
1956, seeds of selected plants have been used to
generate new HT crops in practically the whole island
(Rodrigues et al. 1975; Bettencourt 1981). According
to Gonc¸alves et al. (1978) and Rodrigues et al. (2004),
all current HT accessions have their origin in that
presumable first HT, or from crossings between that
plant and C. arabica.
The supposedly first HT plant was introduced by
vegetative propagation into the ‘‘Centro de Investi-
gac¸a
˜o das Ferrugens do Cafeeiro’’ (CIFC) in Portugal,
receiving the registration number CIFC 4106 (Pereira
et al. 2008). In 1957, seeds of different HT from the
Timor Island were taken to CIFC. The provided plants
were selected for resistance to Hemileia vastatrix
Berk. et Br. Among these, two clones from different
origins, denoted HT CIFC 832/1 and HT CIFC 832/2,
stood out by showing resistance to all isolates of the
pathogen (Rodrigues et al. 1975). These plants, as well
as those derived from their crosses with the main
cultivars of C. arabica, were introduced into almost all
Coffea experimental centers around the world, includ-
ing Brazil (Bettencourt 1973).
HT accessions have been valuable for breeding
programs, as this germplasm shows resistance to
distinct Coffea pathogens, such as H. vastatrix
(Capucho et al. 2009), Colletotrichum kahawae
(Bettencourt 1973; Van der Vossen and Walyaro
1980; Rodrigues et al. 2004), Pseudomonas syringae
(Agwanda et al. 1997) and Meloidogyne exigua
(Rodrigues et al. 2004). These pathogens, especially
H. vastatrix, have constrained ‘‘Arabica’’ coffee
production, which is economically essential for over
50 developing countries (Agwanda et al. 1997). In this
sense, HT has been crossbred with C. arabica lines in
order to generate resistant cultivars (Waller et al.
2007; Capucho et al. 2009).
Since HT has been widely used in coffee breeding
programs, knowledge about the karyotype, ploidy
level, nuclear genome size and base composition of
this plant would contribute to (a) characterization of
accessions true-to-typeness and, consequently, screen-
ing of the desirable cytotypes; (b) physical mapping;
(c) designing of breeding and conservation strategies
(Ochatt 2008); and (d) sequencing projects (Bennett
and Leitch 2005).
However, no study has been conducted in HT
concerning these aspects. Therefore, different HT
accessions were characterized in the present work,
with regard to ploidy, nuclear DNA content and base
composition.
Materials and methods
Plant material
Six HT accessions were used in this study, among
which:
(a) HT CIFC 4106—considered the original HT
plant (C. arabica x C. canephora) obtained in the
Timor Island (Agwanda et al. 1997) and intro-
duced into CIFC (Portugal) by vegetative prop-
agation (Pereira et al. 2008).
(b) HT CIFC 832/1, 832/2 and 1343/269—intro-
duced into CIFC through seeds of selected plants
from Timor Island. In 1970/71, HT clones of
CIFC 4106, 832/1, 832/2 and 1343/269 were
obtained through vegetative propagation by the
CIFC, and brought to the Germplasm Bank of the
Universidade Federal de Vic¸osa, Brazil (Betten-
court 1973).
(c) HT UFV 377-01 and UFV 377-09—accessions
originated from seeds of IIAA 811-7, cultivated
in the ‘‘Instituto de Investigac¸a
˜o Agrono
´mica de
Angola’’ (IIAA) originating from seeds of CIFC
2235; this plant, in turn, was obtained from seeds
of VCE1587, selected in Tanzania (Pereira et al.
2008).
Due to parental origin of HT, C. arabica L.
‘Catuaı
´Vermelho IAC 150and C. canephora Pierre
ex. Froehn ‘Conilon’ were also used. All plants were
cultivated in greenhouse, located at the Universidade
Federal de Vic¸osa, under the same environmental
conditions.
Solanum lycopersicum L. ‘Stupicke
´’ (standard,
2C =2.00 pg Prac¸a-Fontes et al. 2011, and AT =
64.5 % Dolez
ˇel et al. 1992) and Pisum sativum L.
‘Ctirad’ (standard, 2C =9.16 pg Prac¸a-Fontes et al.
2011, and AT =61.4 % Dolez
ˇel et al. 1992) were
chosen as primary references for flow cytometry
(FCM) measurements. Seeds of these species were
kindly supplied by Dr. Jaroslav Dolez
ˇel (Experimental
Institute of Botany, Czech Republic).
Genet Resour Crop Evol
123
FCM analysis
Young leaves of Coffea (sample), HT (sample), S.
lycopersicum and P. sativum (primary standards) were
collected from healthy plants. Leaf fragments (2 cm
2
)
of each sample and primary standard (S. lycopersicum
or P. sativum) were co-chopped and processed for
supplying nuclei suspensions (Clarindo et al. 2012).
The suspensions were analyzed in a Partec PAS
Ò
flow
cytometer (Partec
Ò
GmbH, Munster, Germany),
equipped with a laser source (488 nm) and an UV
lamp (388 nm). FlowMax
Ò
software (Partec
Ò
) was
used for data analyses.
The nuclear genome size and base composition
were assessed for each sample according to Clarindo
et al. (2012). Six independent repetitions were
performed on 3 distinct days for each species and
HT sample, each analyzing over 10,000 nuclei. The
mean values of genome size and AT% were compared
using the Unweighted Pair Group Method with
Arithmetic Mean (UPGMA) method. The statistical
analyses were carried out using the Genes statistical
software (Cruz 2010).
Establishment of cell aggregate suspension
cultures
Young leaves of HT CIFC 4106 were pulverized and,
subsequently, disinfected under laminar flow hood.
