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Rep-PCR Identifies Both Inter- and Intra-Specific Mitochondrial Genome

Differences in Carthamus

Article in Plant Molecular Biology Reporter · October 2013

DOI: 10.1007/s11105-013-0580-5

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Plant Mol Biol Rep
DOI 10.1007/s11105-013-0580-5

ORIGINAL PAPER

Rep-PCR Identifies Both Inter- and Intra-Specific

Mitochondrial Genome Differences in Carthamus
Dinesh Kumar Viswanathaswamy &
Narasimha Rao Nizampatnam

# Springer Science+Business Media New York 2013

Abstract Mitochondria are derived from ancient prokaryotic Keywords Safflower . Carthamus . Mitochondrial genome
endosymbionts, and their genomes exhibit similarities to pro- diversity . rep-PCR
karyote genomes. Therefore, it was hypothesized that the
molecular techniques suitable for distinguishing prokaryotic
genomes could also be used to assess mitochondrial diversity. Introduction
The rep-PCR (repetitive element palindromic-PCR) tech-
nique, based on the repetitive sequences found in bacterial Assessment of plant mitochondrial diversity is of great
genomes, has been used extensively for identifying and interest for both basic and applied research. Organellar
distinguishing bacterial strains. This study was undertaken to genomes—chloroplast DNA (cpDNA) and mitochondrial
evaluate the utility of rep-PCR for identifying mitochondrial DNA (mtDNA)—have been increasingly used in recent
(mt) genome diversity in safflower (Carthamus tinctorius L.) years as markers to assess maternal/or paternal gene flow
and its wild relatives. Using three sets of commonly used because of their uniparental mode of inheritance (McCauley
primers, BOX, ERIC and REP, both inter-specific and intra- 1995). The study of mitochondrial genomes with suitable
specific mt genome diversities in Carthamus were identified. molecular markers will aid in (1) assessing the transfer of
To confirm that the amplicons obtained with rep-PCR were cytoplasm from female parent, especially when wild species
derived from mitochondrial genomes, we cloned and se- are involved in crossing; (2) understanding rearrangements
quenced six randomly chosen bands from rep-PCR gels and in the genome; (3) studying the molecular basis of male
demonstrated that the amplified products were mitochondrial- sterility; (4) ascertaining phylogenetic relationships among
genome-specific. The advantages of rep-PCR in assessing related species; and (5) investigating the origin of cultivated
chondriome variability are discussed. species of safflower. Molecular markers such as RFLP, and
RAPD, PCR-RFLP, which assess genome-wide variability,
have been employed to detect variations across mitochondrial
genomes or to identify specific cytoplasms (Bach et al. 2002;
Electronic supplementary material The online version of this article Engelke and Tatlioglu 2002; Liu et al. 2002; Sane et al. 1997;
(doi:10.1007/s11105-013-0580-5) contains supplementary material,
which is available to authorized users. Yamagishi and Terachi 2003; Yashitola et al. 2004).
Short repeats are common in the mitochondrial genomes
D. K. Viswanathaswamy (*) : N. R. Nizampatnam
of both lower and higher plants (Andre et al. 1992; Alverson
Directorate of Oilseeds Research, Rajendranagar,
Hyderabad 500 030, India et al. 2010, 2011); about 10 % of most sequenced plant
e-mail: dineshkumarv@yahoo.com mitochondrial genomes is constituted by repeats. Most
reported mitochondrial mutations are rearrangements and
D. K. Viswanathaswamy
deletions caused by aberrant recombination between short
e-mail: dineshkumar@dor-icar.org.in
(generally <200 bp) repeats (Odahara et al. 2009). These
Present Address: repeats consist of conserved regions studded with highly
N. R. Nizampatnam variable regions. Therefore, it has been suggested that an
Plant Science Department, South Dakota State University,
alternate strategy to assess the variability in mitochondrial
NPB 137, Northern Plains Biostress,
Brookings SD 57007, USA genomes is to exploit the presence of repeated regions within
e-mail: narasimharao.nizampatnam@sdstate.edu the genome and mutations involving structural rearrangements
 Plant Mol Biol Rep

