Horizontal gene transfer of the algal nuclear gene
psbO to the photosynthetic sea slug Elysia chlorotica
Mary E. Rumphoa,1, Jared M. Worfula, Jungho Leeb, Krishna Kannana, Mary S. Tylerc, Debashish Bhattacharyad,
Ahmed Moustafad, and James R. Manharte
aDepartment
of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, ME 04469; bGreen Plant Institute, Seoul National
University, Gwonseon, Suwon, Gyeonggi 441-853, Korea; cSchool of Biology and Ecology, University of Maine, Orono, ME 04469; dDepartment of Biological
Sciences and the Roy J. Carver Center for Comparative Genomics, Interdisciplinary Program in Genetics, University of Iowa, Iowa City, IA 52242-1324;
and eDepartment of Biology, Texas A&M University, College Station, TX 77843
The sea slug Elysia chlorotica acquires plastids by ingestion of its
algal food source Vaucheria litorea. Organelles are sequestered in
the mollusc’s digestive epithelium, where they photosynthesize
for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations
for the persistence of photosynthesis in the sea slug are (i) the
ability of V. litorea plastids to retain genetic autonomy and/or (ii)
more likely, the mollusc provides the essential plastid proteins.
Under the latter scenario, genes supporting photosynthesis have
been acquired by the animal via horizontal gene transfer and the
encoded proteins are retargeted to the plastid. We sequenced the
plastid genome and confirmed that it lacks the full complement of
genes required for photosynthesis. In support of the second
scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated
into the germline. The source of psbO in the sea slug is V. litorea
because this sequence is identical from the predator and prey
genomes. Evidence that the transferred gene has integrated into
sea slug nuclear DNA comes from the finding of a highly diverged
psbO 3ⴕ flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E.
chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty (‘‘green animals’’) and is explained by
anastomosis of distinct branches of the tree of life driven by
predation and horizontal gene transfer.
symbiosis 兩 Vaucheria litorea 兩 evolution 兩 plastid 兩 stramenopile
S
ymbiotic associations and their related gene transfer events
are postulated to contribute significantly to evolutionary
innovation and biodiversity. This comes from extensive analysis
of organelles such as plastids (e.g., chloroplasts) that originated
via primary endosymbiosis of a free-living cyanobacterium (1, 2).
The cyanobacterial genome was greatly reduced by endosymbiotic gene transfer (EGT) to the host nucleus and wholesale gene
loss, giving rise to the primary lineages of plants and green algae
(streptophytes and chlorophytes), red algae (rhodophytes), and
glaucophytes (3–6) [see the scheme in supporting information
(SI) Fig. S1]. The diverse group of secondary or ‘‘complex’’ algae
(e.g., chromalveolates, euglenids), in turn, arose by secondary
endosymbiosis—the uptake of a eukaryotic alga (green or red
lineage) by a heterotrophic eukaryotic host. In this case, in
addition to EGT, transfer of genes between the unrelated
organisms by lateral or horizontal gene transfer (HGT) and loss
of genes occurred as a result of the ‘‘merger’’ of the two nuclei
(host and endosymbiont) (7). As a result of primary and secondary endosymbiosis, plastid genomes (ptDNAs) encode less
than 10% of the predicted 1,000 to 5,000 proteins required to
sustain the metabolic capacity of the plastid (8, 9).
Examples of HGT between unrelated or nonmating species are
abundant among prokaryotes (10, 11) but less so between prokaryotes and unicellular (12–14) or multicellular eukaryotes (15–
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0804968105
20). Most of these latter examples are associated with parasitism or
phagotrophy, including the elegant studies of HGT from the
␣-proteobacteria Wolbachia to insects and nematodes (16–18), and
the finding of rhizobial-like genes in plant parasitic nematodes (19,
20). The exchange of genetic material between two eukaryotes is
extremely rare, or at least not well documented to date. The
best-studied cases include the transfer of mitochondrial DNA from
achlorophyllous or epiphytic plants to the mitochondrial genome
(mtDNA) of their closely related photosynthetic hosts (21), the
exchange of transposons between two animal (22) or two plant (23)
species, and the presence of plant genes in plant parasitic nematodes
(in addition to the rhizobial genes discussed previously), which are
hypothesized to be ‘‘defense’’ genes whose products protect the
parasite from host detection (20).
The sacoglossan mollusc (sea slug) Elysia chlorotica represents
a unique model system to study the potential for interdomain
HGT between two multicellular eukaryotes—in this case, from
a filamentous secondary (heterokont) alga (Vaucheria litorea) to
a mollusc. This emerald green sea slug owes its coloring and
photosynthetic ability to plastids acquired during herbivorous
feeding (24–29). The plastids do not undergo division in the sea
slug and are sequestered intracellularly in cells lining the finely
divided digestive diverticula. The plastids continue to carry out
photosynthesis, providing the sea slug with energy and carbon
during its approximately 10-month life span (27, 28). Long-term
plastid activity continues despite the absence of algal nuclei (27,
29), and hence a source of nuclear-encoded plastid-targeted
proteins. We hypothesize that the algal nuclear genes encoding
essential plastid proteins are present in the sea slug, presumably
as a result of HGT. Here, we present evidence for such interdomain HGT of psbO, a nuclear gene encoding the plastid
manganese-stabilizing protein (MSP ⫽ PsbO). MSP is a subunit
of the photosystem II complex associated with photosynthetic
oxygen evolution (30, 31), which is, unquestionably, the most
important enzyme complex of oxygenic life.
Results and Discussion
Plastid Genetic Autonomy. The plastids in E. chlorotica are not
transmitted vertically; rather, they must be acquired with each
generation early in development to ensure maturation to the
Author contributions: M.E.R. and J.R.M. designed research; J.M.W., J.L., and K.K. performed
research; J.L., M.S.T. contributed new reagents/analytic tools; K.K., D.B., A.M., and J.R.M.
analyzed data; and M.E.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the GenBank
database [accession nos. EU912438 (V. litorea complete ptDNA), EU599581 (E. chlorotica
complete mtDNA), DQ514337 (V. litorea psbO cDNA), EU621881 (V. litorea psbO DNA), and
EU621882 (E. chlorotica psbO DNA).
