Genet Resour Crop Evol
DOI 10.1007/s10722-013-9976-1
RESEARCH ARTICLE
Cytomolecular characterization of rDNA distribution
in various Citrullus species using fluorescent in situ
hybridization
Umesh K. Reddy • Nischit Aryal •
Nurul Islam-Faridi • Yan R. Tomason
Amnon Levi • Padma Nimmakayala
•
Received: 10 September 2012 / Accepted: 28 January 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract The 18S–28S and 5S rDNA sites are
useful chromosome landmarks and provide valuable
evidence about genome organization and evolution.
This investigation was the first attempt to study the
dynamics, distribution and directionality of rDNA
gains and losses, as well as to understand the
contribution of site number variation in the speciation
of the genus Citrullus. In this study, we employed
fluorescent in situ hybridization (FISH), using the18S–
28S and 5S rDNA gene loci, to evaluate the differences between the (1) cultivated type watermelon
C. lanatus var. lanatus (sweet watermelon), (2) the
‘‘bitter’’ desert watermelon C. colocynthis (colocynth)
that is indigenous to the deserts of northern Africa, the
U. K. Reddy N. Aryal Y. R. Tomason
P. Nimmakayala (&)
Department of Biology, Gus R. Douglass Institute,
West Virginia State University, Institute,
WV 25112-1000, USA
e-mail: padma@wvstateu.edu
U. K. Reddy
e-mail: ureddy@wvstateu.edu
N. Islam-Faridi
U.S. Forest Service, Washington, DC, USA
N. Islam-Faridi
Department of Ecosystem Science and Management,
Texas A&M University, College Station, TX 77843, USA
A. Levi
U.S. Vegetable Laboratory, USDA, ARS, 2875 Savannah
Highway, Charleston, SC 29414, USA
Middle East and Asia, (3) the C. lanatus var. citroides
(citron) ‘‘Tsamma’’ or ‘‘cow watermelon’’ that is
known as and is indigenous to southern Africa, (4) and
C. rehmii that thrive in the Namibian Desert. The FISH
analyses showed that the sweet watermelon and
colocynth have similar rDNA configuration. The
sweet watermelon and colocynth genomes contain
two 18S–28S rDNA gene loci, each located on a
different chromosome, and one 5S rDNA locus which
is co-localized with one of the 18S–28S rDNA gene
loci. On the other hand, the C. rehmii has one 18S–28S
rDNA locus and one 5S rDNA locus positioned on
different chromosomes, while the citron has one18S–
28S rDNA and two 5S rDNA loci, each located on a
different chromosome. A FISH analysis of F1 (citron
9 sweet watermelon) chromosome spreads revealed
uniparental homeologous rDNA gene copies pertaining to the sweet watermelon versus the citron chromosomes, with the sweet watermelon chromosome
containing the 18S–28S and 5S rDNA locus versus the
citron homologue chromosome that has the 5S rDNA
locus, but not the 18S–28S rDNA locus. Genomic
in situ hybridization (GISH) analysis, using the entire
citron genome as a probe to be differentially hybridized on sweet watermelon chromosome spreads,
revealed that the citron genomic probes mainly
hybridize to subtelomeric and pericentromeric regions
of the sweet watermelon chromosomes, suggesting
extensive divergence between the citron and sweet
watermelon genomes. The FISH and GISH cytogenetic analysis here indicate major differences in
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Genet Resour Crop Evol
genome organization between the cultivated watermelon type sweet watermelon and its counterpart
citron that thrive in southern Africa and considered a
useful germplasm source for enhancing disease and
pest resistance in watermelon cultivars.
Keywords Citrullus FISH rDNA organization
5S rDNA 18S–28S rDNA Watermelon
Introduction
Watermelon belongs to the genus Citrullus of the
subfamily Cucurbitoideae, tribe Benincaseae Ser.,
Subtribe Benincasinae (Ser.) C. Jeffrey, of the family
Cucurbitaceae (Robinson and Decker-Walters 1997).
Citrullus was first used as a generic name by Foraskal
for this genus in 1775 and then by Schrader (ex Ecklon
and Zeyher) during 1834. This genus was included in
the list of nomina conservanda by the VIII International Botanical Congress 1954 (Fursa 1972). There
are five recognized Citrullus diploid (n = 11) species
that naturally grow in xerophytic and semi-arid
habitats. Among them, the Citrullus lanatus (Thunb.)