(Atlan and Couvet 1993). A universal PCR technique harvested for mtDNA isolation. For initial experiments,
exploiting conserved regions to detect diversity based on the these wild species were used. To further assess the utility
variable regions of mitochondria would be rapid and of im- of the technique, flower buds from 27 different accessions
mense use. Such a technique would take advantage of the of C. oxyacanthus were used for DNA isolation and
speed and ease of PCR and the high rates of variability analysis.
expected in mtDNA repetitive sequences.
Mitochondria and plastids are derived from ancient pro- DNA Isolation
karyotic endosymbionts and their genomes show homology to
prokaryotic genomes (Dyall et al. 2004; Gray 1999). Mitochondrial DNA was isolated from immature flower
Therefore, we hypothesized that any DNA fingerprinting tech- buds from two accessions of cultivated species Carthamus
nique adopted for diversity analysis in bacteria may also be tinctorius, one accession of C. palaestinus and two acces-
applicable to chondriome studies. The rep-PCR (repetitive sions each of four wild species (C. creticus, C. lanatus, C.
element palindromic-polymerase chain reaction) technique, glaucus and C. turkestanicus) and from 27 accessions of C.
devised for characterization of bacteria, is widely employed oxyacanthus using reported protocols (Kirti et al. 1995)
to distinguish species, strains, serotypes etc. (Adiguzel et al.
2009; Hiett and Seal 2009; Louws et al. 1996; Lupski and rep-PCR
Weinstock 1992; Reyes-Ramirez and Ibarra 2005; van Bekum
1994; Versalovic et al. 1997). In the present study, we tested PCR conditions followed the procedure described by
whether rep-PCR could be extended to analyze plant mtDNA Rademaker and Bruijin (1998). PCR amplification was carried
genomic diversity using Carthamus species. The genus out in a 20-μl reaction mixture consisting of 10 mM TrisHCl
Carthamus includes several species, among which only C. (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.4 mM of each dNTP,
tinctorius is cultivated. Genetic diversity in the safflower 10 pM of each primer, 1.5 U Taq DNA polymerase (Genei,
germplasm has been investigated previously based on Bangalore, India) and 1 ng mtDNA. PCR was performed in a
agromorphological traits (Ashri 1975; Jaradat and Shahid Master Cycler (Eppendorf, Germany) PCR machine. The PCR
2006), biochemical traits (Fernandez-Martinez et al. 1993; conditions were 94 °C for 3 min, followed by 45 cycles of
Han and Li 1992), molecular markers including isozymes DNA amplification [20 s at 92 °C, 1 min at 52 °C for Box A1R
(Han and Li 1992), RAPDs (Amiri et al. 2001; Sehgal and and ERIC primers (1 min at 38 °C for REP primers) and 8 min
Raina 2005; Vijay et al. 2009; Vilatersana et al. 2005), at 68 °C] and a 15 min incubation at 68 °C.
ISSRs (Ash et al. 2003; Yang et al. 2007), AFLPs
(Sehgal and Raina 2005) and EST-SSRs (Mayerhofer Separation of PCR Products
et al. 2010; Naresh et al. 2009; Yamini et al. 2013).
However, mitochondrial diversity studies have not been Amplified products were separated by electrophoresis on
reported in Carthamus and such study is expected to 2 % agarose gel for 7–8 h at constant voltage (2 V/cm).
give better insight into the origin of the cultivated The amplicons were visualized under UV light after staining
species. Here, we report the successful adoption of the rep- with 0.01 % ethidium bromide.
PCR technique to assess both inter-specific and intra-specific
diversity among the mitochondrial genomes of cultivated Cloning and Sequencing of PCR Products
safflower and its wild relatives. It is predicted that if rep-
PCR were adopted with a set of carefully selected accessions To confirm that the amplicons obtained from rep-PCR were
of Carthamus species, it would provide insight into the origin derived from mitochondrial genomes, six representative
of cultivated safflower species. amplicons obtained using the three (BOX, ERIC and REP)
primers, were extracted using a gel extraction kit (Qiagen,
Hilden, Germany) and cloned into the PCR cloning vector
Materials and Methods pTZ57R (InsT/Aclone PCR product cloning kit; Fermentas,
St. Leon-Rot, Germany). Five of the chosen bands were
Plant Material common among the different species of safflower and one
was specific to C. oxyacanthus. The amplicons cloned
Different accessions of cultivated safflower (Carthamus ranged from 350 bp to 2 kb in length. All the clones were
tinctoriusL.) and six wild species (C. palaestinus, C. sequenced using a DNA sequencer (ABI 273A, Applied
oxyacanthus, C. creticus, C. lanatus, C. turkestanicus and C. Biosystems, Foster City, CA) using the universal (M13
glaucus) with different chromosome numbers and geographical forward and M13 reverse) primers available in the vector.
locations, available at the Directorate of Oilseeds Research, The sequences obtained were subjected to BLAST analysis
Hyderabad, India, were grown and immature flower buds were to determine their identity.
Plant Mol Biol Rep

