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Copyright 0 1997 by the Genetics Society of America
The Salmon SmaI Family of Short Interspersed Repetitive Elements
(SINEs): Interspecific and Intraspecific Variation of the Insertion
of SINEs
in the Genomes of Chum and Pink Salmon
Nobuyoshi Takasaki*, Toshifumi Yam&*, Mitsuhiro Hamada*,
Linda Parkt and Norihiro Okada*
*Faculty of Bioscience and Biotechnology, Tokyo Institute of
Technology, Midori-ku, Yokohama 226, Japan, and tNorthwest
Fisheries Science Center, Coastal Zone and Estuarine Studies
Division, Seattle, Washington 981 12-2097
Manuscript received August 16, 1996 Accepted for publication
February 10, 1997
ABSTRACT The genomes of chum salmon and pink salmon contain a
family of short interspersed repetitive
elements (SINEs), designated the salmon SmaI family. It is
restricted to these two species, a distribution that suggests that
this SINE family might have been generated in their common
ancestor. When insertions of the SmaI SINEs at 10 orthologous loci
of these species were analyzed, however, it was found that there
were no shared insertion sites between chum and pink salmon.
Furthermore, at six loci where SmaI SINEs have been
species-specifically inserted in chum salmon, insertions of SINEs
were polymorphic among populations of chum salmon. By contrast, at
four loci where SmaI SINEs had been species- specifically inserted
in pink salmon, the SINEs were fixed among all populations of pink
salmon. The interspecific and intraspecific variation of the SmaI
SINEs cannot be explained by the assumption that the SmaI family
was amplified in a common ancestor of these two species. To
interpret these observations, we propose several possible models,
including introgression and the horizontal transfer of SINEs from "
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pink salmon to chum salmon during evolution.
S HORT interspersed repetitive elements (SINEs; SINGER 1982)
have been isolated from the genomes of many multicellular organisms
from invertebrates to mammals (OKADA 1991a,b; OHSHIMA et al. 1993;
OKADA and OHSHIMA 1995 and references therein), and also from
plants (MOCHIZUKI et al. 1992; YOSHIOKA et al. 1993; DERAGON et al.
1994). Generally, particular SINEs can be unique to a particular
taxonomic rank, for exam- ple, a family, a genus or a few species,
although old SINEs that were widely distributed in mammalian ge-
nomes were recently characterized (for review, see SMIT 1996).
SINEs can be divided into two classes according to their origins.
One class of SINEs is derived from the 7SL RNA (WEINER 1980; ULLU
and TSCHUDI 1984) in the signal recognition particle (SRP) , which
is involved in secretion of polypeptides during protein biosynthe-
sis. The primate Alu family and the rodent type 1 (B l ) family
belong to this class (for review, see SCHMID and MARAIA 1992;
DEININGER and BATZER 1993). The other class of SINEs originated
from specific tRNAs. It ap- pears that all SINEs other than the
primate Alu and rodent B1 SINEs are members of the second class of
SINEs (OKADA 1991a,b; OKADA and OHSHIMA 1995).
Since all SINEs include internal promoters for RNA polymerase
111 within their sequences, they can be tran- scribed independently
of the flanking sequences that surround them. The presence of
flanking direct repeats
Corresponding author: Norihiro Okada, Faculty of Bioscience and
Biotechnology, Tokyo Institute of Technology, 4259 Nagatsutaiho,
Midori-ku, Yokohama 226, Japan. E-mail: [email protected]
Genetics 146: 369-380 (May, 1997)
at the 5' and 3' ends of SINEs, together with a poly(A)- tail at
the 3' end, suggests that their amplification oc- curs by an
RNA-intermediated process. Thus, SINEs are believed to be amplified
by a process of retroposition, whereby RNA transcripts of SINEs are
reverse tran- scribed and reinserted at various sites in the genome
(JAGADEESWAREN et al. 1981; SINGER 1982; ROGERS 1985). By contrast
to DNA transposable elements, which can often be excised quite
precisely, SINEs ap- pear to be inserted irreversibly, and, thus,
they can be used as effective evolutionary and phylogenetic markers
(OKADA 1991b).
The genus Oncorhynchus consists of eight major spe- cies: chum
salmon (Oncorhynchus keta), pink salmon (0. gorbuscha), kokanee
(sockeye salmon; 0. nerka) , chinook salmon (0. tshaurytscha), coho
salmon (0. kzsutch), masu salmon (0. masou), steelhead (0. mykiss)
and cutthroat trout (0. tmtta), all of which live in the Pacific
Ocean (SMITH and STEARLEY 1989). These species have complex life
histories and interesting global distributions. Numer- ous studies
have examined the phylogenetic relation- ships among the species in
this genus, and a consensus exists for most species (UTTER et al.
1973; FERCUSON and FLEMING 1983; THOMAS et al. 1986; SMITH and
STEARLEY 1989; PHILLIPS et al. 1992; SHEDLOCK et al. 1992; MURATA
et al. 1993; for review see STEARLEY and SMITH, 1993). The
exception to this is the relationship among pink, chum and kokanee
salmon: two possible relationships are shown in Figure 1.
Analyses of morphology (STEARLEY and SMITH 1993),
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370
(4
N. Takasaki et al.
genomes exclusively (MURATA et nl. 1993, 1996). This designation
as sister species is not conclusive, however, since no SINEs that
were inserted at orthologous loci in both chum and pink salmon have
been isolated. In the present study, we analyzed in greater detail
the distribu- tion of members of the SmnI family of SINEs in the
genomes of chum and pink salmon, and we demon- strated that the
SmaI family is a unique family, in that it exhibits extensive
interspecific variation for the presence or the absence of SINEs in
the genomes of these fish. These features of the SmaI family of
SINEs suggest the possibility that this family of SINEs was
transferred hori- zontally or was transferred through introgression
from the genome of pink salmon to that of chum salmon during
evolution
7 chum salmon
1 -kokanee
I kokanee L pink salmon 1
chum salmon FIGURE 1.-Possible phylogenetic relationships
among
pink, chum and kokanee salmon. The relationships in a were
obtained from analyses of RFLP of mitochondrial DNA (THOMAS et al.