Matsum. et Nakai), Citrullus ecirrhosus Cogn., Citrullus rehmii De Winter and Acanthosicycos naudinianus (Sond.) C. Jeffrey are indigenous to the arid
sandy regions of southern Africa (Meeuse 1962; Fursa
1972), while C. colocynthis (L.) Schrad. is indigenous
to the deserts of North Africa, near East and SouthWest Asia (Bates and Robinson 1995; Robinson and
Decker-Walters 1997; Jeffrey 2001). The C. rehmii
and C. lanatus are annual species, whereas C.
colocynthis (colocynth), C. ecirrhosus and A. naudinianus are perennial species (Jarret and Newman
2000). C. lanatus includes red sweet watermelon
group (initially named as C. lanatus subsp. vulgaris
and later grouped along with the white flesh types to be
broadly classified as C. lanatus var. lanatus) (here
onwards will be referred as sweet watermelon because
PI 270306 that is in the current study is the red sweet
type). According to Jeffrey (2001), the C. lanatus
includes both var. lanatus and var. citroides. C.
lanatus var. citroides (Bailey) Mansf. ex Greb.
(hereafter citron in the current study) is a group of
ancient cultigens known as ‘Tsamma’ melons of
southern Africa and also called as the ‘cow’ or ‘Citron’
123
melon (Whitaker and Davis 1962; Whitaker and
Bemis 1976; Burkill 1985; Jarret et al. 1997; Laghetti
and Hammer 2007; Mujaju et al. 2010). In contrast
with the sweet red watermelon varieties, the citron
watermelons have white or green flesh with a wide
range of flavors, and high pectin and dietary fibers that
make them a functional food source for native people
in Africa (Dahl Jensen et al. 2011). Among the diverse
opinions on the origin and domestication of watermelon, one theory supports that sweet watermelon was
derived directly from Citrullus colocynthis and the
other theory is about the citron types being the
progenitors (Rubatsky 2001). Though PCR RFLP
study involving non coding chloroplast DNA revealed
that the colocynth is the ancestral progenitor of entire
Citrullus genus (Dane and Liu 2007), there are several
other marker studies (Jarret et al. 1997; Levi et al.
2001; Nimmakayala et al. 2010) that placed subclusters of citron and sweet melon together paving to the
speculation that citron could be the raw material for
the domestication of red sweet watermelon. Phylogenies based on the internal transcribed spacer regions
of the 18S–28S suggested C. rehmii is closure to the C.
lanatus clade as a whole than the colocynth (Jarret and
Newman 2000). Sequence phylogenies of Genetic
mapping studies using progenies derived from the
crosses between citron and sweet watermelon genotypes revealed strong preferential (non-Mendelian)
segregation for several markers, indicating that wide
structural differences among the types of C. lanatus
(Levi et al. 2002, 2006). The current study was
designed to resolve relationships among Citrullus spp.
using molecular cytogenetic analysis. Fluorescence
in situ hybridization (FISH) proved to be useful
cytogenetic tool for elucidating genome features and
organization, and provided valuable information on
homologies between the chromosomal segments of
closely related species (Heslop-Harrison 1991). The
FISH has been extensively employed in identifying
chromosome regions using rDNA sequences, as
probes (Leitch and Heslop-Harrison 1992; Adams
et al. 2000; Islam-Faridi et al. 2007; Martı́nez et al.
2010). The rDNA markers are known to be one of the
robust phylogenetic tools, for inferring genome evolution during speciation (Gu and Xiao 2003; Martı́nez
et al. 2010). The current investigation was to study
rDNA gains and losses in the speciation of the genus
Citrullus.
Genet Resour Crop Evol
Materials and methods
Plant materials and chromosome preparation
Seeds of colocynth (PI 386015; collected in Iran),
C. rehmii (Grif 16135; collected in southern Africa),
sweet watermelon (PI 270306; an Asian type with
globular shape watermelon; collected in the Philippines) and citron (PI 244018; collected in South
Africa) were obtained from Dr. Robert Jarret at the
USDA-ARS, Plant Genetic Resources Conservation
Unit, Griffin, GA 30223. These accessions were
selected as representative for the different Citrullus
spp. since they were used in previous phylogenetic
studies using DNA markers (Jarret and Newman 2000;
Dane and Liu 2007; Dane et al. 2007; Nimmakayala
et al. 2010).