Results primer with accessions of C. lanatus, and BOX primers

with accessions of C. oxyacanthus, C. creticus, C.
Three sets of primers (BOX, ERIC and REP) were used for lanatus, C. turkestanicusand C. glaucus) accessions
the study. Initially, rep-PCR reactions were carried out with within species could be distinguished. Thus, this set of
different amounts of mtDNA (0.1, 1.0, 5.0 and 10 ng per initial experiments proved that rep-PCR could capture
reaction) to identify the optimal template DNA quantity for both inter-specific and intra-specific mitochondrial geno-
rep-PCR and, based on the results, 1.0 ng template DNA per mic variation.
reaction was chosen. DNA amplification was obtained with This technique was extended to analyze the variability in
all three primer sets as well as with all mtDNA templates. mitochondrial genomes within different accessions of C.
The PCR profiles obtained were distinct and reproducible oxyacanthus. The analysis revealed the existence of differ-
for each primer set as well as for each of the species tested. ent mitotypes within these accessions with both BOX and
The number of amplicons in different reactions ranged from ERIC primers (Fig. 2).
8 to 16. In each reaction, most of the amplicons (up to 10) To further verify that the amplicons obtained were derived
were very prominent while others were low to medium in from mitochondrial genomes, six of the PCR bands were
intensity (Fig. 1). The size of the amplicons ranged from cloned and sequenced. Deliberately, we chose to clone one
0.2 kb to >6.0 kb, although bands of >6 kb were faint. Each C. oxyacanthus-specific fragment and five fragments that
primer set yielded sufficient polymorphism to distinguish were common across all species investigated. Sequence anal-
the different species of safflower. The banding pattern ysis showed homology to mitochondrial genome of plants.
consisted of amplicons that were consistent across species; Among the five amplicons analyzed that were common
those that were unique to each of the species were investi- among species, two of the clones corresponded to mitochon-
gated. It was also observed that, in some cases (e.g., ERIC drial maturase, one of them showed very high homology to

Fig. 1a,b Mitochondrial

genome diversity in safflower
and its wild species as revealed
by rep-PCR using two different
sets of primers. a rep-PCR with
BOX primer, b rep-PCR with
ERIC. Lanes: 1 C. palaestinus;
2,3 C. tinctorius; 4, 5 C.
oxyacanthus; 6, 7 C. creticus; 8,
9 C. lanatus; 10, 11 C.
turkestanicus; 12, 13 C.glaucus;
M marker (Hind III and Eco RI
digest of phage l DNA)
 Plant Mol Biol Rep