1986) and of life history (HOAR 1958). The relationships in b were
obtained from analyses of morphology (STEARLEY and SMITH 1993),
allozymes (UTTER et al. 1973) and the sequences of ribosomal DNA
(PHILLIPS et al. 1992) and of mitochondrial control regions
(SHEDLOCK et al. 1992).
allozymes (UTTER et al. 1973) and the sequences of ribosomal DNA
(PHILLIPS et al. 1992) and of mitochon- drial control regions
(SHEDLOCK et al. 1992) suggested a sister relationship between pink
salmon and kokanee (Figure 1b). Alternatively, a sister
relationship between chum and pink salmon (Figure la) was proposed
from analyses of RFLP of mitochondrial DNA (THOMAS et al. 1986) and
of life history (HOAR 1958).
In an attempt to elucidate the possible roles of SINEs in the
evolution of salmonids, we characterized three different families
of SINEs: the salmon SmaI family, the charr FokI family and the
salmonid HpaI family (&DO et al. 1991). The salmonid HpuI
family of repeats appears to be present in all species in the
family Salmonidae (KOISHI and OKADA 1991; DO et al. 1994,1995).
Using insertions of the HpaI SINEs as temporal markers, our group
looked at the phylogenetic relationships among seven species in the
genus Oncorhynchus (KIDO et al. 1991; MURATA et al. 1993, 1996).
Our phylogenetic tree is consistent with the consensus deduced from
various taxonomic studies. With regard to the phylogenetic rela-
tionships among three species described above, our work has
suggested that chum and pink salmon are sister spe- cies because
the salmon SmaI family is present in their
MATERIALS AND METHODS
DNA samples: Individuals from each of the three species of
salmon were sampled from several locations, as shown in Table 1.
Total genomic DNA of each species was extracted by the method of
BIJN and STAFFORD (1976) for large-scale preparation to establish
the genomic library. For the analysis of populations, DNA was
extracted from samples of individual fish as follows. One-half gram
of liver or a whole fry was ho- mogenized on ice in TNE solution,
which contained 10 mM Tris-HC1 (pH 8.0), 100 m~ NaCl and 1 mM EDTA.
Lysis buffer, which contained 500 pg/ml proteinase K, 2% sodium
dodecyl sulfate (SDS), 10 mM Tris-HC1 (pH 8.0), 150 mM NaCl and 10
mM EDTA, then was added to the solution, with incubation at 50" for
2 to 3 hr. DNA was extracted with phenol and chloroform, washed
with chloroform and isoamyl alcohol and collected by ethanol
precipitation.
Construction and screening of genomic libraries, subclon- ing
and sequencing: Total genomic DNA from chum salmon, pink salmon and
kokanee was separately digested with EcoRI for construction of a
genomic library for each species. Digests were size fractionated by
sucrose gradient (10 to 40%, w/v) centrifugation. DNA fragments of
2 to 4 kilobases were ligated with AgtlO arms (Stratagene, LaJolla,
CA) and then packaged in vitro. Scrcening was performed with an
[a-"PIGTP-labeled T7 transcript of cloned DNA, designated R-2, that
contained the tRNA-related region of the SmaI family as probe.
Hybrid- ization was allowed to proceed at 42" overnight in a
solution of 50% (v/v) formamide, 6X SSC (SSC is 0.15 M NaCI, 0.015
M trisodium citrate, pH 7), 1% (w/v) SDS in a final volume of 10
ml. Washing was performed in 2X SSC plus 1% SDS at 55" for 80 min.
Positive phage clones were isolated and their inserts were
subcloned into pUC18 or pUC19. The inserts then were sequenced with
primers that corresponded to or were complementary to the consensus
sequence for the SmaI family.
Dot blotting and hybridization: For dot-blot hybridization,
genomic DNA from each of the three species (20 ng to 10.24 pg) or
linearized plasmid DNA (100 pg to 51.2 ng) was ad- justed to 10.24
pg by the addition of mouse genomic DNA as a carrier. After
denauration in 0.25 NaOH, each sample of DNA was blotted onto a
GeneScreen Plus membrane (Du Pont NEN Products, Boston) with a
dot-blotting apparatus (model DP-96; Advantec, Tokyo). The membrane
was neutral- ized in a solution of 0.5 M Tris-HCI (pH 7.0) and 1 M
NaCl and then it was dried. Hybridization was performed with the
R-2 probe described above and was allowed to proceed under the same
conditions as the screening. Washing was performed first in 2~ SSC
at room temperature for 10 min with constant agitation, and then i
n 2X SSC plus 1% SDS at 55" for 60
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371 Unique SINEs in Chum and Pink Salmon
TABLE 1
Fish species analyzed
Family Genus Species Common name Geographic source
Salmonidae Oncorhynchus keta Chum salmon Sea of Okhotsk The Amur
river in Khabarovsk, Russia The Pacific coast of North America The
Chitose River in Hokkaido, Japan The Tokachi River in Hokkaido,
Japan
The Chenega Creek in Alaska, United States The Duck River in
Alaska, United States Prince William Sound in Alaska, United
The Nishibetsu River in Hokkaido, Japan The Hyoutsu River in
Hokkaido, Japan The Tor0 River in Hokkaido, Japan The Syari River
in Hokkaido, Japan The Abashiri River in Hokkaido, Japan
gorbuscha Pink salmon Japan Sea
States
nerka adonis Kokanee Lake Shikotsu, Hokkaido, Japan
min with constant agitation and finally in 0.1X SSC at room
temperature with constant agitation.
Amplification by PCR When a unit of the family appeared to have
been integrated at a single locus within a genome, we synthesized
5' and 3' oligonucleotide primers (OligolOOO DNA synthesizer;
Beckman, Fullerton, CA). The sequences are shown in Table 2. The
reaction mixtures for amplification by PCR contained Tth buffer
(TOYOBO, Tokyo), 0.2 mM dNTPs (Pharmacia, Uppsala, Sweden), 100 ng
of each primer, 1 pg of genomic DNA and 2 units of Tth DNA
polymerase (TOYOBO) in a final volume of 100 p1. The thermal
cycling involved 30 repeats of denaturation at 93" for 1 min,
anneal- ing at a particular temperature (as shown in Table 2) for 1
min and extension at 72" for 1 min. The products of PCR were
analyzed by electophoresis in 2% (w/v) NuSieve GTG and 1% (w/v)
Seakem GTG agarose gels (FMC BioProducts, Rockland, ME).
