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

Abstract

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.

Full text

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 (a–c)or4
0,60-diamidino-2-phenylindole (d–f). a–c2C-
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). d–fAT%
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
Nı
´vel Superior (CAPES, Brası
´lia, DF, Brazil) for financial
support.
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