Actively growing root tips, about 1 cm long, were
excised from young seedlings growing in potting soil
in a greenhouse and pre-treated in an aqueous solution
of a–monobromonaphthalene (0.8 %, Sigma) for 2 h
at room temperature in the dark. The root tips were
then fixed in 4:1 (95 % ethanol: glacial acetic acid) to
arrest cell division at metaphase. The fixed root tips
were washed thoroughly in distilled and 0.01 M citrate
buffer prior to processing for enzyme digestion (40 %
(v/v) Cellulase (Sigma, USA), 20 % (v/v) Pectinase
(Sigma, USA), 3 % (w/v) Cellulase R10 (SERVA,
Germany), 2 % (w/v) Cellulase RS (SERVA,
Germany), 1 % (w/v) macerozyme (SERVA, Germany),
1.5 % (w/v) Pectolyase Y23 (Kyowa Chemical Products
Co., Ltd.) in 0.01 M citrate buffer, and the chromosome
spreads were prepared as described elsewhere (Jewell and
Islam-Faridi 1994). The chromosome spreads were
checked with a phase contrast microscope (AxioImager
D1, Carl Zeiss, Inc.) and slides containing good
chromosome spreads that are free from cell debris and
almost no overlap were selected for FISH and/or stored at
-80 C until use for FISH.
Germany) using the manufacturer’s instructions. The
genomic DNA from citroides was labeled with BioNick-Translation Mix.
Fluorescent in situ hybridization (FISH)
For FISH, slides previously stored at -80 °C were
dehydrated through 70 and 95 % ethanol, 3 min each,
and air-dried at least 2 h at room temperature. The
slides were then incubated in RNase-A solution
(35 lg/ml) in 29 SSC at 37 °C for 1 h, rinsed twice
in 29 SSC, 5 min each, and dehydrated in a ethanol
series (70, 85 and 95 %, 3 min each) and air-dried
overnight prior to FISH. Standard FISH technique was
utilized as previously reported (Islam-Faridi et al.
2002). Briefly, the hybridization mixture consisted of
50 % deionized formamide, labeled probe (30 ng/
slide), carrier blocking DNA (E. coli DNA, 7.5 lg/
slide), 10 % dextran sulfate, in 2 9 SSC. Genomic
in situ hybridization (GISH) is a molecular cytogenetic technique that employs differential hybridization
of entire genomic probes of one species to the
chromosome spreads of a counterpart species, facilitating gross assessment of chromosome homology or
divergence and physically localizes homologous chromosome regions between related species or subspecies. For GISH, 50 ng of labeled genomic DNA was
used for each slide. Total genomic DNA (10–509 of
the probe DNA) from cucumber was used as a
blocking DNA in the hybridization mixture to prevent
cross hybridization. Probe hybridization sites were
detected with Cy3-conjugated streptavidin (Jacson
Immuno Research Laboratories, West Grove, PA) for
biotin labeled probe and FITC-conjugated anti-digoxygenin (Roche, D-68305 Mannheim, Germany).
The FISH preparations were counterstained with
DAPI (4 lg/ml) in Mcllvaine buffer (pH 7.0) and
mounted with Vectashield (Vector Laboratories, CA)
to prevent photo-bleaching the fluorochromes when
viewing under epi-fluorescence microscope.
Probe DNA preparation
Digital image capture and process
Whole plasmid DNA with 18S–28S rDNA insert of
maize (Zimmer et al. 1988) and 5S rDNA insert of
sugar beet including the spacer region (Schmidt et al.
1994) were labeled with biotin-16-dUTP (Bio-NickTranslation Mix, Roche, Germany) and/or digoxygenin-11-dUTP (Dig-Nick-Translation Mix, Roche,
Digital images were captured using an epi-fluorescence microscope (AxioImager Z1, Carl Zeiss,
Germany) with suitable filter sets (chroma Technology, MA, USA) and CoolCube high performance
CCD camera, and pre-processed with Ikaros and ISIS
V5.1 (MetaSystem Inc., MA, USA). The images were
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Genet Resour Crop Evol
Fig. 1 A bicolor FISH with 18S–28S rDNA (green signals) and 5S rDNA (red signals) probes on watermelon (var. lanatus, PI 270306)
chromosome spreads. a Prometaphase chromosome spread. b Late prophase chromosome spread, and c interphase cell
then further processed with Adobe Photoshop CS v8
(Adobe System, USA).