The nucleotide sequence data reported in this article will
appear in the DDBJ database with the following accession numbers:
AB001877-ABO01881, AB001921-ABOO1923, and AB001964-AB001976.
RESULTS
Characterization of the sequences in the SmaI family of SINEs:
Previous studies in our laboratory showed
that the genomes of chum and pink salmon contain SINEs derived
from tRNA',!', designated the salmon SmuI family (MATSUMOTO et al.
1986; KOISHI and OKADA 1991). The sequences of two genomic clones
from chum salmon, namely, Sma (OK)-2 and Sma (OK)-3 (where OK
stands for 0. ketu) and of two genomic clones from pink salmon,
namely Sma (OC)-l05 and Sma (0G)-107 (where OG stands for 0.
gorbuscha) were reported in a previous article (Figure 2; MATSUMOTO
et al. 1986). Dot hybridization and experiments by PCR in a
previous study also demonstrated that this SINE family is confined
exclusively to the genomes of these two species (KIL>O et al.
1991).
To obtain more information about the SmaI family of SINEs, we
isolated additional clones that contained a SmuI SINE from the
genomic libraries of chum and pink salmon and determined their
sequences. Figure 2, a and b show a total of 15 sequences of SINEs
from chum and a total of 10 sequences of SINEs from pink salmon,
respectively. The consensus sequences de- duced from chum and pink
salmon were identical. In- terestingly, two subfamilies of the SmaI
SINE could be
TABLE 2
Primers for PCR and annealing temperatures
Annealing Locus 5' flanking 3' flanking temperature (")
5' 3' 3' 5' Sma-3 AGGCCCAGAAGCTAGGATAT CCAGTGTGACCACCTGATAC 54
Sma-4 AATCATGAGGAAAGTAGCCG CCCCATACAGAACTAAGACT 55 Sma-21
ATCCTCAGCTTAGGCAACAC ACCCCGTGGTACCCTTTTGA 59 Sma-68
TAGGCTTTAGGAGGGTTCGC CCAGTCTACTTTTGCGCACG 55 Sma-71
TGGCACCTAATTTGAGCCTG TGTCCTTCTCTTCTGTCTAC 59 Sma-80
CCTGTCACATATCGGCCTGT GCAATCAAATGGAGGATACT 55 Sma-105
CTTTCCAGCTCCAGGGTTGGTATTTT GACTTTACGTTCCAAACAGTAGTAC 56 Sma-153
ATGCGCCAACAGTGTGCCTT CTCCCTTTGCAAATGCATAC 60 Sma-171
AACACACGGCACAGGCGTAT TGATCGCAGATACAACGGAA 60 Sma-174
TGCACAACCACCCAACAAAT ACCTCAGTGACACACGTTAG 59
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372 N. Takasaki et nE.
a CONSENSUS
1 20 40 60 80
G G T C ~ ~ T A G C T ~ T ~ C ~ ~ ~ T A G ~ ~ G A ~ ~ C ~ C ~ C
~ T A C - G T A ~ ~ A ~ ~
Sma(0K) -2
-(OK) -3 Sma(0K) -4 SmaIOK) -21 Sma IOK) -39 Sma (OK) -41 Sma
(OK) -43 SIM (OK) -44 Sma (OK) -51
SmaIOK) -54 %(OK) -66 %(OK) -67 &(OK)-68 Sma(OK)-71
Sma(OK)-80
~..............................................-...............T.................-.......-..........
G] ................................ t .............................
c ....................................
ttcttggttgtcat
.........................................................................................................
tgacattgttaatgtgacaa
----..........................................................T.................-..................
gatataggcattgag ~ . . . . .
a......................................... .... ...........ct.
.................................. t g g t c c t t g t g ~ . .
..................................................
...-...-..T-..... .............................. ggaggcagtct
~.................................a.-........... .....
........-.T.. ..................................
atcaactggtctgcc
btaca1..-..**.*....................................................~.
..... ..a........-...... ............ a a a t t g a g t t a t g ~ t
.................................................... ..........T.
................................... ctgcgttcattgtac
~....................................-..-.....a ....
...........~..............a.t-........ .......... atgttctctgcct
............................................... ....
............C.................-.-................
actaacacaaaaaa
...................................................................................................
acatttattgt ..............................................
................ T............ ........................
catttgtttattaggc
.......................................................................................................
accttatatgttcaga
bta---..*.*..-...-..............-.-.--..--......................~................--.-................
100 120 140
CONSENSUS
'IGACTGTAA~TAM+AGCGIT7GCTAAAAGCOICTGCTATA"I!-A"I!-AT
SmaIOK)-2 SmaI0K)-3 slra (OK) -4 -(OK) -21 %(OK) -39 Sma(0K)
-41
Sma(0K) -43 SIM (OK) -44 Sma(OK)-51 Sma (OK) -54
Sma(OK)-66 SmaIOK) -67 %(OK) -68 Sma(OK)-71
Sma)OK) -80
..........
'.....................................--..-..tatattattatattattattattattattatatatatbt
..........
'......-..............................-...-..attattattatat~~~gttttctatggata
....................--.-..*-.-*-***............-...-.-atat attac
gtgtgctctaaaatatqgggtatgtcttgattctq a ..........
a.....................................
--..-..tattattaactgttgttcatcgaatgattaaagtttctaattgtgtgat
................................................-...-..tattattattattattaaactctgcaactgatttaaagtc~~
......................................... t. ............ t a t t ~
a c a t c g c a g g t g g g ~ ~ t g a c a c a c t a a g
................................................t...-..att t
gagcagtct
...
a...........-................................-...-..tatt~tatttcggaagataacacaaaaaca
La .......................................
