Distribution of the DNA transposon family, Pokey in the Daphnia pulex species complex
© Eagle and Crease. 2016
Received: 27 March 2016
Accepted: 13 June 2016
Published: 17 June 2016
The Pokey family of DNA transposons consists of two putatively autonomous groups, PokeyA and PokeyB, and two groups of Miniature Inverted-repeat Transposable Elements (MITEs), mPok1 and mPok2. This TE family is unusual as it inserts into a specific site in ribosomal (r)DNA, as well as other locations in Daphnia genomes. The goals of this study were to determine the distribution of the Pokey family in lineages of the Daphnia pulex species complex, and to test the hypothesis that unusally high PokeyA number in some isolates of Daphnia pulicaria is the result of recent transposition. To do this, we estimated the haploid number of Pokey, mPok, and rRNA genes in 45 isolates from five Daphnia lineages using quantitative PCR. We also cloned and sequenced partial copies of PokeyA from four isolates of D. pulicaria.
Haploid PokeyA and PokeyB number is generally less than 20 and tends to be higher outside rDNA in four lineages. Conversely, the number of both groups is much higher outside rDNA (~120) in D. arenata, and PokeyB is also somewhat higher inside rDNA. mPok1 was only detected in D. arenata. mPok2 occurs both outside (~30) and inside rDNA (~6) in D. arenata, but was rare (≤2) outside rDNA in the other four lineages. There is no correlation between Pokey and rRNA gene number (mean = 240 across lineages) in any lineage. Variation among cloned partial PokeyA sequences is significantly higher in isolates with high number compared to isolates with an average number.
The high Pokey number outside rDNA in D. arenata and inside rDNA in some D. pulicaria isolates is consistent with a recent increase in transposition rate. The D. pulicaria increase may have been triggered by insertion of PokeyA into a region of transcriptionally active rDNA. The expansion in D. arenata (thought to be of hybrid origin) may be a consequence of release from epigenetic repression following hybridization. Previous work found D. obtusa to be very different from the D. pulex complex; mean PokeyA is higher in rDNA (~75), rDNA array size is nearly twice as large (415), and the two are positively correlated. The predominance of Pokey in only one location could be explained by purifying selection against ectopic recombination between elements inside and outside rDNA.
KeywordsDaphnia Transposon Pokey Ribosomal DNA MITE
Transposable elements (TEs) are segments of DNA that can move around the genome. TEs are often detrimental to the host because they can interrupt gene function, their transposition has energy costs (e.g. the repair of double strand breaks), they can cause ectopic recombination , and the epigenetic mechanisms used to control them can shut down surrounding genes . Usually, DNA transposons must undergo horizontal transfer to remain active . However, Pokey, a class II cut-and-paste DNA transposon in the piggyBac superfamily  has been vertically inherited in the subgenus, Daphnia . Pokey inserts at TTAA sites and creates a target site duplication (TSD). It ranges in size from 4.5 to 10 kilobase pairs (kb); the length variation is due to repeat sequences at the 5′ end derived from the ribosomal intergenic spacer (IGS)  and/or the ribosomal internal transcribed spacer (ITS) . Pokey is the only DNA transposon known to insert in a specific location in ribosomal DNA (rDNA); other rDNA elements, such as R1 and R2, are non-Long Terminal Repeat (LTR) retrotransposons . Nevertheless, Kojima and Jurka  recently reported the discovery of class II Dada TEs in organisms as taxonomically diverse as fish, molluscs, annelids, and cladoceran crustaceans (Daphnia). Like Pokey, they show target-site specificity for particular multicopy RNA genes (small nuclear RNA, transfer RNA), encode a putatively cut-and-paste transposase, and create TSDs on insertion. However, unlike other elements that target multicopy RNA genes, Pokey also inserts into TTAA sites outside rDNA in the genomes of North American (NA) D. pulex  and NA D. pulicaria .