1To
whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/cgi/content/full/
0804968105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
PNAS 兩 November 18, 2008 兩 vol. 105 兩 no. 46 兩 17867–17871
EVOLUTION
Edited by Lynn Margulis, University of Massachusetts, Amherst, MA, and approved September 17, 2008 (received for review June 9, 2008)
A
B
C
D
Fig. 1. Laboratory culturing of E. chlorotica. (A) Free-swimming E. chlorotica
veliger larvae. (Scale bar, 100 ␮m.) Under laboratory conditions, the veliger
larvae develop and emerge from plastid-free sea slug–fertilized eggs within
approximately 7 days. The green coloring in the digestive gut is attributable
to planktonic feeding and not to the acquisition of plastids at this stage.
Metamorphosis of the larvae to juvenile sea slugs requires the presence of
filaments of V. litorea. (B) Metamorphosed juvenile sea slug feeding for the
first time on V. litorea. (Scale bar, 500 ␮m.) The grayish-brown juveniles lose
their shell, and there is an obligate requirement for plastid acquisition for
continued development. This is fulfilled by the voracious feeding of the
juveniles on filaments of V. litorea. (Also see Movie S1). (C) Young adult sea
slug 5 days after first feeding. (Scale bar, 500 ␮m.) By a mechanism not yet
understood, the sea slugs selectively retain only the plastids in cells that line
their highly branched digestive tract. (D) Adult sea slug. (Scale bar, 500 ␮m.)
As the sea slugs further develop and grow in size, the expanding digestive
diverticuli spread the plastids throughout the entire body of the mollusc,
yielding a uniform green coloring. (Also see Movie S2.) From these controlled
rearing studies, we were able to conclude that the only source of plastids in
our experimental sea slugs was V. litorea.
adult sea slug (32). Laboratory coculturing studies were carried
out to establish that the alga V. litorea, a derived heterokont alga
that contains secondary plastids of red algal origin (33) (Fig. S1),
was the sole source of plastids in the sea slugs (Fig. 1, Movie S1,
Movie S2, and SI Methods). Subsequent sequencing and mapping
of the V. litorea ptDNA (only the fifth heterokont ptDNA to be
published to date) revealed a very compact 115,341-bp doublestranded circular genome encoding 169 genes, including 139
protein-encoding genes (14 are conserved ORFs [ORFdesignated ycfs] and 2 are unknown ORFs), 27 tRNA genes, and
3 rRNA genes (Fig. 2). The genes are densely arranged with an
average intergenic region of 74bp and 11.1% noncoding DNA.
The overall G ⫹ C content is 28%, which is low compared with
most plastids, including other related heterokonts (34, 35). The
genome is separated into two large single-copy regions (62,002bp
and 43,469bp) by two smaller inverted repeats (both 4935bp). All
the plastid rRNA genes are found on both copies of the inverted
repeat in the highly conserved operon rrs-trnI-trnA-rrl-rrf. Unlike
the four published heterokont ptDNAs that lack introns (34, 35),
V. litorea contains one intron in the trnL UAA gene—an ancient
intron that is also present in cyanobacteria (36). In addition,
V. litorea has retained the genes for the light-independent
chlorophyll biosynthesis pathway: chlB, chlL, and chlN. However, as expected, the V. litorea ptDNA shares more similarity
with heterokonts and red algae than it does with green plastids
(34, 35, 37) (see complete inventory of plastid genes by category
in Table S1).
Examining the genetic autonomy of V. litorea ptDNA revealed
the absence of the major core protein of the oxygen evolving
complex of photosystem II, MSP (encoded by psbO). MSP has
17868 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0804968105
Vaucheria litorea
chloroplast genome
Fig. 2. Map of the ptDNA of V. litorea. Genes on the outside are transcribed
in the clockwise direction, whereas genes on the inside are transcribed in the
counterclockwise direction. Genes are color coded according to their function
as shown. tRNAs are listed by the one-letter amino acid code followed by the
anticodon. The only gene with an intron (L-uaa) is indicated by an asterisk.
been reported to be critical to the stability of the water-splitting
reaction of photosynthesis that generates atmospheric oxygen
(30, 31). The evolutionary conservation of this reaction is
demonstrated by the presence of MSP in all oxygenic photosynthetic organisms (30). Likewise, animal genomes have never
been shown to contain psbO; hence, MSP cannot be made by the
sea slug in the absence of HGT. We have previously demonstrated that oxygen evolution is linked to photosynthetic electron
transport in the sea slug for at least 5 months after being removed
from its algal prey (27), and photosystem II is generally highly
susceptible to photo-oxidative damage requiring de novo synthesis and reassembly of its subunits (38, 39). For these reasons,
we targeted psbO for HGT from V. litorea to E. chlorotica.
HGT and Expression of psbO. Heterologous degenerate primers
(Table S2) were designed based on alignments of published psbO
sequences to amplify an internal fragment using reverse transcriptase (RT)-PCR. A 452-bp fragment was amplified from
both algal and sea slug cDNA (5 months after algal feeding) (Fig.
3A). The translated product was used to blast the GenBank
database, which revealed a relatively high identity (48%–68%)
to several secondary red algal–derived MSP amino acid sequences. The entire V. litorea psbO cDNA sequence was then
obtained using 5⬘- and 3⬘-RACE, and this sequence was used to
design homologous primers to amplify a 963-bp internal fragment of the V. litorea psbO cDNA (Fig. 3C). These same primers
yielded a similar sized PCR product from sea slug cDNA and
DNA templates (Fig. 3C), the sequences of which were identical
to the V. litorea psbO cDNA sequence beginning with the start
codon (there are no introns in the algal gene; Fig. S2).
Although it had been several months since the sea slugs had
been in contact with any algal prey, the possibility of algal
nuclei remaining in the gut of the sea slug and contaminating
the total genomic DNA preparation was eliminated by carrying
out the same PCR on sea slug egg DNA. Because plastids are
Rumpho et al.