.........--..-..atatatttt~gaa ........
t........................g..............-...-..attttatattattaatatagcgatttcaaactgaaattcattgtctttctcagtcaca
.......................................................
atattattata(att)atattattatattattattattagtat6(att)atattactqkgcagt
.......................................
r-.--....-...-..atatt~ttccaactcaaatgacacaaaagtattctattacc~atgtt~gggtaaagaat
..........................................
r.....-...-..tatatattattatctqqktgtagctcaggt
................................................-...-..tatattattategttttktctgttacttgaactctgtgaagcattta
................................................-...-..tattattattgaaaact~ctgacggca
FIGURE 2.-A compilation of sequences of members of the SmaI
family isolated from genomic library of chum salmon and pink
salmon, respectively. The general consensus sequence was deduced
from the alignment of 25 members of the SmuI family and it is shown
at the top of the alignment. The tRNA-related region of the family
is underlined. Nucleotides identical to those in the consensus
sequence are indicated by dots, and gaps introduced to maximize
alignment are indicated by bars. Direct repeats flanking the SmuI
SINE are boxed. a and b show the members of the SmuI family
isolated from the genomic libraries of chum salmon and pink salmon,
respectively. Sma (0G)-105 and Sma (0G)-107 formerly Sma (0G)-5 and
Sma (0G)-7, respectively isolated from pink salmon have been
described previously (KIDO et al. 1991 ).
observed in both species. The SmuIT subfamily contains a T at
position 63, and the SmuIC subfamily has a C at the same position.
The average sequence divergence calculated for the SmuIT subfamily
was 0.67% and that for the SmuIC subfamily was 0.43%, suggesting
that the SmnIT subfamily might be slightly older than the SmuIC
subfamily.
To estimate the number of copies of members of the SmuI family
in the genomes of chum and pink salmon, we performed the dot
hybridization using a T7 tran- script of the tRNA-related region of
the DNA clone, designated R-2, as the probe. As shown in Figure 3,
the intensity of the hybridization signal for chum was almost the
same as that for pink salmon. Moreover, 0.32 pg of DNA from chum or
pink salmon gave a spot of the same intensity as 12.5 ng of R-2
DNA. Assuming that the genome of salmon is 2 X IO9 base pairs (bp)
in length, we can infer that chum and pink salmon each
have 2.6 x lo4 copies of the SmuI SINE, judging from the
intensities of spots on the autoradiogram. In the case of kokanee,
which is the species of Oncorhynchus that is most closely related
to both chum and pink salmon in this genus, only a weak signal was
detected, suggesting that - 100 copies of the SmuI SINE-related
sequence might be present in the genome of kokanee. We isolated
seven DNA clones from a genomic library of kokanee using R-2 DNA as
the probe, and we found that they exhibited weak similarity to the
consensus se- quence of the SmuI SINEs (data not shown; see the
accompanying article by HAMADA et ul. 1997). These results confirm
our previous conclusion that the ampli- fication of the SmuI family
is confined to chum and pink salmon.
All the SmuI SINEs exhibit species-specific insertions in each
species and there were no shared insertion sites between the
genomes of chum and pink salmon: The
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Unique SlSEs in Chr~m and Pink Salmon 373
b CONSENSUS
1 20 40 60 BO
G G T ~ A ~ A G I T G G T A G A G C A T G G C G C I T G T A
S m a ( C G ) -105 Sma (E) -107 % ( ' X ) -144 S m a ( C G )
-145 S m a ( C G ) -152 Sma (E) -153 Sma(CG)-162 Sma ( C G ) -168 9
M ( C G ) -171 S m a ( C G ) -184
CCcaCCt........................................-............t........T......-.-............-....-........
cggaagtttccttctggt......c..............................+..+~...........~..~..~..T.......~~.................+......+.
gaattgttttttgtttac
------.....................................................a..C...................................
agcaaccactgc
~--------........c............................................~...................................
tcacaataggctta
........................................................................................................
catggtgccat ~......-........
...................................................................................
atgatctataagttgtqt................................................a.............C...................................
tcataaaccatc
.......................................................
a..........a..~................................... ag
..............................................................
T...... .............................
tttaatgtgga .........................................
tt...................~................................... 100 120
140
CONSENSUS n;ACPGTAAGPCGCm?T;GATAAAAGCGICII;-CTATA~A~-AT
Sma(E) -105
....................-..........--...-...............-..tattatattagataaaatttcagtttatgacgggatataa
Sm(CG)-107 ......................................... a . . . . .
. . . . . - . . t a t a t t a a a g t t t a c a t a c a c c t t g g
c c a t t t a a Sm(CG) -144 ............
~..-................-...................--.tattatatgattccatgtgtgttatctcatagttttgatgtcttcaccattat
Sma(oG)-145 .......................................................
tatt~gttgaagtcgaagtttacatacat~taggttggagtca w ( x ) - 1 5 2
................................-...............-..--~.22(at)agagagaggctggactgaggqct~ggcc
Sma(X)-153
................................-...............-.-----~tcactgagctcttcagtaatgccaatctactgccaatgtttgt
-(m)-162 .........................-.....-......+tagctatc
Sma(oG)-168
.......................................................
tattattattattattaatactg~~gacatgaacttagacattgtttcgctctctctcat
Sma(oG)-171 ..........................
a....+a...................t..tatatatt~tgtc W ( E ) - 1 8 4
...............-................-...................-..t~qtttttgtttttttaat=acaaa
FK:I'KF. ! ? . - C i ~ t ; n ~ /
distribution of members of the S~ntr I family of SINEs within
chum and pink salmon can be interpreted as an indication that the
Sn~nl family was generated and amplified in a common ancestor of
chum and pink salmon. To examine this hypothesis, we performed PCR
experiments to estimate the time of retroinsertion of each member
of the S?nd family of SINEs. M'e detcr- mined the .i'- and
3"flanking regions of each StnnI se- quence and synthesized two
primers that flanked the unit, as shown in Table 2. M'e then
performed the PCR using genomic DNA from chum, pink and kokancAe
salmon as templates. If the SrntrI SINEs had been gener- ated in a
common ancestor of chum and pink salmon,
wc would expect that SINEs would be commonly pres- ent at
orthologous loci in the genomes of these two species.