In eukaryotes, rDNA is a multigene family arranged in tandem arrays of units each consisting of the 18S, 5.8S, and 28S rRNA genes plus the ITS, the external transcribed spacer (ETS), and the IGS. The number of rDNA units per eukaryotic genome varies from less than 50 to over 25,000  and is usually much higher than the number required for viability . rDNA typically displays the phenomenon of concerted evolution, which means that intraspecific sequence divergence between rDNA copies is generally much lower than interspecific divergence . The mechanisms of concerted evolution are thought to be gene conversion and unequal crossing over , which can cause the number of rDNA units to fluctuate. Due to its multicopy nature and the presence of copies above the number required for viability, some TEs, such as R1, R2, and Pokey, have persisted in rDNA for very long periods of time  even though the homogenizing mechanisms of concerted evolution are expected to continually remove them. In addition, TEs inserted in rRNA genes render the genes non-functional, so selection is expected to favor loss of these elements .
Daphnia are freshwater cladoceran crustaceans with a worldwide distribution . Based on molecular analyses, the D. pulex species complex (subgenus Daphnia) is composed of eight lineages between which hybridization frequently occurs: North American (NA) D. pulex, D. melanica, D. middendorffiana, North American (NA) D. pulicaria, D. tenebrosa, European (EU) D. pulex, European (EU) D. pulicaria , and D. arenata . Although these analyses suggest that the North American and European populations of D. pulex and D. pulicaria should be classified as separate species, they have not been officially described as such . While most cladocerans reproduce by cyclical parthenogenesis (diapausing eggs are produced sexually and direct-developing eggs are produced apomictically), populations of obligate parthenogens (diapausing eggs are also produced apomictically) also occur in some lineages of this species complex .
Eagle and Crease  found that the total haploid number of Pokey in NA D. pulex and NA D. pulicaria is usually less than 20 copies, most of which are found outside rDNA. In addition, Pokey number in rDNA is not correlated with rRNA gene number. In contrast, LeRiche et al.  found Pokey number in another species in the same subgenus, Daphnia obtusa, to be as high as 154 (mean = 75), almost all of which are located inside rDNA. Moreover, Pokey number in rDNA is strongly correlated with rRNA gene number.
Both autonomous (encoding a functional transposase) and non-autonomous (the transposase gene is degraded or partially deleted) copies of Pokey have been found . Further, Miniature Inverted Repeat Transposable Elements (MITEs), which are very short (usually less than 600 nucleotides) , non-autonomous TEs have been observed in D. arenata . Overall, Elliott et al.  discovered four TEs in the D. arenata Pokey family: the original Pokey group discovered in D. pulex, now called PokeyA; a variant containing a transposase gene (complete or partial) that was initially seen in D. obtusa , now called PokeyB; a MITE with terminal inverted repeats (TIRs) similar to those of PokeyA, called mPok1, and a MITE with TIRs similar to those of PokeyB, called mPok2. The mPok elements average 760 base pairs (bp) in length . PokeyA and mPok1 have 16 bp imperfect TIRs, and PokeyB and mPok2 have 12 bp imperfect TIRs . Like PokeyA, PokeyB also contains an open reading frame (ORF) encoding a putative transposase and both ORFs contain an intron . Mean sequence divergence between copies of PokeyA and PokeyB containing a complete or partial transposase gene from D. arenata is 40 %, suggesting that the two TE groups did not diverge from one another recently.
Pokey and mPok number in the D. pulex complex
We used qPCR (Additional file 1: Table S2) to estimate the haploid number of the four TEs in the Pokey family (PokeyA, PokeyB, mPok1, mPok2) in the entire genome (tPokey, tmPok) and in rDNA (rPokey, rmPok) by comparing their PCR amplification rate to the PCR amplification rate of two single copy genes, Gtp (a member of the RAB subfamily of small GTPases) and Tif (a transcription initiation factor) , . We calculated the number of Pokey and mPok outside rDNA (gPokey, gmPok) by subtracting rPokey or rmPok from tPokey or tmPok. We also estimated the number of 18S and 28S rRNA genes in each isolate.