Results
A single root tip yielded several metaphase cells, and
slide with at least 20 or more spreads were selected for
FISH. The FISH analysis using the 18S–28S rDNA and
5S rDNA probes identified considerable differences in
genome organization among the Citrullus species and
subspecies in this study (colocynth, sweet watermelon,
citron and C. rehmii). In sweet watermelon, two major
18S–28S rDNA sites were found and these were
located on two different homologous pairs of chromosomes (Fig. 1a, b, Table 1). A two-color FISH (bicolor
FISH) showed that the 5S rDNA site was located
interstitially and syntenic to one of the 18S–28S rDNA
sites (Fig. 1). The 18S–28S rDNA site and the 5S
rDNA site were quite spaced out to each other as
Table 1 Number of rDNA (18S–28S and 5S) loci/sites
observed in Citrullus spp.
Citrullus species
18S–28S rDNA
5S rDNA
C. colosynthis
2 (4)
1 (2)
C. lanatus,
var. lanatus
var. citroides
C. rehmii
F1, var. lanatus 9 var. citroides
2 (4)
1 (2)
1 (2)
2 (4)
1 (2)
1 (2)
(3)
(3)
The total number of FISH signals observed shown in
parentheses
123
revealed by the interphase FISH (Fig. 1c). In contrast,
only one 18S–28S rDNA site and two 5S rDNA sites
were observed in citron. A bicolor FISH analysis in
citron showed that 5S rDNA sites were located on two
different homologous pairs of chromosomes, while an
18S rDNA site was located on another homologous pair
of chromosomes (Fig. 2a, b). The 18S–28S rDNA site
that was interstitially located along with one of the 5S
rDNA sites on a pair of homologous chromosomes in
sweet watermelon appeared to be lost in citron. The
FISH revealed the presence of the sweet watermelon
chromosome with the 18S–28S rDNA that is colocalized with the 5S rDNA locus, and its counterpart
citron chromosome contains only 5S rDNA, but not the
18S–28S rDNA locus (Fig. 3c).
A total of six FISH signals, three each of 5S rDNA
(Fig. 3a) and 18S–28S rDNA (Fig. 3c) (Table 1) were
observed as expected based on the number 5S and 18S
sites in sweet watermelon and citron. To elucidate the
sweet watermelon versus citron chromosome pairing,
probing of 18S–28S rDNA and 5S rDNA loci were
performed on root tip spreads of F1 plants [sweet
watermelon (PI 270306) 9 citron (PI 244018)]. FISH
analysis in F1 spreads revealed the presence of the
sweet watermelon chromosome, which had one 18S–
28S rDNA site, co-localized with the 5S rDNA site
(Fig. 3c). The citron homologue of this chromosome
had only 5S rDNA site indicating deletion of the 18S–
28S rDNA site. The other 18S–28S rDNA site, which
is common to both sweet watermelon and citron, was
found in homologous pair of F1 chromosomes. The
duplicated 5S rDNA site of citron was hemizygous in
the F1 spreads (Fig. 3a).
Genet Resour Crop Evol
Fig. 2 A bicolor FISH with 18S–28S rDNA (green signals) and
5S rDNA (red signals) probes on citroides chromosome spreads.
a Mid prophase chromosome spread showing two major 18S–
28S rDNA signals, four 5S rDNA signals, and an interphase
nucleus also showing two green signals. b Metaphase chromosome spread showing two major 18S–28S rDNA signals, four 5S
rDNA signals
Fig. 3 18S–28S rDNA,5S rDNA and GISH sites on F1 (cross of
lanatus and citroides). a Single color FISH showing 3 sites of 5S
rDNA(green signal), b FISH showing 3 sites of 18S–28S rDNA
(green signal) and GISH (red signal). c bicolor FISH with both
5S (green signal) and 18S–28S rDNA sites (red signal)
To further substantiate our finding of major chromosomal divergence in citron versus sweet watermelon, we performed GISH on the sweet watermelon
(PI 270306) plant chromosomal spreads using the
whole genomic DNA of citron (PI 244018) (Fig. 6).
The GISH results (Fig. 6) indicated that the citron
genome mainly hybridizes to subtelomeric and pericentromeric regions of the sweet watermelon chromosomes, suggesting that these chromosomal regions
consist of repetitive DNAs, while greater divergence
may exist in genomic regions with low copy gene
sequences. The GISH result in the current analysis
need to be revalidated as the incomplete hybridization
could have caused experimental bias because of
improper blocking as the cucumber DNA was used
for the blocking. This could be because some of the
chromosomal regions in watermelon might have
different repetitive elements, which are not shared
with the cucumber genome.