Ecl (-) Stds
Vli
Ecl
V. lito
rea
B
1.6 kb
1.2 kb
500 bp
400 bp
C
V. litorea
egg
E. chlorotica
adult
adult
~960 bp
PCR
D
RT-PCR
MKVPSALVALSAFSVKTSAFRPAFAGLKTNAKSSSALTMSVQDDI
KTLAVGALTILAGVSILNAPVEAITKDQIESLSYLQVKGTGLANRCP
EVFGTGSIDVNGKTKIVDMCIEPKTFQVLEETSSKRGEAKKEYVNT
KLMTRQTYTLYGIDGSFAPENGKITFREKDGIDYAATTIQLPGGERV
PFLFTVKELVAQATTPGNSVTPGLQFGGPFSTPSYRTGLFLDPKGR
GGSTGYDMAVALPGHQSGIEGDAELFGENNKTFDVTKGNIEFEVN
RVDPSNGEIGGVFVSKQKGDTDMGSKVPKDLLIKGIFYGRLESE
Fig. 3. Expression of psbO in V. litorea and E. chlorotica. (A) RT-PCR using
heterologous primers to psbO amplified a 452-bp fragment from both algal
and adult sea slug cDNA. Water served as the negative control. Standards
were a 1-kb Plus DNA ladder (Invitrogen). Vli, Vaucheria litorea, Ecl, Elysia
chlorotica. (B) Northern blot analysis employing the cloned V. litorea 452
bp psbO product as probe hybridized with a 1.2-kb transcript for V. litorea
and E. chlorotica as well as a 1.6-kb transcript in the sea slug. RNA
Millennium Size Markers (Ambion) were run to estimate transcript size. (C)
Homologous primers were designed from the RACE-amplified sequence of
the V. litorea psbO fragment in A. By PCR, these primers amplified a 963-bp
product from genomic DNA of V. litorea and E. chlorotica eggs and adult
tissue as well as E. chlorotica adult cDNA by RT-PCR. (D) Translation of the
psbO sequences obtained from the 963-bp products in B, for both V. litorea
and E. chlorotica, yielded an identical amino acid sequence with a putative
tripartite targeting signal for MSP. The signal sequence is in red, the transit
peptide is in blue, and the thylakoid targeting domain is in green. Note the
highly conserved phenylalanine residue at the cleavage site of the signal
sequence.
not inherited in E. chlorotica, eggs provide a source of animal
DNA and RNA that is free of algal contamination (27).
Amplification of the sea slug egg DNA with the same primers
resulted in a 963-bp fragment (Fig. 3C) with a sequence
identical to the algal and sea slug psbO fragments (Fig. S2). As
further PCR controls, primers complementary to the V. litorea
internal transcribed spacer region (ITS1; GenBank EF441743)
were used as a positive algal nuclear control (27); no product
was observed from sea slug or sea slug egg DNA templates.
Likewise, attempts to amplify psbO from negative controls
(pufferfish and Dictyostelium DNA) using the same primers
and reagents did not yield a positive PCR product. Finally,
identical PCR results have been obtained from sea slugs
collected from the same site on multiple occasions over a
3-year period (results not shown). Expression of the foreign
gene in the sea slug was further supported by Northern blot
analysis. The V. litorea psbO probe cross-reacted with a 1.2-kb
transcript for both V. litorea and E. chlorotica RNA, along with
a slightly larger transcript (1.6 kb) in the sea slug (Fig. 3B).
The identical translated MSP amino acid sequences for both
V. litorea and E. chlorotica (Fig. 3D) were analyzed by phylogenetic methods (40, 41) incorporating 23 published MSP seRumpho et al.
quences. This revealed nesting of the sequences in the red algal
lineage in a clade containing other heterokonts (Fig. S3). As
expected for this secondary lineage, the V. litorea MSP preprotein contains a tripartite targeting sequence (Fig. 3D; as predicted by refs. 42, 43). This reflects the presence of chloroplast
endoplasmic reticulum around the complex plastids, which must
be traversed by proteins targeted to the plastid (44). What is
interesting is that the MSP preprotein in the sea slug has retained
the entire tripartite targeting sequence (Fig. 3D), especially in
light of the observation that the chloroplast endoplasmic reticulum does not appear to be retained around the engulfed
chloroplasts (28).
Recently, it was reported that nuclear genes encoding plastidlocalized light harvesting complex proteins ( fcp, lhcv1, and
lhcv2) have also been transferred from V. litorea to E. chlorotica
(45). Using a similar PCR approach, identical nucleotide sequences were reported for sea slug and algal fcp and lhcv1, and
only a single base substitution was found between larval lhcv2
and adult sea slug or alga lhcv2. Although evidence from
Southern blotting has not been achieved in the study reported
here or for the light harvesting complex protein genes (45), we
were able to obtain sequence information using genome walking
for the 3⬘ untranslated flanking region of the psbO gene from
both algal DNA and sea slug egg DNA. A nested gene-specific
primer coupled with an adapter-specific primer (Table S2)
yielded a 3⬘ flanking sequence from both organisms that was
identical for the first 81bp corresponding to the 3⬘ end of the
psbO gene and ending with the stop codon (Fig. S4). This
sequence was followed by a highly diverged sequence corresponding to the 3⬘ untranslated region in each genome. These
results support the interdomain transfer of an algal gene to a
mollusc, its expression in the foreign host, and also that the gene
has been inserted into the germline, even though the plastids are
not yet transmitted vertically in the sea slug.
Mechanism and Site of Integration of Transferred Genes. Similar to
many other phagocytic or parasitic relations that lead to presumptive HGT events, the E. chlorotica/V. litorea plastid endosymbiosis involves intimate physical contact between predator
and prey. During the sea slug’s phagocytic feeding, the algal
nuclei come into direct physical contact with the sea slug
digestive epithelium. Upon nuclear rupture in the gut, pieces of
algal chromosomal DNA (and possibly transcripts) may have
been randomly transferred by ‘‘bulk transfer’’ or viral transmission (46) to the sea slug. Two potential sites for insertion of
foreign genes in the sea slug are the nuclear genome and the
mtDNA. Mitochondrion-to-mitochondrion gene transfer is now
recognized as a dominant mode of HGT in plants because of the
larger and more plastic mtDNAs in these taxa (21). The smaller
compact animal or metazoan mitochondrion genome is generally
believed to be a poorer target for foreign gene insertion.
However, some basal metazoans do exhibit greater variation in
mtDNA size and gene content (47). This includes multiple
examples of HGT of group I intron sequences (normally not
found in animals) into the mtDNA of a sponge (48), a sea
anemone (49), and a coral (50).