Haploid number of Pokey, mPok, and rRNA genes in isolates from the Daphnia pulex complex
NAc D. pulex
NAc D. pulicaria
EUc D. pulex
EUc D. pulicaria
112.5 to 440.5
176.0 to 460.0
248.0 to 332.5
97.5 to 339.5
341.0 to 391.5
119.5 to 452.5
234.5 to 576.5
264.5 to 330.5
87.5 to 418.0
341.0 to 354.5
0 to 14.5
3.5 to 48.0
0 to 1.0
1.0 to 10.5
1.0 to 1.5
0 to 1.0
5.0 to 20.0
0 to 1.5
0 to 22.0
0.0 to 8.0
0.0 to 10.5
52.5 to 63.5
0.0 to 3.0
12.5 to 15.0
2.0 to 6.0
5.0 to 9.0
54.5 to 61.0
3.5 to 7.0
5.0 to 8.0
7.0 to 8.5
0 to 2.0
0 to 1.0
25.0 to 38.5
0.5 to 1.5
2.0 to 2.0
The mean number of rPokeyA is lowest in D. arenata (Table 1, Fig. 3) and highest in NA D. pulicaria. The high mean in NA D. pulicaria is due to the unusually high number in two isolates (PC1.2, PC2.1), as previously described by Eagle and Crease . These isolates were chosen to determine whether the high number of rPokeyA could be explained by recent transposition (see below). rPokeyA is absent in three of the four D. arenata isolates, but is only absent in three of the other 41 isolates (Additional file 1: Table S3). This pattern is reversed for rPokeyB in which the mean number is highest in D. arenata (Table 1, Fig. 3), but the element is absent from rDNA in 35 of the other 41 isolates (Additional file 1: Table S3).
The MITEs, mPok1 and mPok2 are primarily found in D. arenata (Fig. 3). Indeed, the only MITE we detected in the other four lineages is gmPok2, but it is present in no more than 2 copies per haploid genome (Table 1), and absent in most isolates (Additional file 1: Table S3). mPok2 was detected both inside (mean = 6) and outside (mean = 32) rDNA in D. arenata (Table 1, Fig. 3), but mPok1 was only detected outside rDNA (mean = 7.5) (Table 1, Fig. 3).
rRNA gene number in the D. pulex complex
Correlation between haploid rPokey family and rRNA gene number in isolates from five lineages of the Daphnia pulex complex
NA D. pulex a
6.3 x 10 −16
Additional file 2: Figure S1
4.9 x 10 −9
Additional file 2: Figure S1
NA D. pulex
Additional file 2: Figure S2
Additional file 2: Figure S2
Variation in partial PokeyA sequences in North American D. pulicaria
Haploid number of Pokey, mPok, and rRNA genes in four isolates of North American Daphnia pulicaria
mean number of differences from consensusc
0 to 97
0 to 128d
0 to 6
12 to 78
Pokey family distribution in NA and EU D. pulex and D. pulicaria
In general, Pokey number is similar in four of the five lineages we examined; NA and EU D. pulex and NA and EU D. pulicaria. The mean number of PokeyA plus PokeyB is less than 20 and both TEs are primarily located outside of rDNA. Our PokeyA numbers (Table 1) are similar to estimates from Eagle and Crease  for NA D. pulex (mean rPokeyA = 2.1 and mean gPokeyA = 9.6 based on 43 isolates) and NA D. pulicaria (mean rPokeyA = 6.6 and mean gPokeyA = 9.5 based on 26 isolates). However, the NA D. pulicaria rPokeyA mean in the current study (17.6) is higher than the previous estimate because we only included five isolates, two of which have unusually high rPokeyA number, and were chosen for that reason.
Both the current and previous estimates of gPokeyA and gPokeyB are within the range of other DNA transposons found in the Daphnia genome sequence . For example, there are approximately 31 copies per Tc1/mariner family (217 copies from 7 families) and approximately 6 copies per hAT family (33 copies from 6 families). In contrast, the mean numbers of rPokeyA and rPokeyB (Table 1) tend to be low compared to the mean number of rDNA-specific non-LTR retroelements, R1 (34) and R2 (12) in D. melanogaster .