Leaf fragments (1 cm
2
)wereculturedinsemi-solidcalli
induction medium, supplemented with 5 lM 6-benzyl-
aminopurine (BAP), for 30 days, in the dark, at 24 °C.
Subsequently, the calli were cultured, for the same
period, in medium containing 5 lM BAP and 10 lM
2,4-dichlorophenoxyacetic (2,4 D). For establishment
of cell aggregate suspension (CAS) cultures, 0.25 g of
calli was transferred to liquid medium containing the
same concentrations as the latter (i.e., 5 lM BAP and
10 lM 2,4-D). The flasks were maintained on shaker at
100 rpm and 24 °C, under a 16/8 h light/dark regime,
with 36 lmol m
-2
s
-1
light radiation. For all media,
the composition of salts and supplements was that
described by Clarindo et al. (2012).
Cytogenetic analysis
Cytogenetic procedure from CAS was performed
according to Clarindo et al. (2012). After, the aggre-
gates were washed, fixed, and enzymatically
macerated. Slides were then prepared using the cell
dissociation and air-drying techniques. Subsequently,
the slides were stained with a 5 % Giemsa solution
(Merck
Ò
) in phosphate buffer (pH 6.8), for 5 min,
washed twice in distilled water, air-dried, and finally
placed on a hot plate at 50 °C, for 3 min.
Images of metaphase chromosomes were captured
with a Media Cybernetics
Ò
Camera Evolution
TM
charge-coupled device (CCD) video camera, mounted
on a Nikon 80i microscope (Nikon, Japan).
Results and discussion
FCM histograms showed G
0
/G
1
nuclei peaks exhib-
iting coefficients of variation (CVs) between 2.75 and
4.15 %. As found by Clarindo et al. (2012), G
0
/G
1
peaks exhibited adequate CV values, indicating reli-
able and reproducible nuclear genome size as well as
AT% measurements. Differently from other FCM
studies in Coffea (Cros et al. 1995; Noirot et al. 2003),
variation in 2C-value and AT% was not observed in
every sample (Table 1). Thus, the FCM procedure
used here seems to reduce the interference of second-
ary metabolites on nuclear chromatin staining.
The G
0
/G
1
peak channel of S. lycopersicum and HT
CIFC 4106 were very close. As the G
0
/G
1
peak of the
standard should not overlap with the peak of the
sample (Greilhuber et al. 2007), P. sativum was also
used as primary reference standard (data not shown).
Table 1 Mean nuclear DNA content and base composition
(±standard deviation) of the Coffea species and HT cytotypes
measured by FCM using S. lycopersicum (2C =2.00 pg, and
AT =64.50 %) as internal standard
Coffea or HT 2C value
(pg)
1C bp
(910
9
)*
AT%
C. arabica 2.71 ±0.04 1.33 63.84 ±0.08
C. canephora 1.46 ±0.02 0.71 64.46 ±0.16
HT CIFC 4106 2.10 ±0.01 1.03 65.66 ±0.06
HT CIFC 832/1 2.62 ±0.03 1.28 69.78 ±0.07
HT CIFC 832/2 2.81 ±0.01 1.37 66.58 ±0.19
HT CIFC 1343/269 2.68 ±0.01 1.31 63.89 ±0.08
HT UFV 377-01 2.88 ±0.02 1.41 60.71 ±0.23
HT UFV 377-09 2.86 ±0.02 1.40 62.65 ±0.02
* 1C mean values converted to bp (base pairs), considering that
1 pg of DNA corresponds to 0.978 910
9
bp (Dolez
ˇel et al.
2003)
Genet Resour Crop Evol
123
The mean values of nuclear genome size and AT% of
each species and HT accessions were identical in
relation to values measured from S. lycopersicum.
Based on the G
0
/G
1
peak of primary standard (S.
lycopersicum or P. sativum) and of each sample
(Fig. 1), genome size and AT% values were calculated
for C. arabica,C. canephora, as well as for each HT
accession (Table 1). C. arabica and C. canephora
showed mean 2C and AT% values identical to those
reported by Clarindo et al. (2012). The mean 2C-
values of different HT varied from 2.10 pg (HT CIFIC
4106) to 2.88 pg (HT UFV 377-01), and the mean
AT% ranged from 60.71 % (HT UFV 377-01) to
69.78 % (HT CIFC 832/1).
The previous mean values were compared by
UFGMA, providing a dendrogram with two clusters:
one composed by C. canephora and HT CIFC 4106,
and another constituted by C. arabica, HT CIFC 832/1,
CIFC 832/2, CIFC 1343/269, UFV 377-01, and UFV
377-09 (Fig. 2).
HT CIFC 4106 showed mean 2C =2.10 pg and
AT% =65.66 %. This genome size value corre-
sponded to the DNA ploidy level of a triploid plant.