We performed single and bicolor FISH analysis to
understand site number variations of 18S–28S rDNA
and 5SrDNA sites in the ancestral species colocynth
(PI 386015). The study revealed that the colocynth
possessed a similar site number of 18S–28S rDNA and
5SrDNA sites as noted in the sweet watermelon,
indicating that the cultivated watermelon has similar
chromosomal organization to the ancestral progenitor
colocynth (Fig. 4).
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Genet Resour Crop Evol
Fig. 4 FISH probed with 18S–28S and 5S rDNA clones on C.
colocynthis metaphase chromosome spread. a Species colocynthis contained two sites of 18S–28S rDNA (red signals) and they
are on two different homologous pairs of chromosomes. b FISH
showing a site of 5S rDNA (green signals) and located
interstitially and syntenic to one of the 18S–28S rDNA (may
be the robust one) site. c superimposed image of ‘‘a’’, ‘‘b’’ and
‘‘d’’. d chromosome spread of C. colocynthis stained with DAPI
Fig. 5 FISH probed with 18S–28S and 5S rDNA clones on rehmii chromosome spread. a Species rehmii contained one site each of
18S–28S rDNA (red signals) and 5S rDNA (green signals) located on two different chromosomes
FISH analysis was carried out on the root tip
chromosome spreads of C. rehmii to note that this
species contained only one each of 18S–28S rDNA
and 5S rDNA sites (Fig. 5). Apparently, as noticed in
FISH analysis of citron, the 18S–28S rDNA site that
was observed to be syntenic to the 5S rDNA site as in
colocynth and sweet watermelon also lost in C. rehmii.
123
The duplication event of the 5S rDNA site that was
observed in var. citroides was not found in C. rehmii,
indicating that the duplication of the 5S rDNA site in
citron could be relatively recent (Fig. 6). Results
pertaining to major structural rearrangements and site
number variation in various species of the genus
Citrullus are summarized in a flow chart (Fig. 7).
Genet Resour Crop Evol
Fig. 6 Genomic DNA in situ hybridization using citroides DNA as probe on F1
Fig. 7 Flow chart showing rDNA distribution between C.
colocynthis, C. lanatus var. lanatus, C. rehmii and C. lanatus
var. citroides
Discussion
Physical mapping and characterization of 18S–28S
rDNA and 5S rDNA sites have provided a number of
useful chromosome markers in several plant species
(Hizume et al. 1992; Brown and Carlson 1997; IslamFaridi et al. 2007). PCR selection and variation in site
number among polymorphic sequences present in a
genome can lead to a biased representation for
different paralogues (Álvarez and Wendel 2003;
Fukushima et al. 2008). Current FISH analyses in
Citrullus accessions were undertaken to eliminate
PCR bias and characterize site number of 18S–28S
rDNA and 5S rDNA.
In the current study, the FISH analysis demonstrated that the rDNA gene loci (18S–28S and 5S) are
useful cytogenetic markers, showing wide differences
among the Citrullus species and subspecies. Dane and
Lang (2004) and Jarret and Newman (2000) proposed
that C. rehmii might be the ancestral to sweet
watermelon. The results in this study clearly showed
the close relationship of C. rehmii with both citron and
sweet watermelon complementing previously
assumed phylogenetic relationship.
Similar to the other diploid plant species (Hanson
et al. 1996; Linares et al. 1996; Murata et al. 1997), our
research revealed that the number of major 18S–28S
rDNA loci in Citrullus genus ranged between 1–4, and
the 5S rDNA loci tended to be localized at one or two
positions. This study confirmed that the FISH is a
powerful tool that can be useful to further study the
Citrullus spp. chromosomes, possibly by using low
copy BACs or other protein coding gene families, as
has been shown in other plant species, such as Iris
(Martı́nez et al. 2010). In sweet watermelon and
colocynth, our study has shown that the two major
18S–28S rDNA loci are on different chromosomes,
while the 5S rDNA locus positioned interstitially and
syntenic to one of the 18S–28S rDNA loci (Hanson
et al. 1996).
The two major 18S–28S rDNA sites of sweet
watermelon and colocynth, which were found on two
123
Genet Resour Crop Evol
different homologous pairs, are not located in citron in
the same configuration. On the contrary, only one
18S–28S rDNA site was found in citron and C. rehmii.