Overall, PokeyA outnumbers PokeyB, which could be due to a higher transposition rate, weaker purifying selection at the level of the host, and/or lower rates of deletion of PokeyA. Due to the fact that they both insert into the same TTAA site in rDNA, it seems unlikely that the strength of purifying selection against these two TEs at the level of the host, or their rate of deletion differs. Therefore, we suggest that the higher number of PokeyA may be due to a higher transposition rate, which could be tested using yeast excision assays.
Although Pokey number and distribution is similar in the four D. pulex and D. pulicaria lineages, it was necessary to set gPokeyA to 0 in six isolates because rPokeyA exceeds tPokeyA. The tPokeyA primer pair is located at the 5′ end of the transposase gene, and can only amplify autonomous PokeyA. Conversely, it was necessary to locate the rPokeyA forward primer near the 3' end of the gene, even though it was not possible to design primers in that region to target only rPokeyA or rmPok1. Consequently, the rPokeyA forward primer amplifies both autonomous and non-autonomous rPokeyA, as well as rmPok1. However, the higher number of rPokeyA compared to tPokeyA cannot be due to the presence of rmPok1 because no copies of mPok1 were detected in these isolates by end-point PCR using internal, mPok1-specific primers. Thus, the higher number of rPokeyA compared to tPokeyA in some isolates is most likely explained by copies of rPokeyA that did not amplify with the tPokeyA primers (Table 1, Fig. 3), but further study would be required to confirm this.
The number of mPok we observed in NA and EU D. pulex and NA and EU D. pulicaria is very low (0 to 2 haploid copies per isolate) compared to other piggyBac MITEs, for which numbers range from 50 to 2x104 in metazoans including nematodes, insects, and vertebrates . Indeed, the absence of any mPok1 in the 41 isolates we examined suggests that this MITE may not occur in these four lineages. In addition, the absence of mPok2 in rDNA and the low number outside rDNA (maximum of two copies) suggests that the activity of mPok2 is very low in these lineages.
Pokey family distribution in D. arenata
While mPok1 and mPok2 were both detected in D. arenata, the former was only detected outside rDNA and in low numbers suggesting its activity is low. The mean p-distance between copies of mPok1 and the mPok1 consensus sequence is only 2 % in D. arenata isolate AR1.1 (Additional file 2: Figure S3). This, along with the fact that it does not occur in the other lineages, suggests that mPok1 could be of recent origin. In contrast, mPok2 occurred both inside and outside rDNA, with a mean haploid number of genomic copies greater than 30. This is at the top of the range for gPokey in the other four lineages (Fig. 3), suggesting that mPok2 may be active in D. arenata. Recent activity is also consistent with a p-distance less than 3 % between the mPok2 consensus sequence and 75 % of the mPok2 in isolate AR1.1 (Additional file 2: Figure S3). However, the p-distance ranges from 4 to 22 % for the other copies of mPok2 (Additional file 2: Figure S3). This, and the fact that mPok2 is found in other lineages in the D. pulex complex, suggests that this MITE is not of recent origin . Further work is required to determine the distribution of mPok in the D. pulex complex, and in other species in the subgenus Daphnia.
The number of Pokey and mPok, except rPokeyA, is substantially higher in D. arenata than the other four lineages, resulting in a very different distribution (Fig. 3). Daphnia arenata is endemic to western Oregon  and may have diverged from NA D. pulex during the Pleistocene glaciation, during which it was isolated from other refugial populations south of the glaciers by the Cordilleran ice sheet . This restriction to a small geographic region could have resulted in a smaller effective population size, such that the fate of slightly deleterious mutations (such as some TE insertions) could have been primarily determined by genetic drift instead of purifying selection .
Another explanation for the high Pokey number in D. arenata is a much higher transposition rate compared to the other lineages. Such an increase could be the result of changes in Pokey, such as mutations in the transposase that increase transposition rate, or mutations in the terminal repeats that increase recognition by the transposase. These hypotheses could be tested using yeast excision, yeast one-hybrid, and/or electrophoretic mobility shift assays.