Considering that the 2C-value of HT CIFC 4106 is
close to the sum of 1C-value of C. arabica
(1C =1.355 pg) and C. canephora (1C =0.73 pg),
the FCM data suggest that HT CIFC 4106 originated
from the fusion of one reduced reproductive cell of C.
arabica (n =2x =22 chromosomes) with another of
C. canephora (n =x=11 chromosomes).
Regarding the FCM result for HT CIFC 4106 as
well as its origin, CAS culture was established for this
HT. In liquid medium, these calli were used as
chromosome source. Owing to the enhanced cytoge-
netic procedure making use of CAS, metaphases
were obtained showing individualized chromosomes,
Fig. 1 Representative FCM histograms exhibiting G
0
/G
1
peaks
provided from nuclear suspensions stained with propidium
iodide (ac)or4
0,60-diamidino-2-phenylindole (df). ac2C-
value measured using S. lycopersicum as internal standard
(channel 200, 2C =2.00 pg). aHT CIFC 4106 (channel 210,
2C =2.10 pg). bHT CIFC 832/1 (channel 262, 2C =2.62 pg).
cHT UFV 377-01 (channel 288, 2C =2.88 pg). dfAT%
measured using S. lycopersicum as internal standard (channel
200, AT =64.50 %). dHT CIFC 4106 (channel 224,
AT =65.66 %). eHT CIFC 832/1 (channel 323,
AT =69.78 %). fHT UFV 377-01 (channel 232,
AT =60.71 %)
Genet Resour Crop Evol
123
flattened on the slide, without chromatin deformations
and cytoplasmic background noises, and exhibiting
well-defined primary constriction. From these meta-
phases, the chromosome number of HT CIFC 4106
was evidenced as 2n =3x =33 (Fig. 3).
Pereira et al. (2008) reported that HT CIFC 4106
represents the original HT obtained in Timor Island.
According to different authors (Bettencourt 1973;
Rodrigues et al. 1975,2004; Carvalho et al. 1989;
Agwanda et al. 1997; Capucho et al. 2009), this HT
originated from a cross between C. arabica and C.
canephora. Regarding these facts, in addition to the
FCM and cytogenetic results, HT CIFC 4106 can be
considered an allotriploid plant.
Karyotypic and nuclear 2C-value data also showed
that all analyzed HT CIFC 4106 plants were triploids
(3x =33 chromosomes and 2C =2.10 pg). There-
fore, since 1917–1918, vegetative propagation has
enabled the clonal multiplication and germplasm
conservation of the true-to-type HT CIFC 4106
cytotype.
Interspecific hybridation plays a relevant role in
plant evolution and crop breeding, by enabling the
generation of new cytotypes or species (Frankel et al.
1995; Leflon et al. 2006). However, the establishment
of a hybrid depends on the chromosome pairing during
meiosis (Leflon et al. 2006). Chromosome paring in
auto- and allotriploids is characterized by occurrence
of trivalents, bivalents and univalents (McClintock
1928; Kosmala et al. 2006; Thonnalak et al. 2010),
which results in irregularities during anaphasic dis-
junction (Ramsey and Schemske 1998). Conse-
quently, reproductive cells generally present an
unbalanced chromosome number, leading to sterility
(Ramsey and Schemske 1998).
As reported for other triploid plants (Ramsey and
Schemske 1998), HT CIFC 4106 also produces very few
seeds (Pereira et al. 2008), being thus denoted as a semi-
fertile cytotype. The semi-fertility condition in triploids
Fig. 2 Dendrogram
generated from mean values
of nuclear genome size and
AT% compared using
UPGMA statistical method.
Two groups were clustered:
one composed by C.
canephora and HT CIFC
4106, and another
constituted by C. arabica,
HT CIFC 832/1, CIFC
832/2, CIFC 1343/269, UFV
377-01, and UFV 377-09
Fig. 3 HT CIFC 4106 karyotype showing well-individualized
chromosomes, flattened on the slide, without chromatin
deformations and cytoplasmatic background noises. This
analysis confirmed that this HT possesses 2n =3x =33
chromosomes. Bar 5lm
Genet Resour Crop Evol
123
is related to high similarity between progenitor genomes
(Leflon et al. 2006). Indeed, the progenitors of HT CIFC
4106, C. canephora and C. arabica, show a similar
genome. Moreover, C. canephora hasbeenconsidereda
possible progenitor of C. arabica, the only allotetraploid
Coffea species (Clarindo et al. 2012).
The remaining HT accessions showed distinct 2C-
values and/or AT% (Table 1), being thus considered
different cytotypes. The UPGMA dendrogram from
these data presented two clusters. The first was
composed by C. canephora and HT CIFC 4106,
which showed the lowest mean nuclear 2C-value and
similar mean AT%. The second cluster comprised C.
arabica and the remaining HT cytotypes, which
showed mean nuclear 2C-values higher than that of
HT CIFC 4106 (Table 1; Fig. 2).
HT CIFC 832/1, CIFC 832/2, CIFC 1343/269, UFV
377-01, and UFV 377-09 were obtained from self-
crossing of HT CIFC 4106, or from crossing between
this HT and C. arabica. Later, these plants were sent to
Brazil and multiplied by vegetative propagation
(Bettencourt 1973). Therefore, these HT arose from
aneuploid reproductive cells of the allotriploid HT
CIFC 4106. The different mean values of nuclear
DNA content and AT% also indicate that the repro-
ductive cells produced by HT CIFC 4106 exhibited
assorted chromosomes.
The data from FCM and cytogenetic procedures
allowed to characterize the genome of HT accessions.
As these cytotypes have been used for the develop-
ment of C. arabica cultivars, the present findings are
relevant for the design of crossings in Coffea breeding
programs. In addition, these data represent the first
step to understand the origin and genome evolution of
HT.
Acknowledgments The authors are grateful to Conselho
Nacional de Desenvolvimento Cientı
´fico e Tecnolo
´gico (CNPq,
Brası
´lia, DF, Brazil), Fundac¸a
˜odeAmparoa
`Pesquisa do Espı
´rito
Santo (FAPES, Vito
´ria, ES, Brazil), Fundac¸a
˜odeAmparoa
`
Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte,
MG, Brazil), and Coordenac¸a
˜o de Aperfeic¸ oamento de Pessoal de
´vel Superior (CAPES, Brası
´lia, DF, Brazil) for financial
support.