To determine if the mtDNA of E. chlorotica serves as a target
for any foreign genes, including psbO, we used PCR and primer
walking to obtain the complete sequence and map the 14,132-bp
mtDNA from sea slug eggs (Fig. 4). The genome was found to
encode the standard 37 genes found in other typical animal
mitochondria (see ncbi.nlm.nih.gov/genomes/ORGANELLES/
33208). No introns were identified, and only 0.0125% of the
DNA was noncoding. By measuring the G ⫹ C content over
adjacent windows of 500 nt with 200-nt overlaps, the values were
found to be uniformly distributed across the windows, suggesting
homogeneity in GC content of the mtDNA and not supporting
PNAS 兩 November 18, 2008 兩 vol. 105 兩 no. 46 兩 17869
EVOLUTION
Vli
A
+
any physical contact, must be considered formally possible. This
is especially true in the context of genetically modified organisms. The implications for evolution and speciation through
acquisition of foreign parts and selected genes to produce new
lineages, as proposed by Margulis (2), are heightened by this
unusual photosynthetic mollusc.
Methods
Experimental Materials. E. chlorotica was collected from Martha’s Vineyard
Island in Massachusetts and maintained without algae in aquaria containing
aerated artificial seawater (925 mosmol; Instant Ocean, Aquarium Systems) at
12 °C during a 14-h photoperiod (27). After 3 months, eggs produced by E.
chlorotica were used to initiate culturing experiments as described in SI
Methods. V. litorea CCMP2940 filaments were maintained in a modified f/2
medium (27).
Nucleic Acid Preparation. DNA and RNA were isolated from sea slugs (5 months
after feeding or collection), sea slug eggs, and algal filaments using DNAzol or
DNAzol extra strength (Molecular Research Center, Inc.) and the RNeasy mini
kit (Qiagen), respectively, unless noted differently. RNase and DNase were
added during the extraction process for DNA and RNA, respectively, and
negative controls were run on each. First-strand cDNA was synthesized using
SuperScript II ribonuclease H⫺ RT (Invitrogen) and oligo d(T) priming on
DNase-treated RNA.
-
+
Fig. 4. Map of the mtDNA of the sacoglossan mollusc E. chlorotica. Genes
transcribed clockwise are shown on the outside of the circle, whereas those
transcribed counterclockwise are shown to the inside of the circle. Names
of tRNA genes are indicated by the three-letter amino acid code with the
two leucine and two serine tRNAs differentiated by ⫹ and ⫺ signs, recognizing codons UAG and UAA for leucine and AGN and UCN for serine,
respectively.
the existence of a chimeric region (Fig. S5). To assess further the
possibility of HGT in E. chlorotica mitochondria, we did phylogenetic analyses with nucleotide data generated using a sliding
window approach with the genome data (i.e., DNA sequences
that are independent of gene structure) and using the complete
translated ORFs. The maximum likelihood phylogenetic trees
inferred with these alignments showed that the E. chlorotica
sequences are monophyletic with molluscs, consistent with a
vertical evolutionary history for E. chlorotica mtDNA (e.g., see
the phylogenetic tree of cytochrome b in Fig. S6). These analyses
point to an intact and ‘‘typical’’ animal mtDNA in E. chlorotica.
We, however, do not argue absolutely against the possibility of
a partial DNA insertion from an algal or other source in this
genome; rather, that if such an insertion exists, it is not detectable using the approaches described here. In any case, it is
undoubtedly more likely that large-scale gene insertion would be
more readily accommodated in sea slug nuclear DNA than in
mtDNA, and high-throughput genome sequencing will be necessary to prove this idea.
Conclusions
Molecular evidence is presented supporting eukaryotic multicellular interdomain HGT (including into the germline) using a
mollusc model and expression of an essential algal nuclear gene
required for photosynthesis. Many questions remain to be answered, however; for example, the chromosomal location and
additional flanking sequences of the psbO gene in the sea slug.
Key will be to establish how this gene was activated in the mollusc
and to identify the mechanism of plastid protein targeting. It is
also very likely that HGT contributes to the long-term survival
and functioning of V. litorea plastids in E. chlorotica and that
many more algal nuclear genes have been transferred in the sea
slug. In light of these findings, the prospect of natural HGT
taking place between distantly related organisms, especially with
17870 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0804968105
PCR Amplification and Northern Blotting. Degenerate primers (psbO R and
psbO L2; see Table S2) were designed to amplify an internal fragment of V.
litorea psbO based on the conserved regions of several heterokont and red
alga psbO sequences (for list, see SI Methods). This 452-bp psbO fragment was
then used as a probe for Northern blot analysis with the Northern Max
kit (Ambion) and RediprimeII random prime labeling system (Amersham
Biosciences).
RACE and Phylogenetic Analysis of psbO. The complete V. litorea psbO gene
was obtained by rapid amplification of cDNA ends (RACE) using the GeneRacer Kit (Invitrogen) and primers listed in Table S2. Homologous primers
(psbO L5 and psbO R8) were then designed to amplify a larger (963-bp)
internal fragment of the V. litorea psbO cDNA. Phylogenetic analysis of
psbO (MSP) was based on amino acid sequences of 25 mature proteins (for
list, see SI Methods) and carried out using maximum parsimony in PAUP
4.0b10 (41).
ptDNA Sequencing. ptDNA was purified from V. litorea filaments as described (51). ptDNA was digested with the restriction endonucleases PstI,
HindIII, and EcoRI. The PstI and HindIII fragments were cloned, and a
restriction site map using all three enzymes was produced as described by
Lehman and Manhart (52). A total of 104 kb was obtained from cloned
restriction fragments. The remainder of the genome (11 kb) was obtained
by PCR amplification (53). Thirty-eight oligonucleotide primers were used
to fill gaps between cloned restriction fragments and to check fragment
connections, using all possible combinations of these primers. Fragments
were sequenced by primer walking (53).
Genome Walking. Clontech’s Genome Walking Kit was used with gene-specific
and adapter primers (sequences presented in Table S2) to amplify the 3⬘ end
of the psbO gene and the flanking untranslated region using algal DNA and
sea slug egg DNA (see SI Methods for additional details).
mtDNA Sequencing. Universal primers (ref. 54; Table S3) were used to amplify
fragments of the mitochondrial rrnL, cob, and cox1 regions from sea slug egg
DNA and then in various combinations to amplify the entire mtDNA. The
mitochondrial sequence was annotated using Dual Organellar GenoMe Annotator (DOGMA) (55), and the map was drawn using OrganellarGenomeDRAW (OGDRAW) (56). Additional information, including analysis for HGT, is
provided in SI Methods.