Fig. 2a,b Mitochondrial

genome diversity among different
accessions of C. oxyacanthusas
revealed by the rep-PCR
technique. a rep-PCR with BOX
primer, b rep-PCR with ERIC
primer. Lanes: 1–27 Different
accessions of C. oxyacanthus, M
molecular size markers (Hind III
and Eco RI digest of phage l
DNA, 100 bp marker)

tRNA for cysteine, one to mitochondrial genomic sequences Discussion

and the other showed homology to both mitochondrial ge-
nome sequence and the 16S rRNA genes of chloroplasts. This Recent advances in DNA analysis have made it possible to
may not be an aberration as mitochondrial genomes are identify genetic variation in chloroplast and mitochondrial
known to acquire sequences from the chloroplast genome genomes of plants and to analyze this variation at the pop-
through horizontal gene transfer (Alverson et al. 2010). The ulation level. A review of population genetic theory for
amplicon, which was specific to C. oxyacanthus, did not show these uniparentally inherited non-recombining genomes in-
any homology to known sequences. A summary of the dicates that they are likely to show low levels of intra-
BLAST result is presented in Table 1. specific sequence variation in coding regions and that
Thus our experiments proved that rep-PCR can be targeting of alternative forms of variation (duplications,
employed to study variability in mt genomes and further rearrangements) of non-coding regions of the genome with
showed that this technique can distinguish both intra- and higher mutation rates may be necessary to detect useful
inter-specific chondriomes. genetic markers. Techniques of DNA analysis have made

Table 1 Summary of BLAST search of sequenced clones. mt Mitochondrial

Sample no. Clone description Clone size Sequence homology

1 CB-1, common amplicon of BOX primer 400 bp Perfect homology to mt maturase gene
2 CB-2, common amplicon of BOX primer 350 bp Perfect homology to maize mt genomic DNA
and chloroplast 16S rRNA
3 CE-1, common amplicon of ERIC primer 2 kb Perfect homology to mt tRNA gene
4 CE-2, common amplicon of ERIC primer 700 bp Partial homology to mt genomic DNA
5 CR-1, common amplicon of REP primer 600 bp Perfect homology to mt tRNA gene
6 SB-1, amplicon specific to C. oxyacanthus of BOX primer 500 bp No homology to any sequence
Plant Mol Biol Rep