The 18S–28S rDNA site that was interstitially located
along with the 5S rDNA site in sweet watermelon and
colocynth was apparently deleted in citron and C.
rehmii. The biological significance of this loss and its
potential adaptive significance is unclear. FISH analysis of sweet watermelon and citron F1 progeny
revealed occurrence of uniparental homologous rDNA
gene copies pertaining to sweet watermelon chromosome with the 18S–28S rDNA site, which is closely
linked to a 5S rDNA site and the citron homoeologue
of this chromosome that had only 5S rDNA site
showed the deletion of the 18S–28S rDNA site. Many
earlier studies indicated that the 18S–28S sites present
differentially in closely related species, which may be
because of changing the positions (Berjano et al.
2009).
In our study, we identified polymorphic levels of 5S
rDNA sites because of the apparent duplication of
these sites in citron. Previous studies indicated the 5S
rDNA site is a stable chromosome marker (PedrosaHarand et al. 2006; Berjano et al. 2009). The
duplication event of 5S rDNA site in citron should
be further analyzed using PCR studies to characterize
the intra and interspecific length variation as well as to
categorize pseudo and functional 5S gene units. The
5S rDNA is generally crossing over causing molecular
drives for genome evolution (Ohta 1984; Dover 1986).
Classical taxonomic study (Robinson and DeckerWalters 1997), molecular markers (Jarret et al. 1997;
Levi et al. 2001; Nimmakayala et al. 2010); isozymes
(Zamir et al. 1984; Navot and Zamir 1987), internal
transcribed spacer regions (ITS1 and ITS2) of 18S–
28S nuclear ribosomal DNA (Jarret and Newman
2000) and detailed chloroplast analysis (Dane and
Lang 2004; Dane et al. 2004; Dane and Liu 2007)
established that the cultivated watermelon is phylogenetically sister to citron. All the species in Citrullus
genus are freely crossable in varying degrees, producing semi sterile to fertile progenies, which made
elucidation of domestication process in cultivated
watermelon complicated to understand. Our cytogenetic analysis showed that sweet watermelon has
exactly the same rDNA distribution, number of loci
and configuration as in the ancestral species colocynth,
and differs drastically when compared with its sister
123
citron. This result highlights the chromosome structural differences in rDNA site number variation
between citron and sweet watermelon, explaining
why preferential segregation could occur in F2 and
BC1 populations derived from crosses between sweet
watermelon 9 citron, indicating that broad differences
may exist in genome organization of these two
subspecies (Levi et al. 2002, 2006). The accessions
of various taxa used in the current research were
chosen based on their use by previous researchers for
analysis of chloroplast phylogenies and molecular
markers. The citron accession PI 244018 with cream
color seeds with black and grayish black spots from
Transvaal, South Africa was characterized as chloro
type 3 by Dane and Liu (2007). The cultivated sweet
watermelon accession PI 270306 (Philippines) with
red flesh color and black seeds did not cluster with
citron and colocynth, when subjected to principal
component analyses using a large number of shared
markers, indicating it could be a representative model
of the sweet watermelon taxon (Nimmakayala et al.
2010). Colocynth (PI 386015) from Iran was characterized to possess haplotype II of G3pdh (Dane et al.
2007), and the C. rehmii (Grif 16135) accession was
the same as used by Jarret and Newman (2000) for
internal transcribed spacer sequence analysis to conclude C. rehmii was the progenitor for C. lanatus. In
particular, the number and localization of 5S rDNA
loci is often a distinguishing feature between even
closely related genomes, as has been shown by ecotype
specific polymorphisms in Arabidopsis thaliana
(Fransz et al. 1998). Based on this, we conclude the
present study should be extended to the other characterized chlorotypes (citron 1 and citron 2) of citron
(Dane and Liu 2007) as well as colocynth accessions
from various phylogeographic clusters (Dane et al.
2007) and sweet watermelon accessions that were
characterized to belong to different clusters (Nimmakayala et al. 2010). The information pertaining to
rDNA site distribution and number variation will help
to choose parents from various species for introgression of wild genes into cultivated watermelon.
Acknowledgments The authors are grateful to Dr. Jarret,
Plant Genetic Resources Conservation Unit, USDA-ARS,
Griffin, GA, 30223 for providing the seeds of germplasm
accessions. Funding support is provided by NSFEPSCOR#1003907, Gus R. Douglass Institute and USDANIFA Research (2010-02247).
Genet Resour Crop Evol
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