ACKNOWLEDGMENTS. This research was supported by National Science Foundation grants IBN-9808904 (M.R. and J.M.) and IOS-0726178 (M.R. and M.T.);
the American Society of Plant Biologists’ Education Foundation (M.R. and
M.T.); the Maine Technology Institute (M.R.); Ministry for Food, Agriculture,
Forestry, and Fisheries, Korean Government, Korea Research Foundation (J.L.);
the National Institutes of Health (grant R01ES013679 to D.B.), and the University
of Maine (M.R.). This is manuscript no. 3024 of the Maine Agriculture and Forestry
Experiment Station Hatch Project no. ME08361-08MRF (NC 1168).
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Supporting Information
Rumpho et al. 10.1073/pnas.0804968105
SI Methods
Sea Slug and Algal Culturing. Sea slug egg masses were cultured in
Petri dishes containing sterile autoclaved artificial sea water
(Instant Ocean) and incubated under room conditions of temperature and lighting. Veliger larvae emerged from egg masses
after 5–7 days and were fed aliquots of Rhodomonas salina or
Isochrysis galbana. Within 16–22 days, the veligers started to
develop pigmented striations on their shells; at this point,
filaments of V. litorea were provided to induce metamorphosis.
Cultures were supplemented with additional V. litorea on a daily
basis to promote growth and establishment of the symbiotic
association.
ptDNA Sequencing. ABI 3100 automated sequencers (Applied
Biosystems) at the University of Maine Sequencing Facility and
the Institute for Plant Genomics and Biotechnology, Texas
A&M University, were used to sequence the ptDNA fragments.
Sequences were assembled with Sequencher version 4.0, and
genes were identified using DNA and protein sequences submitted to BLAST and Genetic Computer Group (www.accelrys.com/products/gcg/). The map was constructed using Canvas
version 6.0.
Primer Design and PCR Amplification of psbO. Conserved regions of
several heterokont and red alga psbO sequences were aligned
using Clustal X (1) to design primers, including Karenia brevis
(GenBank AY116667), Isochr ysis galbana (GenBank
AY116669), Heterocapsa triquetra (GenBank AY116668), Heterosigma akashiwo (GenBank AY191862), Phaeodactylum tricornutum (GenBank AY191862), and Cyanidium caldarium (2).
The resulting primers, psbO R and psbO L2, amplified an internal
region of the V. litorea psbO gene. A list of all primers used can
be found in Table S2. PCR conditions included 1 ⫻ enzyme
reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix (Invitrogen),
primers (0.5 ␮M each), 1 to 10 ng of DNA, and 2.5 U of platinum
Taq polymerase (Invitrogen). Cycle conditions were 94°C for 2
min, 25 cycles of 94°C for 30 sec, 58.7°C for 30 sec, and 72°C for
1 min, with a final elongation step at 72°C for 10 min. Products
were separated using a 1% agarose gel in 1 ⫻ Tris borate EDTA
buffer (TBE) and visualized by staining with ethidium bromide.
5ⴕ- and 3ⴕ-RACE. The complete V. litorea psbO transcript was
obtained by RACE using the GeneRacer Kit (Invitrogen) and
oligo d(T) and random hexamer priming. Amplification of the 5⬘
end was performed with the following primary primer sets: psbO
Rev1 and GeneRacer 5⬘ primer and psbO For1 and GeneRacer
3⬘ primer (Invitrogen). The full-length V. litorea psbO sequence
was used to generate homologous primers (psbO L5 and R8),
which amplified a larger (⬃963-bp) psbO fragment. A list of all
primers used can be found in Table S2.
Northern Blotting. The procedures for Northern Max (formalde-
hyde-based system for Northern blots; Ambion) were followed
for Northern blot analysis of the psbO transcripts using 4 ␮g of
DNAse-treated total RNA for each sample. RNA Millennium
Size Markers (Ambion) were run in an adjacent gel lane to
estimate transcript size. RNA was transferred to Hybond-n ⫹
nylon membranes (Amersham Biosciences) using the Nytran
SuPerCharge Turboblotter (Schleicher & Schuell) and the
Northern Max 20 ⫻ SSC transfer buffer, and was UV crosslinked. The psbO probe was prepared using the Rediprime II
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
random prime labeling system (Amersham Biosciences) and
[32P] dCTP (50 ␮Ci). Prehybridization was performed with
ULTRAhyb (Ambion) preheated to 42°C for 1 to 2 h before
hybridizing overnight at 42°C. Following hybridization, the membrane was washed with 2 ⫻ SSC/0.1% SDS for 5 min at room
temperature, followed by two washes in 0.1 ⫻ SSC, 0.1% SDS for
15 min at 42°C. The blot was exposed to x-ray film (Image Plus;
Diagnostic Imaging, Inc.) at ⫺80°C for 5 days before manual
development.
Genome Walking. Algal and sea slug egg genomic DNA was
isolated using DNAzol extra strength (Molecular Research
Center, Inc.). The egg DNA was further purified on a CsCl
gradient to remove inhibiting mucopolysaccharides (3). All
DNA (2.5 ␮g) was restriction digested with 80 U of MscI, PvuII,
or SspI overnight at 37°C; precipitated; and concentrated to 50
ng/␮l in deionized H2O. Genome walking was performed following the method detailed in Clontech’s Genome Walking Kit.
Briefly, 0.2 ␮g of restriction-digested DNA, 47.5 pmol modified
adapter (T ⫹ N mixed 1:1), and 3 U of T4 DNA ligase were
mixed and incubated at 16°C overnight. Adapter-ligated DNA
was precipitated and used in sequential inverse PCR reactions.