A third explanation for high Pokey number in D. arenata is a loss of epigenetic regulation by the host. The most well-known example of such a loss is the phenomenon of P-M hybrid dysgenesis in Drosophila, which occurs when a male from a strain with P-elements is mated to a female from a strain without P-elements . The female usually provides the epigenetic regulation, but if she does not have P-elements, then the paternal P-elements are able to transpose without regulation in the offspring , leading to effects such as male recombination, sterility, mutation, chromosomal aberrations, and nondisjunction .
Interspecific hybridization is also known to stimulate increased TE activity due to the disruption of host defenses, although this is not always the case (see Ungerer and Kawakami  for an example). Labrador et al.  found that the Osvaldo LTR retrotransposon has a higher transposition rate in hybrids of Drosophila buzzatii and Drosophila koepferae compared to non-hybrids. Similar increases in transposition rate in interspecific hybrids have been seen for other elements in rice and Drosophila (e.g. [27–31]).
Vergilino et al.  suggested that D. arenata may be a hybrid between NA D. pulex and D. tenebrosa or EU D. pulicaria because the position of D. arenata varies on phylogenetic trees generated from different nuclear loci [32–34]. NA D. pulex is likely the maternal parent based on analysis of mitochondrial DNA [13, 21]. If D. arenata does have a hybrid origin, release from host regulation could explain the relatively high number of Pokey and mPok2 and possibly the occurrence of mPok1. Further work is required to establish the distribution of mPok in other Daphnia species, determine the origin of mPok1, confirm the hybrid origin of D. arenata, and test the hypothesis that the high Pokey load in D. arenata is a consequence of increased transposition in a hybrid lineage. If this is the case, we would also expect the number of active TEs from other superfamilies to be higher in D. arenata compared to other lineages in the D. pulex complex, but this remains to be tested.
Distribution of Pokey in the subgenus, Daphnia
Our results are very different from those obtained for D. obtusa in which rPokey number is much higher (mean = 75 for PokeyA and 9 for PokeyB) and gPokey number is much lower (mean = 0.5 for PokeyA) . Moreover, rPokeyA is significantly positively correlated with 28S number in D. obtusa (R2 = 0.54). The mean haploid rRNA gene number in lineages of the D. pulex complex is 240, with values ranging from 90 to nearly 580. Conversely, the mean haploid number in 21 isolates of D. obtusa is 415, with values ranging from 180 to 960. LeRiche et al.  suggested that rPokeyA number could be higher in D. obtusa due to the larger size of its rDNA array, which may be a consequence of higher rates of sister chromatid exchange . A large rDNA array provides more Pokey insertion sites, and a high rate of sister chromatid exchange provides more opportunity for the element to increase via recombination, even if the transposition rate is low.
Although the number of Daphnia species whose Pokey distribution has been studied is modest, the emerging pattern is that these elements tend to be concentrated in one genomic location (inside or outside of rDNA) and occur in low number in the other. In the D. pulex complex, Pokey is concentrated outside rDNA and in D. obtusa it is concentrated inside rDNA. Moreover, with the exception of D. arenata, gPokey in the D. pulex complex (less than 30 copies) tends to be much lower than rPokey in D. obtusa (up to 154 copies). Even so, the lower number of gPokey in the D. pulex complex is consistent with the number of other DNA elements observed in the Daphnia genome sequence as mentioned above .
One explanation for the tendency of Pokey to be concentrated in only one genomic location is purifying selection against chromosomal rearrangements caused by ectopic recombination between copies inside and outside rDNA. The ectopic recombination model  suggests that TEs should accumulate in genomic regions of low recombination due to the deleterious effects of exchange between elements at non-homologous sites. Although we do not know the location of gPokey in Daphnia genomes, their relatively low number suggests that their accumulation is resisted by selection. In contrast, Eickbush  suggested that ectopic recombination between TEs inserted in rDNA may not have severe effects because the outcome is similar to the effects of concerted evolution. On the other hand, ectopic recombination between gPokey and rPokey could severely impact genome organization and result in strong purifying selection, such that Pokey is generally not able to persist in substantial numbers in both locations in the same genome. Daphnia arenata is a notable exception to this general pattern as PokeyB and mPok2 numbers are unusually high both inside and outside rDNA. If Pokey has been recently transposing in D. arenata, as suggested above, then there may not have been sufficient time for purifying selection to limit the expansion of Pokey in one or the other genomic location.