To our surprise, all the S m d SINEs at 10 genomic loci that we
examined exhibited species-specific inser- tions in each species
and there were no shared insertion sites betwcen thcsc hvo species
(Figures 4 and .5). For example, Figure 4a shows the results of PCR
for the Smtr-4 locus, isolatcd from the genomic library of chum
salmon. The primers amplified a DNA fragment of 310 bp whcn thc S m
I unit was present (a black arrowhead in Figure 4a) or a fragment
of 160 bp in the absence of thc SINOI unit (a white arrowhead in
Figure 4a). I n
10.24 5.12 2.56 1.28 0.64 0.32 0.16 0.08 0.04 0.02 pg
(51.2) (25.6) (12.8) (6.4) (3.2) (1.6) (0.8) (0.4) (0.2) (0.1)
ng
FKX.RF. 3.-Dot-hlot hybridization for the dctcrmination of the
numbers o f copies of . Y r r w l sequences in chum salmon, pink
salmon and kokanee. Dot hyhritlization experiments were performed
using I~~hc~lccl RNA that had heen transcribed by T i RNA
polymerase from t h e tRS .h" l ;md rcgion ol'Sma (OK)-!?. The
prohe was tlrsignatrtl R-2 (residues 1-68). The numhers indicated
ahow the photograph show the amount of' gcnomic DNA o f rach
sprcics t h a t was usrd. Thc nwnhcrs in parenthcses show the
amor~nts of the K-2 plasmid, which was used as the standard.
-
374 N. Takasaki e( nl.
sia: 310- I
FIGURE 4.-(:llar~tctc.1-iz;lrion 01' the t S m d SINES that were
species-specifically retroinserted in the genome of chum salmon.
Products of PCR were analyzed hy electrophoresis in agarose gels.
Loci of Smn-4 (a), Smn-3 (h), Smn-PI (c), Sma- 68 (d), Smn-71 (e)
and Smn-80 (0 contained members of the Smnl family of SlNEs that
had been specifically integrated into the genomes of chum salmon.
Black and white arrowheads indicate positions of DNA with and
without a unit of the SmnI SINE, respectively. Lengths of DNA
fragments are indicated in hase pairs.
this case, only the genome of chum contained the SmaI SINE at
this locus. Similarly, at the other five loci iso- lated from the
genomic library of chum, namely, Sma- 3, -21, -68, -71 and -80,
each member of the SmaI family of SINEs was found to be present
specifically only in the genome of chum (Figure 4, h-0. By
contrast, at four loci isolated from a genomic library of pink
salmon, namely, Smn-105, -153, -I 71 and -184, the SmaI SINEs were
found to be present specifically and exclu-
(a) Sma-105
S l o p 3 6 O P
1 2 3 4 5 1 2 3 4 5
(b) Sma-153
460) 210 P
: 4 420 r FIGURE 5.-Retroinsertion of Smd SINEs specific to
pink
salmon. Loci ol' Sma-IO5 (a), Sma-153 (h), Smn-I71 ( c ) and
Smn-184 (d) all contained memhers of the SmnI family of SINEs that
had been specifically integrated into the genomes of pink salmon.
Black and white arrowheads indicate positions of DNAwith and
without a unit of the Smnl SINE, respectively. Lengths of DNA are
indicated in base pairs.
sively in the genome of pink salmon (Figure 5, a-d). The
sequences at each Sma-105 locus in both chum salmon and kokanee
were determined, and they con- firmed the species-specific
retroposition in pink salmon (data not shown).
In all, we analyzed 23 genomic loci isolated from chum and pink
salmon as described above, but we failed to isolate any orthologous
loci at which SmaI SINEs were commonly found in both chum and pink
salmon (data for the remaining 13 loci were not shown). We
certainly did not expect such extensive interspecific variation of
insertion of the SmaI SINEs. Although our results do not
necessarily exclude the possibility that the SmaI family might have
been generated in a common ancestor of chum and pink salmon, they
do suggest a new scenario wherein the SmaI SINEs were amplified
independently in the respective lineages of chum salmon and pink
salmon after their divergence (see DISCUSSION).
The SmaI SINEs specific to chum are dimorphic among populations
of this species: At three loci (Sma- 3, -71 and -8O), the
insertions of SmaI SINEs were dimor-
-
Unique SINEs in Chum and Pink Salmon 375
1 2 3 4 5 6 7 8 9101112
B Sma-80
FIGURE &-The SINE unit that was specifically retroin- serted
in chum salmon appears to be polymorphic among populations of this
species. Loci of Sma-4 (a) and Sma-28 (b) contained or did not
contain members of the Smd family of SINEs that had been
specifically integrated into the genomes of 10 individual chum
salmon. Ten individual chum salmon were collected from the Amur
River in Khabarovsk, Russia, in 1993 (lanes 1-10). Black and white
arrowheads indicate positions of DNA with and without a unit of the
SmnI SINE, respectively. Lengths of DNA are shown in base
pairs.