Gene-specific primers and adapter primers (Table S2) were used
in four sequential nested amplifications starting with PsbO GW
3⬘-3. Amplified products were band isolated using the Qiagen
Gel Extraction Kit and cloned into pGEM-TEZ vector (Promega), plated on LB agar plates containing 50 ␮g ml⫺1 Amp, and
grown overnight at 37°C. Inserts were amplified with Sp6 and T7
primers and analyzed by gel electrophoresis. Amplified product
(5 ␮l) was mixed with 2 ␮l of ExoSap-IT (USB Corp.) to remove
residual dNTP and primers and was incubated for 15 min at 37°C
before inactivating at 80°C for 15 min. Samples were sequenced
by the University of Maine DNA Sequencing Facility.
Cloning and Sequence Analysis. Unless stated otherwise, the TOPO
TA Cloning Kit for Sequencing (Invitrogen) was used to clone
DNA fragments excised from 1% agarose gel slices by S.N.A.P.
(Invitrogen) column centrifugation. Plasmids were isolated using
the Qiaprep Miniprep Spin Kit (Qiagen), and inserts were
sequenced in both directions by the University of Maine DNA
Sequencing Facility. A minimum of two totally independent
PCR reactions and cloning events and three to six different
plasmids were sequenced for all PCR products reported here.
Consensus nucleotide sequences [manually identified after aligning with ClustalX (1)] and translated amino acid sequences were
used to search the GenBank database (www.ncbi.nlm.nih.gov/
BLAST/).
Phylogenetic Analysis of psbO (MSP). The psbO phylogeny was
constructed using maximum parsimony in PAUP 4.0b10 (4). Maximum parsimony heuristic searches included 1,000 replications of
RANDOM addition and tree bisection-reconnection. Sets of
equally parsimonious trees were summarized using strict consensus.
Bootstrapping (5) was implemented in PAUP using 1,000 replicates
of heuristic searches with SIMPLE addition sequence and tree
bisection-reconnection. Sources for the sequences used are as
follows: green lineage: Spinacia oleracea (S00415), Oryza sativa
(NP㛭001043134), Chlamydomonas reinhardtii (CAA32053), Volvox
carteri (AAD55562), Bigelowiella natans (AAP79149), and Euglena
gracilis (BAA03529); red lineage: Karenia brevis (AAM77464),
Isochrysis galbana (AAM77466), Heterocapsa triquerta
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(AAM77465), Guillardia theta (ABD51936), Porphyra yezoensis
(AAW33888), Cyanidioschyzon merolae (BAD36767), Phaeodactylum tricornutum (AAO43192), Thalassiosira pseudonana (thaps1/
scaffold㛭39:162585–163984 from genome.jgi-psf.org/), Heterosigma
akashiwo (AAN11311), and Vaucheria litorea (DQ514337); mollusc: Elysia chlorotica (EU621882); glaucophyta: Cyanophora paradoxa (CAH04962); and prokaryotes: Nostoc punctiforme PCC
73102 (ZP㛭00111456), Trichodesmium erythraeum (YP㛭723422),
Thermosynechococcus elongatus BP-1 (BAC07996), Crocosphaera
watsonii WH 8501 (ZP00515383), Cyanothece sp. ATCC 51142
(AAF13997), Synechococcus sp. WH 8102 (CAE06818), and
Gloeobacter violaceus PCC 7421 (BAC91632).
mtDNA Sequencing. After 3 months in culture in the absence of
any algae, the sea slugs produced eggs and these were used for
DNA extraction. Universal primers (6, 7) were used to amplify
fragments of the mitochondrial rrnL and cox1 regions, and cob
primers were designed based on published mollusc sequences
(ref. 8; Table S3). Standard PCR mixtures contained 10 mM
Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.5 ␮M each primer,
template DNA (30–40 ng), and Taq Polymerase (1 U; New
England Biolabs) in a final volume of 50 ␮l. They were subjected
to an initial denaturation cycle at 94°C for 2 min; 25 cycles at
94°C for 30 sec; annealing at 42°C for rrnL, 51°C for cob, and
48°C for cox1, each for 30 sec; and then extension at 72°C for 1
min. All PCR products were separated in a 1% agarose gel, and
fragments were isolated using the QIAquick Gel Extraction Kit
(Qiagen). Fragments were ligated using the pGEM-T Easy
Vector system (Promega) and transformed into NEB (New
England Biolabs) 5␣ competent cells. Three to five colonies of
each cloned PCR product were selected, and the cloned fragments were sequenced using T7 and SP6 universal primers.
Following identification of primers that yielded known mitochondrial products from sea slug egg DNA, combinations of
these primers (Table S3) were used to generate longer products
to amplify the entire circular mtDNA. The mitochondrial gene
order of the mollusc Roboastra europaea was used as a guide (10).
Two cox1 primers were used with two cob primers in four
different combinations of separate PCR reactions. These reac1. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX
windows interface: Flexible strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Res 24:4876 – 4882.
2. Tohri A, et al. (2002) Comparison of the structure of the extrinsic 33 kDa protein from
different organisms. Plant Cell Physiol 43:429 – 439.
3. Rumpho ME, Mujer CV, Andrews DL, Manhart JR, Pierce SK (1994) Extraction of DNA
from mucilaginous tissues of a sea slug (Elysia chlorotica). BioTechniques 17:1097–
1101.
4. Swofford DL, et al. (2001) Bias in phylogenetic estimation and its relevance to the
choice between parsimony and likelihood methods. Syst Biol 50:525–539.
5. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791.
6. Palumbi S, et al. (1991) The Simple Fool’s Guide to PCR (Department of Zoology,
University of Hawaii, Honolulu).
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
tions yielded two products of approximately 7,000bp each. The
long PCR products were directly sequenced by primer walking.
The two cob primers were also used in combination with the two
rrnL primers, and the resulting fragments were directly sequenced following gel extraction. The remainder of the genome
was obtained in the same manner using a combination of two
rrnL and cox1 primers (see Table S3). For long PCR reactions,
the Phusion High-Fidelity PCR Kit (New England Biolabs) was
used and PCR mixtures were subjected to an initial denaturation
at 98°C for 30 sec, 35 cycles of denaturation at 98°C for 20 sec,
annealing for 20 sec, and extension at 72°C for 5 min. The
annealing temperature was adjusted in accordance with the
melting temperature (Tm) of the primer pairs. The resulting
fragments were extracted from an agarose gel as described
previously and sequenced by primer walking on both strands.