Variation among partial PokeyA sequences in North American D. pulicaria
Eagle and Crease  suggested that the transposition rate of rPokeyA may have recently increased in some populations of NA D. pulicaria because its number is unusually high in some isolates from populations PC1 and PC2. Sequence divergence between TE copies that have recently transposed is expected to be low as there has been little time for them to accumulate differences from their parent copy. Our results are consistent with this hypothesis as the sequences from isolates with high PokeyA number (PC1.2 and PC2.1) show significantly less deviation from their consensus sequence than do sequences from isolates with average PokeyA numbers in the same population (PC1.1 and PC2.2, Table 3). Moreover, all but two of the sequences encode a potentially functional transposase. However, we cannot rule out the possibility that recent recombination within the rDNA array has also played a role in this rPokeyA expansion.
A recent increase in transposition rate is often a consequence of an element’s ability to evade host silencing, which has been suggested to depend on the distribution of elements that occur in rDNA. For example, Eickbush et al.  found that rates of R2 transcription are higher when the elements are spread throughout the rDNA array than when they are clustered, and indirect evidence suggests that Pokey insertions are clustered in the rDNA of NA D. pulex . Eickbush et al.  concluded that the largest continuous block of uninserted rDNA units tend to be transcribed, while other units remain silent. However, when R2 insertions are spread across the rDNA array, or have recently transposed into the midst of a large continuous block of uninserted units, they are more likely to be transcribed. Similarly, it is possible that recent insertion of PokeyA into a large continuous block of uninserted rDNA units in a NA D. pulicaria individual (either by recombination or transposition) triggered an increase in PokeyA transcription. The hypothesis that transcription rate is higher in individuals with high PokeyA number could be tested using nuclear run-on transcription assays and Reverse Transcription-PCR . In addition, fiber-FISH assays could be used to determine the arrangement of Pokey within rDNA arrays. This technique has been used to show that Pokey is dispersed in isolate AR1.1 (Figure S11 in ).
The goals of this study were to determine the distribution of the Pokey family (PokeyA, PokeyB, mPok1 and mPok2) inside and outside rDNA in lineages of the D. pulex complex, and to test the hypothesis that rPokeyA expansion in NA D. pulicaria is the result of recent transposition. We found the distribution to be similar in four of the lineages; D. pulex and D. pulicaria from North America (NA) and Europe (EU) and in general, Pokey is more common outside than inside rDNA. PokeyA expansion in NA D. pulicaria rDNA appears to be recent and we suggest it could have been triggered by a change in rDNA distribution that reduced the host's ability to regulate Pokey transcription.
The Pokey family distribution in D. arenata is very different from the other four lineages. In particular, the mean number of both PokeyA and PokeyB outside rDNA is five to six times higher in D. arenata and the two types of mPok are nearly exclusive to this lineage. We suggest that the proliferation of Pokey and mPok in D. arenata may be a consequence of release from epigenetic repression as a result of interspecific hybridization, although a hybrid origin for this lineage requires confirmation.
The mean number of rRNA genes and rPokeyA is much larger in D. obtusa compared to the five D. pulex lineages. However, both PokeyA and PokeyB are absent or nearly so outside D. obtusa rDNA. Overall, these results suggest that Pokey primarily occupies only one or the other genomic location within a species. We suggest that this may be a consequence of purifying selection against ectopic recombination between elements in different genomic locations, which could cause severe genomic rearrangements.