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... The semi-fertile HT 'CIFC 4106', a vegetatively propagated accession derived from the original HT plant, shows 2n = 3x = 33 chromosomes. Therefore, it was considered an allotriploid (Clarindo et al. 2013). We classified the chromosomes, assembled the HT 'CIFC 4106' karyogram and showed it for the first time now. ...
... However, the low germination and low metaphase index observed in Coffea species (Conagin and Mendes 1961) are common bottlenecks, which might be circumvented using plant tissue culture techniques. CAS have been used as an alternative source of mitotic cells for different Coffea species, such as C. canephora, C. congensis, C. arabica Carvalho 2006, 2009), C. eugenioides (Sanglard et al. 2019) and HT 'CIFC 4106' (Clarindo et al. 2013). CAS maintained in liquid medium display a high frequency of cell division, providing a suitable index of prometaphases and metaphases (Fowler 1984;Clarindo and Carvalho 2006). ...
... Therefore, the HT 'CIFC 4106' chromosome 9 was probably inherited from a reduced cell of C. arabica (C a E a ), precisely from the E a subgenome. In addition, the nuclear DNA content of HT 'CIFC 4106' (1C = 2.10 pg, Clarindo et al. 2013) is equivalent to the sum of the mean 2C nuclear genome size of C. canephora (2C = 1.41 pg, 1C = 0.705, Clarindo and Carvalho 2009) and the 1C nuclear value of C. eugenioides (2C = 1.38 pg, 1C = 0.690 pg, Sanglard et al. 2019) and HT 'CIFC 4106' chromosome number (2n = 3x = 33) corresponds to the fusion of one reproductive cell of C. arabica (C a E a ; n = 2x = 22) and one of C. canephora (C; n = x = 11). These data also support the CC a E a hypothesis. ...
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Main conclusion Coffea karyotype organization and evolution has been uncovered by classical cytogenetics and cytogenomics. We revisit these discoveries and present new karyotype data. Abstract Coffea possesses ~ 124 species, including C. arabica and C. canephora responsible for commercial coffee production. We reviewed the Coffea cytogenetics, from the first chromosome counting, encompassing the karyotype characterization, chromosome DNA content, and mapping of chromosome portions and DNA sequences, until the integration with genomics. We also showed new data about Coffea karyotype. The 2n chromosome number evidenced the diploidy of almost all Coffea, and the C. arabica tetraploidy, as well as the polyploidy of other hybrids. Since then, other genomic similarities and divergences among the Coffea have been shown by karyotype morphology, nuclear and chromosomal C-value, AT and GC rich chromosome portions, and repetitive sequence and gene mapping. These cytogenomic data allowed us to know and understand the phylogenetic relations in Coffea, as well as their ploidy level and genomic origin, highlighting the relatively recent allopolyploidy. In addition to the euploidy, the role of the mobile elements in Coffea diversification is increasingly more evident, and the comparative analysis of their structure and distribution on the genome of different species is in the spotlight for future research. An integrative look at all these data is fundamental for a deeper understanding of Coffea karyotype evolution, including the key role of polyploidy in C. arabica origin. The ‘Híbrido de Timor’, a recent natural allotriploid, is also in the spotlight for its potential as a source of resistance genes and model for plant polyploidy research. Considering this, we also present some unprecedented results about the exciting evolutionary history of these polyploid Coffea.
... HT CIFC 4106 is a natural allotriploid with 2C=2.10 pg and 2n=3x=33 chromosomes, formed from the crossing between C. arabica and C. canephora (Clarindo et al. 2013). HT CIFC 4106 plants are used in Coffea breeding programs as genetic sources of resistance to economically important diseases and pests (Romero et al. 2014). ...
... pg, Praça-Fontes et al. 2011). For this, nuclei suspensions were prepared from leaves of each plantlet and the explant donor plant, according to the procedures described by Clarindo et al. (2013) and Sanglard et al. (2019). The nuclei suspensions were analyzed in a Partec PAS cytometer (Partec GmbH, Münster, Germany). ...
... The chromosome number was determined from root meristems of the recovered plantlets. To achieve this, the roots were excised, individually washed in dH 2 O for 15 min, treated with 3 µM amiprophos-methyl for 4 h at 30 C, fixed in 3 : 1 methanol : acetic acid, and stored at 20 C. After at least 12 h, the roots were washed in dH 2 O, then macerated for 2 h at 36 C in an enzymatic pool [4% cellulase (Sigma), 0.4% hemicellulase (Sigma), 1% Macerozyme Onozuka R10 (Yakult), 100% pectinase (Sigma)] diluted in dH 2 O in the proportion 1 : 8 (enzyme pool : dH 2 O), washed in dH 2 O, fixed in 3 : 1 methanol : acetic acid, and stored at 20 C (Clarindo et al. 2013). Slides were prepared by cellular dissociation and air-drying techniques, followed by staining with 5% Giemsa. ...
Article
“Híbrido de Timor” (HT) ‘CIFC 4106’ is a natural allotriploid formed from Coffea arabica×C. canephora, which has been used to elucidate morphogenic in vitro responses and to regenerate new individuals, owing to its resistance to coffee pathogens and its semifertile condition. However, seedlings have not been efficiently regenerated from somatic embryogenesis hitherto. This study aimed to adapt a new indirect somatic embryogenesis procedure for HT ‘CIFC 4106,’ and to evaluate the genetic stability of plantlets regenerated. Leaf explants were inoculated in semisolid (M1) and liquid (M2) media for friable calli induction. During six subcultures (three months), the semisolid system yielded more friable calli, with visually greater cell mass. Subsequently, the friable calli of the two systems were randomly transferred to semisolid (M3) or liquid (M4) media for somatic embryo regeneration. The highest somatic embryo regeneration rate was observed in M1–M4. All recovered plantlets showed stable allotriploidy. We reinforce the relevance of adjusting in vitro conditions for indirect somatic embryogenesis establishment to provide plantlets of the different Coffea germplasms. In addition, the proposed indirect somatic embryogenesis protocol in a liquid system enables the propagation of HT ‘CIFC 4106’ plantlets, overcoming the seminal propagation barriers.