phic in the individual chum salmon sampled from the Okhotsk Sea
(Figure 4b, e and 0. To examine whether the insertions of six SmaI
SINEs described in the previ- ous section were fixed or dimorphic
among each popu- lation of chum, we analyzed DNA from 10 individual
chum salmon from four regions: the Amur River in Khabarovsk,
Russia; a variety of locations on the Pacific coast of North
America; the Chitose River in Hokkaido, Japan; and the Tokachi
River in Hokkaido, Japan. As examples, we show in Figure 6, a and
b, respectively, the results of PCR with the Sma-4 locus and with
the Sma-80 locus for individual chum salmon from the Amur River. In
the case of the Sma-4 locus (Figure 6a), individuals were scored as
homozygous (+/+; lanes 2, 6 and IO), or heterozygous (+/-; lanes
1,4, 5 and 7) for the presence of the 310-bp band (+) or homozygous
(-/-; lanes 3,s and 9) for the presence of the 160-bp band (-). The
results indicated that the SmaI SINEs at the Sma-4 locus were
highly dimorphic among individu- als in the Amur River in
Khabarovsk, Russia. To confirm that orthologous loci were
faithfully amplified in these cases, we determined, for example,
sequences for the 160-bp band (-) in lanes 1 and 3, as shown in
Figure 7. In the case of the Sma-80 locus (Figure 6b), three of 10
individuals were scored as heterozygous (+/-; lanes 2 , s and 10)
and the other seven were scored as homozy- gous (-/-; lanes 1, 3,
4, 5, 6, 7 and 9). In this case,
the extent of fixation of the SmaI unit was low as com- pared
with that of the Sma-4 locus. The intraspecific polymorphism of the
SmaI SINEs at the Sma-4 locus and at the Sma-80 locus was also
observed among the other populations of chum, although the extent
of fixation differed among populations (see the columns labeled
Sma-4 and Sma-80 in Table 3). Moreover, all the SmaI SINEs at the
other four loci were polymorphic among the four populations of chum
that we examined. The genotypes, expected values and allele
frequencies of chum-specific loci were determined by PCR analysis,
and they are summarized in Table 3. Of these six loci, two, namely,
the Sma-68and Sma-3 loci, are of consider- able interest. In the
case of the Sma-68 locus, the fre- quency of insertions in the
population in the Amur River was 0.2, whereas the value for the
population from the Pacific coast of North America was 0.9,
revealing a significant difference in the extent of insertions be-
tween the two populations. In the case of the Sma-3 locus, there
were no insertions of the SmaI SINEs in the populations from the
Chitose and Tokachi Rivers in Hokkaido, Japan, whereas the
frequency of insertions in the population from the Pacific coast
reached 0.4. These results suggest the possibility that analyses of
the frequencies of insertions of the SmaI SINEs might be useful for
population studies of chum salmon (see DIS CUSSION).
The SmaI SINEs specific to pink salmon are fixed among
populations of this species: We collected sam- ples of pink salmon
from various locations in Japan and Alaska (United States), and we
assayed for the presence of SINE insertions at four
species-specific loci (Sma-I 71, - 184, -105 and -153). The results
of PCR demonstrated that these units were fixed in all the
populations of pink salmon examined (Figure 8 and data not shown).
Pink salmon have a rigid 2-yr anadromous life cycle and excep tions
to this pattern are exceedingly rare. The temporal separation
resulting from the life cycle of this species has actually produced
two genetically distinct lines, which spawn in even and in odd
years, respectively (GHARRETT d al. 1988). However, we found no
genetic variations among the four SmaI loci that were specific to
pink salmon between even- and odd-year populations of pink salmon
(data not shown). In contrast to the SmI SINEs that are specific to
chum, all SmaI SINEs specific to pink salmon that were examined
appear to be fixed among populations of this species.
DISCUSSION
The present study confirmed our previous conclusion that the
SmaI family of repeats is confined to the two species in the genus
Oncorhynchus, namely, chum and pinksalmon (&DO et al. 1991;
KOISHI and OKADA 1991). The number of copies of SmaI SINEs in the
genomes of the two species is almost identical. These two sets of
observations suggest that the SmaI SINEs were gener- ated in a
common ancestor of chum and pink salmon.
-
376 N. Takasaki et al.
Sma (OK) -4 m G A A G G -
TTTTGGGCCCTTTTTTAGACTTAAACATACCTCCTGTACCTCCTGTAATGTAGACCCTATGATCTGTGAACTGTACAT
TCTTGGGCCC-TTTTTAGACCTAAACA----------TAATGTAGACCCTATGATCTGTGAAC~TACAT
TTTTGGGCCC-TTTTTAGACCTAAACA----------TAAGACCCTATGATCTGTGAACTGTACAT
Amur-1 Amur-3 .........................
............................. Sma (OK)-4 G G A A C A G T T A A C T
T C T T G G T C T C A T T A T A C T C C T T C T G T A T T Amur-1
GGAATGTACAGTTAACATCTTGGTGTCATTATACTCATTATACTCCT----------------------------------------------------
Amur-3
GGAACAGTTAACATCTTGGTCATTATACTCCT----------------------------------------------------
.................................. Sma (OK)-4
CGATCCCCGGGACCACCCATACGTAGAATGTATGTATGCACACATGACTGTAATGTAGTCGCTTTGGATARRAGCGTCTGCT~T~CATATA
Amur-1 Amur-3
...........................................
...........................................
Amur-1 Sma (OKI-4
TTATTATATATATATTACAGTGTGCTCTAAAATATGGGGTATGTCTTGATTCTGA
Amur-3 """""""_ TACAGTGTGCTCTARRATAT
TACAGTGTGCTCTARRATAT """""""_ .................... FIGURE 7.-The
short DNA fragments were faithfully amplified by PCR from the
orthologous loci. Amur 1 and Amur 3
indicate the sequences of 160-bp fragment DNA in lanes 1 and 3
of Figure 6a. The sequence for Sma-4, which contains a unit of the
SmaI SINE, was shown at the top line. Gaps are indicated by bars,
and dots indicate identical nucleotides among these three
sequences. Primer sequences are underlined.
We were therefore surprised to find no shared insertion sites
between chum and pink salmon. The fact that we found the chum
salmon loci to be polymorphic for SINE insertions and that the pink
salmon loci were not only added to the mystery. Thus, at present,
we cannot conclude that the SmuI family was generated in the common
ancestor of the two species. We can, however, propose several
possible models to explain the interspe- cific and intraspecific
variation of the insertion of SmuI SINEs in chum and pink
salmon.