it possible to extend our knowledge of population genetic These primers amplify genomic regions located between
structure not only by using nuclear DNA markers (RAPD, repetitive sequences. Polymorphism among samples
AFLP, microsatellite) but also by exploiting our ability to arises either due to differences in the distribution of
detect genetic variation in the two organelle genomes in repetitive sequences in the genome or due to changes
plants, i.e., those of chloroplasts and mitochondria. in the regions between the repetitive sequences. These
Mitochondrial genome recombination involves both large- primers have proven extremely useful in the study of
sized (greater than 1 kb) repeats that mediate high-frequency microbial diversity (Versalovic et al. 1994) and have been
reciprocal DNA exchange to produce subgenomic DNA mol- employed for the identification and classification of bacteria
ecules (Mackenzie and McIntosh 1999), and intermediate- and for molecular epidemiological studies of human and plant
sized repeats that participate in low-frequency DNA ex- pathogens (Adiguzel et al. 2009; Hiett and Seal 2009; Louws
change. Structurally, mitochondrial genomes are internally et al. 1996; Reyes-Ramirez and Ibarra 2005; van Bekum
recombining circular chromosomes with variations arising as 1994; Versalovic et al. 1997).
a result of substantial intrachromosomal recombinantion at Mitochondrial genomes are considered to be of pro-
sites of internal repeats in the genome (Atlan and Couvet karyotic origin. Variable numbers of tandem repeats
1993). Structural differences between genomes from closely (VNTRs) analogous to those found in bacteria have been
related taxa can be substantial as a consequence of complex found in plant mtDNA (Nishizawa et al. 2000; Yazdankhah
rearrangements (Palmer 1992). Such rearrangements have and Lindstedt 2007). We hypothesized that rep-PCR, which is
been the targets for detecting variation among mt genomes. employed extensively to study diversity in bacterial genomes,
It has been well established that plant mt genomes have could also be used for mt genome diversity studies. We tested
different kinds of repetitive elements that contribute substan- the applicability of rep-PCR for distinguishing mitochondrial
tially to the size of the mt genome as well as give additional genomes of the cultivated safflower (C. tinctorius) and its wild
scope for rearrangements in the chondriome (Alverson et al. relatives. Our results demonstrated clearly that the tech-
2010; Aono et al. 2002; Ennos et al. 1999; Lily and Havey nique is useful in distinguishing different mitochondrial
2001; Mackenzie and Mcintosh 1999). genomes of the genus Carthamus. However, unlike in
There are several reasons for the adoption of mtDNA as a bacteria where rep-PCR yields a large number (10–50)
marker of choice. Experimentally, mtDNA is relatively easy of bands, using Carthamus mtDNA as template yielded
to amplify because it is present in multiple copies in the cell. fewer bands (up to 16). This could be due to the relatively
Mitochondrial DNA is the most convenient and cheapest small size of the mitochondrial genomes of plants (generally
solution when a new species in the wild has to be explored in the range of 300 kb compared to bacterial genomes, which
genetically. RFLP techniques have been employed to detect are more than 1,000 kb). However, the mitochondrial genome
such rearrangements (Fukunaga and Kato 2003). But RFLP size of Carthamus is not known and so the correlation be-
deployment to study variation is limited by high technical tween number of amplicons obtained and genome size could
and resource demands. RAPD has been shown to be useful not be ascertained.
in the characterization of different CMS sources in rice To date, there is only one report of the use of rep-PCR to
where the RFLP technique was inadequate. RAPD and study the variability in mitochondrial genomes of plants
gene-specific PCR techniques have also been adopted for (Ashutosh et al. 2005). In higher plants, cytoplasmic male
diversity studies in mt DNA (Amini et al. 2008; Wang et al. sterility (CMS) is determined by the mitochondrial genome.
1996). However, RAPD requires that a large number of The rep-PCR technique was employed to identify different
random primers be screened to identify those that are poly- CMS lines of Brassica juncea. The amplified products were
morphic. Wijnants et al. (2001) suggested the use of RAGE- converted into SCAR markers, which clearly distinguished
PCR primers designed to amplify inter-genic regions of CMS Catholica (W) from other lines (Ashutosh et al. 2005).
mitochondrial genome for phylogenetic analysis of mt ge- Analysis of genetic diversity in safflower using RAPD and
nomes. But the large number of primers (97 pairs) that may nuclear genes (universal markers for Asteraceae family) has
have to be employed is a severe limitation. These observa- been reported using different types of molecular markers
tions suggest that a PCR-based method that targets the (Amini et al. 2008; Chapman et al. 2010; Yamini et al.
variability in interspersed repeat sequences found in mito- 2013). Nuclear DNA as well as chloroplast DNA diversity
chondrial genomes could be of immense use in assessing have been employed to understand the phylogenetic relation-
chondriome diversity and could also be of use in a variety of ships among Carthamus species (Chapman and Burke 2007;
other studies. Sehgal et al. 2008) and there are contrasting views regarding
The rep-PCR technique was devised by Lupski and the progenitor of the cultivated safflower (Carthamus
Weinstock (1992) and uses three specific primers, designated tinctorius L.). However, so far there are no reports of using
BOX, ERIC and REP, which were designed to match con- mt-genome-based markers for understanding the phylogenetic
served sequences distributed in diverse bacterial genomes. relationships among different Carthamus spp.
 Plant Mol Biol Rep

Conclusion Aono N, Shimizu T, Inoue T, Shiraishi H (2002) Plaindromic repetitive

elements in the mitochondrial genome of Volvox. FEBS Lett
521:95–99
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