... However, natural or synthetic polyploids are not always more productive and vigorous than their progenitors, since the modifications induced by polyploidization in the genome, epigenome, transcriptome and other "-omes" can also be neutral or even deleterious (Renny-Byfield and Wendel 2014). Recently, our research group induced the hexaploidy (2C = 4.20 pg and 2n = 6x = 66 chromosomes) from the allotriploid "Híbrido de Timor" 'CIFC 4106' (HT, 1C = 2.10 pg and 2n = 3x = 33 chromosomes, Clarindo et al. 2013) through the treatment of friable calli with colchicine (Sanglard et al. 2017). The allotriploid HT originated from the natural interspecific crossing between Coffea arabica L. and Coffea canephora Pierre ex Froehner. ...
... The SE were separately placed in distilled water and cut into 2 cm 2 fragments. These materials were chopped (Galbraith et al. 1983) in nuclei extraction buffer (Otto 1990), and the nuclear suspensions were filtered and stained with propidium iodide (Clarindo et al. 2013). After 20 min in the dark, the nuclear suspensions were analyzed with a Partec-PAS® flow cytometer (Partec® Gmbh, Munster, Germany) and the DNA ploidy level was determined. ...
... The slides were then prepared with cell dissociation and air-drying techniques. Afterwards, the slides were placed on a hot plate at 50 °C for 5 min, stained with 5% Giemsa (Merck®) for 5 min, rinsed with dH 2 O and air-dried (Clarindo et al. 2013). The images of the chromosomes were captured using a 100× objective and a CCD camera (Nikon EvolutionTM) accopled to a Nikon 80i microscope (Nikon, Japan). ...
Article
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Autoallohexaploid plantlets of “Híbrido de Timor” (HT, Coffea) were recently regenerated by chromosome set doubling from allotriploid HT. Besides the in vitro propagation and chromosome doubling set, indirect somatic embryogenesis allows us to investigate the short-term polyploidy consequences. We aimed to establish and compare the indirect somatic embryogenesis in natural allotriploid and synthetic autoallohexaploid HT, evaluating the role of the in vitro environment, ploidy level and global level of 5-methylcytosine on somatic embryo regeneration and karyotype stability. Our results showed that the autoallohexaploid needed more time for induction and proliferation of friable calli, showing lower mean numbers of responsive explants and a higher mean level of 5-methylcytosine. Autoallohexaploid also showed a higher 5-methylcytosine level during the somatic embryo regeneration, but both hybrids exhibited the same mean value of mature cotyledonary somatic embryos for friable callus. Regarding the in vitro environment, the activated charcoal at 8 and 16 g L−1 increased global level of 5-methylcytosine and the mean number of abnormal somatic embryo in autoallohexaploid. Based on these results, the autoallohexaploid exhibited a lower in vitro response compared to its allotriploid ancestor, despite having at least twice as many copies of in vitro response genes. Besides, our data evidenced that the in vitro environment, ploidy level and global 5-methylcytosine level influence the Coffea indirect somatic embryogenesis, showing the outcome of the short-term induced polyploidy. Therefore, we showed the euploidy influence during indirect somatic embryogenesis, demonstrating the relevance of the “omics” evaluations of the new Coffea germplasm in relation to their ancestor.
... This natural allopolyploid originated ~ 100 years ago on the Timor Island (Bettencourt 1973). HT 'CIFC 4106', the probable first HT plant, is an allotriploid (anorthoploid-odd number of chromosome sets) with 1C = 2.10 pg and 2n = 3x = 33 chromosomes (Clarindo et al. 2013), and has been recently propagated via direct somatic embryogenesis (Sattler et al. 2016) and ISE (Sanglard et al. 2017). ...
... Leaf fragments of each Coffea explant donor plant and of the internal standard Solanum lycopersicum L. (2C = 2.00 pg, Praça-Fontes et al. 2011) were co-chopped in nuclei extraction buffer (Otto 1990). The nuclei suspensions were processed, stained (Otto 1990;Clarindo and Carvalho 2009;Clarindo et al. 2013) and analyzed in a Partec PAS ® flow cytometer (Partec ® GmbH, Muenster, Germany). The 2C value was measured considering the G 0 /G 1 nuclei peak of the Coffea samples and S. lycopersicum. ...
... The mean nuclear 2C DNA content value of all explant (three ISE experiments) donors was identical to previous data: 2C = 1.38 ± 0.060 pg for C. eugenioides (Fig. 1a), 2C = 1.41 ± 0.012 pg for C. canephora (Fig. 1b), 2C = 2.62 ± 0.043 pg for C. arabica (Fig. 1c), and 1C = 2.10 ± 0.003 pg for HT 'CIFC 4106' (Fig. 1d). Thus far, our research group had expressed the nuclear genome size of HT 'CIFC 4106' in 2C (Clarindo et al. 2013;Sattler et al. 2016). However, since the karyotype characterization and karyogram assembly were not performed, chromosome pairs were not identified for this allotriploid. ...