In general, a SINE is believed to be generated in the genome of
one individual and to spread into the population through sexual
reproduction and genetic
drift. If the two species diverged before a site became fixed
for the SINE insertion, that site might be polymor- phic between
the resulting species. If such were the case for chum and pink
salmon, and if the time required for fixation in the genome of chum
salmon were long because of a large population size, as illustrated
in Fig- ure 9a, there might be no shared insertion sites between
chum and pink, and SINEs might also be intraspecifi- cally
polymorphic in the genome of chum salmon. In this model, we
hypothesize that most SmuI SINEs were amplified in a common
ancestor of pink and chum. However, we recently demonstrated both
that many members of the HpuI family of repeats, another family
TABLE 3
Distribution of members of the SmaI family that are specific to
chum salmon
Population Locus Sma-3 (C) Smu-4 (C) Sma-21 (T) Sma-68 (T)
Sma-71 (C) Sma-80 (C)
The Amur River in Khabarovsk, Russia ++ 0 (0.625) (0.25) 3 (2.5)
(0.5) 1 (1.6) (0.4) 0 (0.4) (0.2) 0 (0.225) (0.15) 0 (0.2251
(0.15)
+- 5 (3.75) 4 (51 6 (4.8) 4 (3.2) 3 (2.55) 3 (2.55) " 5 (5.625)
(0.75) 3 (2.5) (0.5) 3 (3.6) (0.6) 6 (6.4) (0.8) 7 (7.225) (0.85) 7
('7.225) (0.85)
The Pacific coast of North America ++ 0 (1.6) (0.4) 4 (3.6)
(0.6) 3 (2.5) (0.5) 9 (8.1) (0.9) 0 (0.225) (0.15) 1 (1.225)
(0.35)
+- 8 (4.8) 4 (4.8) 4 (51 0 (1.8) 3 (2.55) 5 (4.55) " 2 (3.6)
(0.6) 2 (1.6) (0.4) 3 (2.5) (0.5) 1 (0.1) (0.1) 7 (7.225) (0.85) 4
(4.225) (0.65)
The Chitose River in Hokkaido, Japan ++ 0 IO) (0) 1 (0.9) (0.3)
3 (3.6) (0.6) 6 (6.4) (0.8) 0 (0.1) (0.1) 0 (1.2251 (0.35)
10 (10) (1) 5 (4.9) (0.7) 1 (1.6) (0.4) 0 (0.4) (0.2) 8 (8.1)
(0.9) 3 (4.2251 (0.65) +- 0 (0) 4 (2.1) 6 (4.8) 4 (3.2) 2 (1.8) 7
(4.55) "
The Tokachi River in Hokkaido, Japan ++ 0 (01 (0) 4 (3.025)
(0.55) 1 (2.5) (0.5) 0 (2.5) (0.5) 0 (0.225) (0.15) 0 (0.625)
(0.25)
10 (10) (1) 3 (2.025) (0.45) 4 (2.5) (0.5) 0 (2.5) (0.5) 7
17.225) (0.85) 5 (5.625) (0.75) +- 0 (0) 3 (4.95) 5 (5) 10 (5) 3
(2.55) 5 (3.75) "
Each genotype is followed by expected number in braces and
allele frequency in parentheses. The letters C and T after the name
of locus indicate members of the SmaIC subfamily and the SmaIT
subfamily inserted at each locus, respectively.
-
Unique SINEs in Chum and Pink Salmon 377
(s & e 8nnnnnnr-V p 9
< e 49
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 21 22 23 2425
26 27 28- Sma-171
380,
FIGURE %-The SINE unit that was spccifically retroinserted in
pink salmon appears to he fixed among populations from various
locations. Individual pink salmon were collected from the Cllenega
Creek (lanes 2-6) and the Duck River and Prince William Sound in
Alaska (lanes 7-11); and the Nishihetsu River (lanes 12-14), the
Hyoutsu River (lanes 15-17), the Toro River (lanes 18-20), the
Syari River (21-23) and the Ahashiri River (lanes 24-26) in
Hokkaido, Japan. Lengths of DNA are shown in base pairs. Black and
white arrowheads indicate positions of DNA with and without a unit
of the SmaI SINE, respectively.
of SINEs that is present in all species in the subfamily
Salmoninae ( I n 0 et ul. 1991), have been amplified species
specifically in chum and pink, and that they are all fixed among
each species (TAKASAKI et al. 1994, 1996). Therefore, we can
conclude that the period be- tween the divergence of these two
species and the pres- ent time was long enough for SINEs to have
become fixed among populations. In other words, the SINEs that
exhibit the intraspecific polymorphism in the ge- nome of chum
salmon are relatively young so that they were not those amplified
in a common ancestor of chum and pink salmon. Namely, the model
shown in Figure 9a can not explain consistently both data that we
obtained in the present study and those in previous studies.
An alternative scenario (Figure 9b) is a modification of the
first model. The active gene of the SmuI family of repeats was
generated in a common ancestor of the two species as was postulated
in the first model. In the lineage of chum salmon, however,
amplification events occurred successively until quite recently,
causing the intraspecific polymorphism of SINEs in chum. How- ever,
in the lineage of pink salmon, the amplification of SINEs ended at
an early stage after divergence, such that all the SINEs remained
fixed in all populations. To prove this hypothesis, we must isolate
and characterize a few SmaI SINEs that were amplified in a common
ances- tor of the two species and that are commonly present at
orthologous loci in chum and pink salmon.
The models described in Figure 9, c and d, involve horizontal
transfer or introgression of SINEs from one species to the other.
Horizontal gene transfer means the transfer of a gene from one
species to the other without sexual reproduction, whereas
introgression in- volves hybridization of two different species,
followed
by transfer of nuclear DNA or mtDNA. In these scenar- ios, the
SmuI SINEs were first amplified species specifi- cally in the
lineage of pink salmon, and all the units were fixed among the
populations of pink salmon, since the amplification was such an
ancient event that the SINEs were fixed in the populations.
Relatively recently during evolution, the SmaI SINEs were
transferred from pink to chum salmon through horizontal gene
transfer or introgression. The intraspecific polymorphism of SINEs
in chum can be explained by this recent transfer.
With respect to introgression, several individuals gen- erated
by hybridization between chum and pink salmon were isolated from
the Pacific ocean (K. NUMACHI, un- published results), so it is
possible that SINEs were transferred also through introgression.