Elliott et al.  suggested that Pokey originated as a genomic element and subsequently invaded Daphnia rDNA, which makes it unique compared to other DNA elements. Moreover, Pokey has been vertically inherited in the subgenus Daphnia since it diverged from the other Daphnia subgenera, Ctenodaphnia and Hyalodaphnia , which may have occurred as long ago as 100 million years . It seems likely that the persistence of Pokey in Daphnia over such a long period of time is at least partially due to the potential for copies from one genomic location to reinvade the other if it is lost . However, the factors that determine whether Pokey is primarily a genomic or an rDNA element within each species, and whether this configuration is stable over time are unknown and warrant further research.
Daphnia isolates and DNA extractions
We analyzed a total of 45 cyclically parthenogenetic Daphnia isolates from five lineages in the D. pulex complex (Fig. 1, Additional file 1: Table S1). Twenty-six of these isolates were NA D. pulex. One to three isolates were sampled from ten populations in the Midwest United States and southern Ontario. In addition, 19 isolates from the other lineages were included: four D. arenata, five NA D. pulicaria, eight EU D. pulex, and two EU D. pulicaria. Daphnia individuals were collected and clonally propagated (isolates) as described in Eagle and Crease . DNA was extracted from multiple individuals from each isolate using phenol:chloroform or the Aquagenomics kit (MultiTarget Pharmaceuticals LLC, Salt Lake City, Utah, USA) as in Eagle and Crease . DNA concentrations, which ranged from 13 to 4400 ng/μL (Additional file 1: Table S1), were measured using a NanoDrop® ND-8000 spectrophotometer (ThermoScientific).
Estimation of Pokey family and rRNA gene number
We used the SYBR green real-time qPCR and ΔCT relative quantification method as described in Eagle and Crease  to estimate the haploid number of each type of gene. CT, or cycle threshold, is the point at which the amplification curve crosses a set threshold. We estimated the number of the four TEs in the Pokey family (PokeyA, PokeyB, mPok1, and mPok2) in the entire genome (tPokey, tmPok) and in rDNA (rPokey, rmPok), as well as the number of 18S and 28S rRNA genes in all 45 Daphnia isolates.
Primer pairs used for qPCR of the Pokey family and rRNA genes in the Daphnia pulex complex
Amplicon Size (bp)
18S 1864 F
5'-ccg cgt gac agt gag caa ta
18S 1913 R
5'-ccc agg aca tct aag ggc atc
28S 2508 F
5'-gcc tgc tcg tac cga tat cc
28S 2558 R
5'-cta gag gct gtt cac ctt gga ga
Pok 3720 F
5'-cag ttc aaa gag tgg ctc ctc c
Pok 3770 R
5'-cgg gtc tga ctt ctg gtt cg
PokB 1375 F
5'-aaa gag gag aag aat gac ccg g
PokB 1425 R
5'-tca gaa gag cac cct acc ttg g
mPok1 524 F
5'-gga cac cta tgg cgg gat t
mPok1 574 R
5'-cgc tga ggt ctg tcg gga
mPok2 714 F
5'-ggt cag ttg gct ccg aca a
mPok2 759 R
5'-aa ccc ttt atc gac gcg aag a
T. Elliott, unpublished
rPokeyA + rmPok1
Pok 6561 F
5'-caa tcg aat ccg acc atc g
28S 3073 R
5'-tga cga ggc att tgg cta cc
PokB 4283 F
5'-aat ttc agt caa gca cgg cc
28S 3073 R
5'-tga cga ggc att tgg cta cc
mPok 714 F
5'-ggt cag ttg gct ccg aca a
28S 3073 R
5'-tga cga ggc att tgg cta cc
5'-gac atc atc ctg gtt ggc ct
5'-aac gtc agc ctt ggc atc tt
5'-tat tca gca tgg aga gac ggc
5'-gat gtc gac tga cgc tgg aa
qPCRs had a final volume of 20 μL and contained 1X PerfeCTa SYBR Green FastMix, ROX (Quanta BioSciences) and 0.25 μM of each primer. Reactions were run on a StepOne Plus instrument (Applied Biosystems) using the following protocol: 95 °C for 10 min; followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Melt curves were run by heating the amplicons at 95 °C for 15 s, decreasing the temperature to 60 °C for 1 min, then heating to 95 °C in 0.3 °C increments. The DNA template was either serial dilutions of amplicons generated from plasmid DNA (standard curve plates) or 10 ng of genomic DNA (experimental plates). Samples were run in triplicate with the exception of the tmPok1 and rmPok2 primer pairs for samples in which previous end-point PCR produced no amplicon (Additional file 3). In such cases, only a single reaction was done. Negative control reactions were run for each primer pair on every standard curve plate. Negative control reactions were also run for each primer pair on 15 out of the 22 experimental plates (Additional file 3). The StepOne Software (Applied Biosystems) set the baseline for each reaction. The threshold was set based on amplicon size as in Eagle and Crease  (Table 4). A threshold of 0.2 was used for 50 bp amplicons, and the threshold for larger amplicons was determined using the formula, 0.2 x 2^[1-(50/length in bp)].