Article
Full-text available
Indirect somatic embryogenesis (ISE) is required for plant propagation and a prerequisite for applications that may provide new germplasms. Genetic, epigenetic and physiological features of the explant donor are barriers for ISE establishment, hindering its wide use. Despite the identification and/or expression analysis of genes during ISE, no approach to establish the karyotype aspects has been performed so far. So, this study aims to establish the ISE and compare the in vitro responses between diploid (Coffea canephora and Coffea eugenioides), allotriploid (“Híbrido de Timor”—HT) and true allotetraploid (Coffea arabica) Coffea in a taxonomic and evolutive scenario. Under the same in vitro conditions, the four Coffea differed from each other during ISE. Leaf explants of the true allopolyploids yielded the highest mean number of friable calli (FC) in relative short time and visually exhibiting more pronounced length. FC of the allotetraploid C. arabica presented the highest mean number of mature cotyledonary somatic embryos (MCSE), which were also recovered faster in this species. However, MCSE mean number in HT was the same or lower than diploid Coffea. Besides, intraspecific variation related to the ISE responses was observed in each Coffea, mainly the mean number of FC obtained from ex vitro and in vitro C. arabica and C. eugenioides explants. So, epigenetic and physiologic features may also have influenced the ISE responses. The findings provide the basis for performing other approaches considering the ploidy level, epigenetic and physiological backgrounds. Besides, the data also contributed for understanding about the consequences of polyploidy.
... Cenicafé, desde la década de 1970, desarrolla un programa de selección por resistencia a CBD en ausencia del patógeno, utilizando como fuentes de resistencia el Híbrido de Timor (HT) y Rume Sudan, cruzándolas con la variedad Caturra. El HT es producto de un cruzamiento interespecífico natural entre C. arabica y C. canephora, y presenta regiones de introgresión que le han conferido resistencia a la roya del cafeto (Hemileia vastatrix), a CBD, Pseudomonas syringae y al nematodo Meloidogyne exigua (Clarindo et al., 2013). Cruzamientos entre C. arabica var. ...
Article
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La enfermedad de las cerezas del café (CBD), antracnosis causada por el hongo Colletotrichum kahawae subsp. kahawae, ha sido registrada solo en África y puede ocasionar pérdidas de producción hasta del 80%. Cenicafé desarrolla variedades resistentes a las enfermedades más limitantes para el cultivo, aún en ausencia de los patógenos en Colombia, usando al Híbrido de Timor (HT) como la principal fuente de resistencia a la roya del cafeto y a CBD. El propósito de este estudio fue conocer la presencia del gen Ck-1 de resistencia a CBD en las principales variedades de café cultivadas en Colombia, establecer la correlación entre la presencia de Ck-1 y la respuesta a inoculación de hipocótilos y explorar las bases genómicas de la resistencia. Los marcadores moleculares ligados a Ck-1 se ubicaron en el cromosoma 1 de Coffea canephora, región genómica con quince genes de resistencia a enfermedades. Se encontraron marcadores para Ck-1 en todas las líneas mejoradas derivadas del HT-1343 y ausentes en las variedades Típica, Borbón y Caturra, y en líneas derivadas del HT-832/1. No hubo correlación entre las formas alélicas de resistencia a CBD y la resistencia medida por inoculación de hipocótilos. La alta frecuencia de formas alélicas asociadas con resistencia a CBD en materiales seleccionados por resistencia a roya sugiere cosegregación de genes de resistencia para ambas enfermedades. La estrategia de variedades multilínea desarrolladas por Cenicafé, que actualmente corresponde a cerca del 80% del café sembrado en el país, hace que la población en general esté protegida ante la eventual llegada del patógeno a Colombia.
... For example, CLR resistance (C. canephora × C. arabica; Clarindo et al., 2013;Avelino et al., 2015) and leaf-miner resistance (C. arabica × C. racemosa ;Medina Filho et al., 1977b) for Arabica coffee. ...
Article
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Coffea arabica (Arabica) and C. canephora (robusta) almost entirely dominate global coffee production. Various challenges at the production (farm) level, including the increasing prevalence and severity of disease and pests and climate change, indicate that the coffee crop portfolio needs to be substantially diversified in order to ensure resilience and sustainability. In this study, we use a multidisciplinary approach (herbarium and literature review, fieldwork and DNA sequencing) to elucidate the identity, whereabouts, and potential attributes, of two poorly known coffee crop species: C. affinis and C. stenophylla. We show that despite widespread (albeit small-scale) use as a coffee crop species across Upper West Africa and further afield more than 100 years ago, these species are now extremely rare in the wild and are not being farmed. Fieldwork enabled us to rediscover C. stenophylla in Sierra Leone, which previously had not been recorded in the wild there since 1954. We confirm that C. stenophylla is an indigenous species in Guinea, Sierra Leone, and Ivory Coast. Coffea affinis was discovered in the wild in Sierra Leone for the first time, having previously been found only in Guinea and Ivory Coast. Prior to our rediscovery, C. affinis was last seen in the wild in 1941, although sampling of an unidentified herbarium specimen reveals that it was collected in Guinea-Conakry in 2015. DNA sequencing using plastid and ITS markers was used to: (1) confirm the identity of museum and field collected samples of C. stenophylla; (2) identify new accessions of C. affinis; (3) refute hybrid status for C. affinis; (4) identify accessions confused with C. affinis; (5) show that C. affinis and C. stenophylla are closely related, and possibly a single species; (6) substantiate the hybrid C. stenophylla × C. liberica; (7) demonstrate the use of plastid and nuclear markers as a simple means of identifying F1 and early-generation interspecific hybrids in Coffea; (8) infer that C. liberica is not monophyletic; and (9) show that hybridization is possible across all the major groups of key Africa Coffea species (Coffee Crop Wild Relative Priority Groups I and II). Coffea affinis and C. stenophylla may possess useful traits for coffee crop plant development, including taste differentiation, disease resistance, and climate resilience. These attributes would be best accessed via breeding programs, although the species may have niche-market potential via minimal domestication.