The gene for ATPase8 was annotated by alignment with other
opisthobranch ATPase8 genes, including Aplysia californica
(NC㛭005827), Roboastra europaea (NC㛭004321), and Pupa strigosa (AB028237), using CLUSTAL X (1). Two trnS genes that
were not identified by tRNA-SE Scan or DOGMA programs
were identified by their ability to form characteristic secondary
structures. Both nucleotide and amino acid sequence alignments
were used to define the start and stop codons for each gene.
Analysis of the mtDNA for HGT. The E. chlorotica mitochondrial
nucleotide windows (500 nt each) were analyzed by compiling a
database of 1,429 RefSeq (11) complete mtDNAs (animals,
heterokonts, green plants and algae, red algae, alveolates, and
fungi) for homologous segments to each of the windows using
Washington University (WU)-BLAST. Each window was then
aligned along with its homologues using MUSCLE (12); inferred
maximum likelihood topologies were then determined using
PhyML (13). In the case of translated ORFs, a database was
assembled of 24,722 RefSeq (11) proteins from the mitochondria
of 24 species spanning animals (including molluscs), heterokonts, green plants and algae, fungi, alveolates and slime
molds, and six bacteria. Then, each ORF was analyzed by
searching for homologues, aligning, and inferring trees (using the
same set of tools as for the nucleotide windows, adjusting the
parameters appropriately for amino acid sequences).
7. Folmer O, Black M, Hoeh R, Lutz R, Vrijenhoek R (1994) DNA primers for amplification
of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.
Mol Mar Biol Biotechol 3:294 –299.
8. Merritt TJS, et al. (1998) Universal cytochrome b primers facilitate intraspecific studies
in molluscan taxa. Mol Mar Biol Biotechnol 3:7–11.
10. Grande C, Templado J, Cervera L, Zardoya R (2002) The complete mitochondrial
genome of the nudibranch Roboastra europaea (Mollusca: Gastropoda) supports the
monophyly of opisthobranchs. Mol Biol Evol 19:1672–1685.
11. Pruitt KD, Tatusova T, Maglott DR (2007) NCBI reference sequences (RefSeq): A curated
non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids
Res 35:D61–D65.
12. Edgar RC (2004) MUSCLE: A multiple sequence alignment method with reduced time
and space complexity. BMC Bioinformatics 5:113.
13. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large
phylogenies by maximum likelihood. Syst Biol 52:696 –704.
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Fig. S1. Schematic illustrating the evolutionary origin of secondary plastids in V. litorea and tertiary plastids in E. chlorotica. The drawing highlights the red
algal secondary endosymbiotic origin of V. litorea. Four membranes surround the algal plastids as a result of the two endosymbiotic events, with the outermost
membrane being continuous with the nuclear envelope. The bottom panel with two sea slug digestive epithelial cells illustrates that only two membranes, the
typical plastid double envelope, are typically seen around the plastids in the sea slug. The plastids are colored red in the drawing to reflect the red algal origin.
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Fig. S2. Nucleic acid sequence comparison of psbO from V. litorea cDNA and genomic DNA, E. chlorotica DNA, and E. chlorotica egg DNA. An initial 452-bp
fragment was obtained from V. litorea by RT-PCR, and the complete cDNA for psbO was obtained by 5⬘ and 3⬘ RACE. This sequence was used to design primers
that amplified a ⬃963-bp fragment from DNA or cDNA of both organisms. The psbO gene does not contain an intron. The black dots identify identical base pairs
in all four sequences beginning with the start codon, and the dashes indicate that the sequence is not available for comparison. Base pair numbering is according
to the V. litorea cDNA sequence.
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
4 of 13
Fig. S3. Maximum parsimony tree based on amino acid sequences of 25 psbO-encoded MSPs. Strict consensus of nine trees (1,530 steps, consistency index ⫽
0.650, retention index ⫽ 0.576) using maximum parsimony in PAUP 4.0b10 (1, 2). Numbers are shown above branches for all boot strap values ⬎90. Weighted
branches are indicative of branches with boot strap support values ⬎70. For a complete list of sources, see SI Methods.
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Fig. S4. Nucleic acid sequence comparison of the psbO 3⬘ flanking region of V. litorea and E. chlorotica DNA. Genome walking using a nested gene-specific
primer with an adapter-specific primer yielded 3⬘ psbO flanking sequence data from both V. litorea and E. chlorotica. The sequences were identical for the first
81bp corresponding to the 3⬘ end of the psbO gene and ending with the stop codon (bold text). This sequence was followed by the highly diverged sequence
corresponding to the 3⬘ untranslated flanking region in each organism.
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Fig. S5. Distribution of E. chlorotica G ⫹ C content over the sliding window of 500 nucleotides with overlaps of 200 nucleotides. The values were found to be
uniformly distributed across the windows with a linear trend described by the model y ⫽ 0.0026x ⫹ 36.25. The average G ⫹ C content of the mtDNA of E. chlorotica
was 36.19%.
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Fig. S6.
Maximum likelihood phylogenetic tree of cytochrome b. The tree was inferred using PhyML (13) with 100 bootstrap replicates.
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Movie S1. Young sea slug sucking plastids out of Vaucheria litorea filaments. A juvenile sea slug is observed feeding on filaments of the heterokont alga
V. litorea. There is an obligate requirement at this stage for plastid acquisition for continued development to the adult stage. This is fulfilled by juveniles
puncturing the siphonaceous filaments and sucking out the cellular contents. Only the plastids are retained by the sea slug in cells lining the digestive epithelium.
Movie S1 (MOV)
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Movie S2. Mature “solar-powered” sea slug Elysia chlorotica. An adult sea slug is observed feeding on the heterokont alga V. litorea. Algal chloroplasts are
retained in the digestive epithelium in an endosymbiotic association yielding an emerald green “solar-powered” sea slug. The sea slug can sustain itself for its
entire life-span of about 10 months photoautotrophically requiring only light and air as a source of carbon dioxide. Adult animals range from about 1.5 to a
maximum of 6 cm in length.