Hybridization and gene introgression, which sometimes follow poly-
ploidization and generate alterations in the genetic constitution
of populations, are generally more wide- spread in plants than in
animals. However, the number of reported cases of introgression in
animals is steadily increasing (HARRISON 1990; ARNOLD 1992; SMITH
1992). SMITH (1992) and STEARLEY and SMITH (1993) suggested that
introgression of mtDNA occurred be- tween chum and pink salmon
during evolution on the basis of the incongruence between the
phylogenetic relationships deduced from the analysis of mtDNA (the
phylogenetic tree shown in Figure la; THOMAS et al. 1986;
GINATULINA et al. 1988) and that deduced from analysis of
morphology and allozymes (the phylogenetic tree shown in Figure 1
b; U-ITER et dl. 1973). Hybridiza- tion has generally been examined
by contrasting the phylogenies inferred from analyses of allozymes
and mtDNA because mtDNA is apparently more susceptible to
interspecific gene flow than is nuclear DNA (AUBERT and SOLICNAC
1990; RIESEBERC and SOLTIS 1991; Dow-
-
N. Takasaki et al.
(c) m
L f chum salmon chum salmon
horizontal transmission or
introgression
pink salmon pink salmon
t- kokanee kokanee
m + t + + t + chum salmon (a) kokanee
+t+
+ w + + pink salmon U pink salmon U 1 horizontal transmission or
introgression
kokanee I chum, salmon
FIGURE 9.-Possible models explaining the interspecific and
intraspecific variation of the SmaI family of SINEs. Black arrows
indicate the time of amplification of the SmaI SINEs. Square
brackets indicate the time required for the SINEs to be fixed
within a species. The phylogenetic trees shown in a-c reflect the
data obtained by analysis of mtDNA (THOMAS et ul. 1986; GINATULINA
et al. 1988) and of life history (HOAR 1958). The phylogenetic tree
shown in d reflects the data from analysis of morphology (STEARLEY
and SMITH 1993) and allozvmes (UTTER et al. 1973). White thin
arrows in c and d, respectively, indicate the direction of transfer
of members of the SmuI family. '
LING and DEMARAIS 1993). However, hybridization has not
prevented the introgession of nuclear DNA. In Daphnia, evidence of
nuclear introgression has been found together with evidence of the
introgression of mtDNA (TAYLOR and HEBERT 1992). In general, it is
difficult to detect the extensive introgression of nuclear DNA by
analysis of allozymes except in the case of hy- bridizing
populations in a hybrid zone. Studies with random amplified
polymorphic DNA (RAPD) markers have demonstrated examples of the
introgression of nuclear DNA (STEIT et ul. 1994). Moreover,
evidence of intergenomic introgression was recently reported from a
study of an unusual ribosomal DNA sequence in allo- polyploid
cottons (WENDEL et ul. 1995). For the reasons stated above and in
view of the existence of the chum- pink hybrids captured in the
wild, it appears possible that introgression of nuclear DNA, in
general, which could not be detected by analyses of allozymes, oc-
curred between chum salmon and pink salmon through hybridization
and, moreover, that the Smd family of repeats generated in the
lineage of pink salmon could have been transferred into the genome
of chum salmon. If such is the case, because of the fixation of the
SmuI SINEs among the populations of pink salmon
and the intraspecific polymorphism of the SmuI SINEs in chum
salmon, we can assume that the introgression of the SmuI family of
repeats occurred from pink salmon to chum salmon. This assumption
is consistent with the direction of introgression of mtDNA that
SMITH (1992) suggested on the basis of the sequence divergence of
mtDNA. At present, the possibilty of in- trogression allows us to
interpret all the phenomena related to the SmuI family that are
described in the present report without any incongruity.
If horizontal transfer or introgression of the Smd SINEs
occurred from pink to chum salmon, the sister relationship between
chum and pink salmon (Figures l a and 9c), which was assumed from
the presence of the SmuI SINEs in both species (MURATA et ul.
1993), must be reconsidered. In this case, a phylogenetic rela-
tionship in which pink salmon and kokanee have a sister
relationship (Figures l b and 9d) is also possible. Such a
relationship was suggested from analyses of osteologic data (SMITH
and STEARLEY 1989; STEARLEY and SMITH 1993) and allozymes (UTTER et
uZ. 1973). To elucidate the phylogenetic relationships of chum,
pink and ko- kanee salmon, more detailed studies are required.
In general, within the many SINE sequences of sev-
-
Unique SINEs in Chum and Pink Salmon 379
era1 families of SINEs examined to date, intraspecific
polymorphism of SINEs is very rare and most SINEs are found to be
fixed in all the populations of one species (TAKASAKI et al. 1994,
1996). On the basis of this feature of SINEs, we can reconstruct
phylogenetic relationships using SINE insertions (MURATA et al.
1993,1996). It has been reported, however, that the insertion of a
very few members of the Alu family in the human lineage is
polymorphic among human populations ( BATZER and DEININCER 1991;
BATZER et al. 1991, 1994; PERNA et al. 1992), and this polymorphism
might be useful for stud- ies of the structure of human
populations. In the pres- ent study, we found that insertion of the
SmaI SINEs was polymorphic among the populations of chum salmon.
Extensive intraspecific polymorphism of the SmaI SINEs in chum
salmon might also provide a useful and conve- nient method for the
determination of population structures of this species. In
paticular, the Sma-3 SINE was found in genomes of chum salmon from
the Amur River in Russia and the Pacific coast or North America,
whereas it was absent from genomes of chum salmon from Japanese
rivers. Since it is known that chum salmon return to their natal
rivers with greater reliabil- ity than other salmon species (GROOT
and MARGOLIS 1991), this kind of locus might be useful for
identifying the origin of chum salmon caught in the Pacific
ocean.
The authors are grateful to Drs. MINEO SANEYOSHI and SHIGEHIKO
URAWA for gifts of salmon samples. This work was supported by a
Grant-in-Aid for Specially Promoted Research from the Ministry of
Education, Science and Culture ofJapan.
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YOSHIOKA, Y., S. MATSU~KITO, S. KOIIMA, K. OHSl?IMA, N.
OKr\L>A