... The genetic diversity of C. arabica available within the collections outside of Ethiopia is currently exploited for breeding programs to cope with the challenges of climate change around the world. While a global coffee genetic resource conservation consortium is certainly needed to ensure that existing coffee genetic resources are preserved in Ethiopia, the low level of genetic diversity in C. arabica compared to the much higher diversity in the present-day populations of its progenitors suggests that introgression events into the allotetraploid species from the diploid species such as those exploited to achieve coffee rust resistance in derivatives of the Timor hybrid 46,47 are of paramount importance to broaden substantially the genetic diversity in the cultivated germplasm and increase environmental, economic and social sustainability of coffee cultivation. ...
Article
Full-text available
The genome of the allotetraploid species Coffea arabica L. was sequenced to assemble independently the two component subgenomes (putatively deriving from C. canephora and C. eugenioides) and to perform a genome-wide analysis of the genetic diversity in cultivated coffee germplasm and in wild populations growing in the center of origin of the species. We assembled a total length of 1.536 Gbp, 444 Mb and 527 Mb of which were assigned to the canephora and eugenioides subgenomes, respectively, and predicted 46,562 gene models, 21,254 and 22,888 of which were assigned to the canephora and to the eugeniodes subgenome, respectively. Through a genome-wide SNP genotyping of 736 C. arabica accessions, we analyzed the genetic diversity in the species and its relationship with geographic distribution and historical records. We observed a weak population structure due to low-frequency derived alleles and highly negative values of Taijma’s D, suggesting a recent and severe bottleneck, most likely resulting from a single event of polyploidization, not only for the cultivated germplasm but also for the entire species. This conclusion is strongly supported by forward simulations of mutation accumulation. However, PCA revealed a cline of genetic diversity reflecting a west-to-east geographical distribution from the center of origin in East Africa to the Arabian Peninsula. The extremely low levels of variation observed in the species, as a consequence of the polyploidization event, make the exploitation of diversity within the species for breeding purposes less interesting than in most crop species and stress the need for introgression of new variability from the diploid progenitors.
... Today, robusta comprises c. 40% of global coffee trade (1), although its true proportion in the global market is probably higher, given that it is used to adulterate Arabica coffee (13). Moreover, robusta has been vital for breeding CLR-resistant cultivars of Arabica coffee, via backcrossing with Arabica-robusta hybrids (14), the most notable of these being the Timor hybrid (15). Robusta coffee has therefore been responsible for overcoming most of the key issues for coffee sector sustainability, either by direct replacement or through use in breeding new cultivars, rendering the development and use of other coffee species unnecessary. ...
... Timor hybrid lineages, which are natural hybrids of C. arabica crossed with C. canephora (Clarindo et al. 2013), have a valuable germplasm that is resistant to the root-knot nematodes Meloidogyne exigua and M. paranaensis, coffee leaf rust, cercosporiosis, and coffee berry disease (Silva et al. 2006;Gichuru et al. 2008;Salgado et al. 2014;Peres et al. 2017;Rezende et al. 2017). The cultivars derived from this germplasm could also produce high quality coffee (Sobreira et al. 2015). ...
Article
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The root-knot nematode Meloidogyne paranaensis is considered the major threat in Coffea arabica plantations. Due to the aggressiveness of this nematode, cultivation is virtually impossible without a resistant cultivar. The main goal of this research was to identify resistant progenies derived from crossings of Catuaí and Timor hybrid cultivars in a greenhouse experiment. Additionally, genetic parameters for resistance were estimated to trace selection strategies. Coffee plants were inoculated with 9000 M. paranaensis eggs and second-stage juveniles (J2). Eighty-six plants previously selected in a Meloydogine exigua infested field gave rise to 86 progenies that were evaluated based on the dry matter of aerial parts (DMAP), fresh matter of extracted root (FMER), height (H), and diameter (DIA). The responses of the progenies to nematodes were assessed using the gall index and thickness (GIT), number of eggs J2/g root (NEJGR), and reproduction factor (RF).The cultivars Mundo Novo IAC 379-19, Catuaí Vermelho IAC 99, Paraíso MG H 419-1, and IPR 100 were used as susceptible and resistant controls. The GIT is genetically correlated with NEJGR and RF, and could be used in plant selection programs. Five progenies were resistant to M. paranaensis, with two of them not segregated to susceptibility. These progenies are considered multiple resistant to root-knot nematodes because they are also resistant to M. exigua.
... Today, robusta comprises c. 40% of global coffee trade (1), although its true proportion in the global market is probably higher, given that it is used to adulterate Arabica coffee (13). Moreover, robusta has been vital for breeding CLR-resistant cultivars of Arabica coffee, via backcrossing with Arabica-robusta hybrids (14), the most notable of these being the Timor hybrid (15). Robusta coffee has therefore been responsible for overcoming most of the key issues for coffee sector sustainability, either by direct replacement or through use in breeding new cultivars, rendering the development and use of other coffee species unnecessary. ...
Article
Full-text available
Wild coffee species are critical for coffee crop development and, thus, for sustainability of global coffee production. Despite this fact, the extinction risk and conservation priority status of the world’s coffee species are poorly known. Applying IUCN Red List of Threatened Species criteria to all (124) wild coffee species, we undertook a gap analysis for germplasm collections and protected areas and devised a crop wild relative (CWR) priority system. We found that at least 60% of all coffee species are threatened with extinction, 45% are not held in any germplasm collection, and 28% are not known to occur in any protected area. Existing conservation measures, including those for key coffee CWRs, are inadequate. We propose that wild coffee species are extinction sensitive, especially in an era of accelerated climatic change.