Movie S2 (MOV)
Rumpho et al. www.pnas.org/cgi/content/short/0804968105
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Table S1. Complete listing of Vaucheria litorea chloroplast genes by category
Protein-encoding genes
Photosynthesis
Photosystem I
139
Photosystem II
18
Chlorophyll biosynthesis
Cytochrome
ATP synthase
Rubisco
Transcription/translation/
replication
RNA polymerase
Translation factors
Replication helicase
Ribosomal proteins
Small subunits
Large subunits
Miscellaneous proteins
Maintenance
Transport
Amino acid biosynthesis
Other proteins
Conserved ORFs
Unidentified ORFs
RNA-encoding genes
Ribosomal RNAs
Transfer RNAs
10
4
9
8
3
psaA, psaB, psaC, psaD*, psaE, psaF, psaI, psaJ, psaL,
psaM
psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ,
psbK, psbL, psbN, psbT, psbV, psbX, psbY, psbZ, psb28
chlB‡, chlI, chlL‡, chlN‡
petA, petB, petD, petF, petG, petJ§, petL, petM, petN
atpA, atpB, atpD, atpE, atpF, atpG, atpH, atpI
rbcL, rbcS, cfxQ
4
2
1
rpoA, rpoB, rpoC1, rpoC2
tufA, tsf¶
dnaB
18
rps1§, rps2, rps3, rps4, rps5, rps7, rps8, rps9, rps10, rps11,
rps12, rps13, rps14, rps16, rps17, rps18, rps19, rps20
rpl1, rpl2, rpl3, rpl4, rpl5, rpl6, rpl9‡, rpl11, rpl12, rpl13,
rpl14, rpl16, rpl18, rpl19, rpl20, rpl21†, rpl22, rpl23,
rpl24, rpl27, rpl29, rpl31, rpl32, rpl33, rpl34, rpl35,
rpl36
27
4
5
2
8
14
2
35
6 (3⫻2)
29
clpC, dnaK, ftsH, groEL
secA, secY, sufB, sufC, tatC
ilvB§, ilvH§
acpP储, acsF§, ccsA, ccs1, ftrC§, ycf17 (hlip§), thiG, thiS
ycf3, ycf4, ycf12, ycf19‡, ycf33, ycf36§, ycf37‡, ycf39,
ycf41, ycf42, ycf54§, ycf60‡, ycf65†§, ycf66†
rrl, rrs, rrf
trnA(ugc)X2, trnC(gca), trnD(guc), trnE(uuc), trnF(gaa),
trnfM(cau), trnG(gcc), trnG(ucc), trnH(gug), trnI(cau),
trnI(gau)X2, trnK(uuu), trnL(uaa)**, trnL(uag),
trnM(cau), trnN(guu), trnP(ugg), trnQ(uug), trnR(acg),
trnR(ccg), trnR(ucu), trnS(gcu), trnS(uga), trnT(ugu),
trnV(uac), trnW(cca), trnY(gua)
*Genes not found in Streptophyta or Chlorophyta chloroplast genomes are bold; those found in Chlorophyta but not embryophytes (land plants) are
single-underlined.
†Gene found in streptophytes but not chlorophytes.
‡Gene not found in other published heterokont chloroplast genomes (including Odontella sinensis, Phaeodactylum tricornutum, Thalassiosira pseudonana, and
Heterosigma akashiwo).
§Gene found only in Heterosigma akashiwo.
¶Gene found only in Phaeodactylum tricornutum.
储Gene found in Odontella sinensis, Phaeodactylum tricornutum, and Heterosigma akashiwo but not in Thalassiosira pseudonana.
**Gene-containing intron.
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Table S2. Primers for psbO*
Probe
PCR and RT-PCR
psbO R
psbO L2
psbO L5
psbO R8
5⬘ RACE
psbO Rev1
GeneRacer RNA oligo (Invitrogen)
3⬘ RACE
psbO For1
Oligo d(T) (Invitrogen)
Genome Walking
PsbO GW 3⬘-1
PsbO GW 3⬘-2
PsbO GW 3⬘-3
PsbO GW 3⬘-4
Adapter Primer 1
Adapter Primer 2
Genome Walker AdapterT
Genome Walker AdapterN
Product size
Sequence (5⬘ to 3⬘)
452bp
RCC DCG KCC YTT SGG RTC MAG GAA
ARG GGH WSH GGY YTB GCV AAC
GAA GGT CCC ATC TGC TTT GGT C
ATT CGC TCT CAA GCC TTC CAT AG
963bp
CCA CGT CCT TTG GGG TCA AGG
AAG GGA AGN GGT TTG GCC AAC AG
GGGGAGATTGGAGGAGTCTTTGTTTCG
GGAGACACTGATATGGGCTCTAAAGTCC
CACCTTGTACGGGATTGACGGCTCTTTCG
GAAAGACGGGATTGATTATGCTGCCACTAC
GTAATACGACTCACTATAGGGC
ACTATAGGGCACGCGTGGT
GTAATACGACTCACTATAGGGCACGCGTGGTC
GACGGCCCGGGCTGGT
(P)ACCAGCCC(L)
*All primers were synthesized by Integrated DNA Technologies, Inc. unless stated otherwise. L, forward primer, R, reverse primer.
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Table S3. Primer pairs for Elysia chlorotica mitochondrial genome*
Primer
A. Initial primers
RrnL (6)†
Cox1 (7)
Cob (8)
B. Final primer
combinations
Primer
Cobp3
Cox1f1
Cobp4
16SarL
16SbrH
Cox1f2
Primer sequence
Gene amplified
F: GAAAAAAGACGAGAAGACCC
R: GGGTCTTCTCGTCTTTTTTC
F: GGTCAACAAATCATAAAGATATTGG
R: TAAACTTCAGGGTGACCAAAAAATCA
F: TGTGGRGCNACYGTWATYACTAA
R: AANAGGAARTAYCAYTCNGGYTG
rrnL
Primer sequence
TGTGGRGCNACYGTWATYACTAA
GGTCAACAAATCATAAAGATATTGG
AANAGGAARTAYCAYTCNGGYTG
CGCCTGTTTAACAAAAACAT
CCGGTCTGAACTCAGATCACGT
TAAACTTCAGGGTGACCAAAAAATCA
cox1
cob
Regions amplified
Part of cob, coxII, ATP8, ATP6, rrnS, nad3, nad4, coxIII, nad2, part of coxI
Part of cob, nad4L, nad1, nad5, nad6, part of rrnL
Part of rrnL, part of cox1
*All primers were synthesized by Integrated DNA Technologies, Inc.
†Number in parentheses refers to supplementary reference number. F, forward primer, R, reverse primer.
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