Article
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Progênies do café Híbrido de Timor e F2-F4 oriundas de cruzamentos desse café com outros cultivares resistentes ou não a Hemileia vastatrix e cruzamentos entre outras fontes de resistência ao patógeno, foram avaliadas em três experimentos, em Campinas, para observação de sua produtividade, em relação a alguns cultivares de Coffea arabica tomados como testemunhas. As progênies do Híbrido de Timor apresentaram pequena produtividade, indicando baixa adaptação, com exceção daquelas de prefixos C 1737, C 1738 e C 1699. As progênies derivadas de cruzamentos do Híbrido de Timor com cultivares de porte pequeno, como Caturra Vermelho e Vila Sarchi de Coffea arabica, mostraram-se, também, pouco produtivas. Destacou-se apenas a progênie C 1669, rústica. Das combinações do Híbrido de Timor com outros cultivares de C. arabica com resistência a H. vastatrix, apenas a progênie C 1698 se revelou melhor. As progênies F2 derivadas de cruzamentos do cultivar S 795 portador do fator SH3 de resistência com Mundo Novo, deram produções bastante razoáveis. Notou-se, de modo geral, acentuada variabilidade na produção das progênies, o que é indicado pelos elevados valores dos coeficientes de variação obtidos nos três experimentos. Os dados desses experimentos mostraram a dificuldade de aproveitamento das progênies e dos derivados do Híbrido de Timor analisados. Tratando-se, no entanto, de material de elevado grau de resistência às raças de H. vastatrix, novas hibridações deverão ser sintetizadas, com cultivares comerciais, a fim de se conseguirem linhagens resistentes, vigorosas e mais produtivas.
Article
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O objetivo deste trabalho foi caracterizar a herança da resistência do Híbrido de Timor UFV 443-03 à ferrugem-do-cafeeiro (Hemileia vastatrix). Para isso, a raça II e o patótipo 001 de ferrugem foram inoculados em 246 plantas da população F2, 115 plantas do retrocruzamento suscetível (RCS) e 87 plantas do retrocruzamento resistente (RCR), originadas do cruzamento entre o genótipo suscetível cv. Catuaí Amarelo IAC 64 e a fonte de resistência Híbrido de Timor UFV 443-03. Para ambos os inóculos, a cv. Catuaí Amarelo IAC 64 foi suscetível, enquanto o Híbrido de Timor UFV 443-03, a planta representante da geração F1 e as plantas do RCR foram resistentes. As plantas F2, quando inoculadas com a raça II, apresentaram dois padrões de segregação significativos: 15:1 e 61:3. A herança da resistência foi confirmada pela inoculação das plantas do RCS, que segregaram na proporção de 3:1, padrão esperado para herança condicionada por dois genes. A hipótese de segregação 7:1 para três genes foi rejeitada. Resultados semelhantes foram obtidos para o patótipo 001. Dois genes dominantes e independentes conferem a resistência genética do Híbrido de Timor UFV 443-03 à raça II e ao patótipo 001 de H. vastatrix.
Article
Price collapse and oversupply have made coffee a high-profile crop in recent years: never has efficient production and crop protection been more important for reducing costs and increasing quality. Packed with illustrations, this book covers the origins, botany, agroecology and worldwide production statistics of coffee, and the insect pests, plant pathogens, nematodes and nutrient deficiencies that afflict it. With emphasis on integrated crop management, this book reviews control measures suitable for any coffee pest or disease and will enable agriculturists to design and implement sustainable pest management systems. © J.M. Waller, M. Bigger and R.J. Hillocks 2007. All rights reserved.
Article
Meiotic behavior was investigated in the microsporocytes of six wild bananas with AA and BB genomes and 46 cultivated bananas with AA, AAA, BBB, AAB and ABB genomes. All banana material was collected from the Pak Chong Research Station, Kasetsart University, Nakhon Ratchasima, Thailand. Most of the wild bananas showed normal synapses with a high chromosome pairing frequency of 11 bivalents per pollen mother cell (PMC). The cultivated bananas exhibited many variations in chromosome association at metaphase-I. Cultivars with the AA genome had more univalents than those of wild species with the same AA genome. Univalent, bivalent and trivalent occurrences were also common in all triploid cultivated bananas, but quadrivalents were very rare. The univalents were high in the AAB and ABB genomes, whereas trivalency was high in the AAA and BBB genomes. Micronuclei were observed in the tetrad of all cultivated bananas, but were very low in wild bananas. Many types of tetrad were shown in different orientation patterns at metaphase-II. The occurrence of the parallel disposition pattern was high, but the perpendicular and lineal disposition patterns were not common in any of the bananas.
Article
Karyotype and nuclear 2C-value data are con-sidered important in taxonomic and evolutionary approa-ches in Coffea. Still, new methods are needed to further support such studies, especially to determine the progeni-tors of Coffea arabica. In this work, new cytogenetic and flow cytometry data were used to compare Coffea arabica, Coffea canephora and Coffea congensis. These data cor-roborate the hypothesis that C. canephora and C. congensis originated from a single ancestor, whose basic chromo-some number was x = 11. In agreement with the obser-vations of other authors, the karyotype and mean 2C-values confirm that C. arabica is a true allotetraploid originating from two diploid Coffea species with similar genomes. Although C. canephora and C. congensis have been con-sidered potential progenitors of C. arabica, karyotype comparison revealed that only one of these species may be parental to C. arabica. These accurate cytogenetic and flow cytometry data contribute to expand our knowledge of the Coffea genome, as well as of possible progenitors of C. arabica.
Article
Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4′,6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.