CT values were used to estimate gene number, using the formula: 2-ΔCT where ΔCT is ([CT x PAE multicopy gene] - [CT x PAE single-copy gene]), as in Eagle and Crease . If the standard deviation for mean CT for a gene was greater than 0.2, then the most extreme value was omitted (Additional file 1: Table S2) from further analysis. If there was no clear outlier among the three CT values, then all three values were used. The two or three CT values for each multicopy gene were compared to the two or three CT values for both single copy genes producing 8 to 18 estimates of gene number. The Tif-Gtp ratio was 1.66 in isolate PX1.2 and 1.42 in isolate PX2.2; these two isolates likely have three copies of the Tif gene instead of the expected two. Therefore, the estimates of gene number using Tif as the single copy gene were multiplied by 1.5 in these isolates. In addition, both tmPok1 and rPokeyA + rmPok1 amplified in one isolate, AR3.1. To determine if the estimate of rPokeyA + rmPok1 included rPokeyA, rmPok1 or both, this isolate was screened for rmPok1 with end-point PCR (Additional file 3), but no amplicon was detected.
gPokey/gmPok number was calculated as [tPokey – rPokey or tmPok – rmPok]. In six isolates (EPX1.1, EPX1.2, EPX2.1, EPX2.2, PC2.1, and PX6.3), the calculation of gPokeyA produced a negative number in which case gPokeyA was set to zero, and tPokeyA was assumed to be the same as rPokeyA.
We used paired t-tests for means, two-sample t-tests assuming unequal variances with the sequential Bonferroni correction , and linear regressions (Microsoft Excel, Richmond, Washington, USA) to examine the relationship between 18S and 28S rRNA gene number. We also estimated the correlation between rRNA genes and Pokey family number using regression analysis.
Sequencing partial PokeyA copies from North American D. pulicaria
We cloned and sequenced partial PokeyA elements from four NA D. pulicaria isolates from two populations. From each population, an isolate with high number of PokeyA (PC1.2, PC2.1) and an isolate with average number of PokeyA (PC1.1, PC2.2) were selected (Table 3). The average number of PokeyA for NA D. pulicaria was based on the results of Eagle and Crease . Partial PokeyA sequences were amplified from approximately 50 ng of genomic DNA in a 25 μL reaction, which contained 1X Phusion HF Buffer (NEB), 0.4 mM dNTPs, 0.08 μM each of the Pok-2904 F (5’ ggg aca tag gtg tcc cgg) and Pok-6178R (5’ tcg acc agg ggt ctt tcc agt c) primers, and 1 units of Phusion DNA Polymerase. Reactions were run on either a PTC-100 Thermocycler (MJ Research) or a T100 Thermocycler (Bio-Rad Laboratories, Inc.) using the following protocol: 3 min initial denaturation at 98 °C; 35 cycles of 10 s denaturation at 98 °C, 30 s annealing at 55 °C, and 2 min elongation at 72 °C; followed by a final elongation at 72 °C for 10 min. The 3.3 kb amplicons were verified by electrophoresis on a 1 % TAE agarose gel, stained with GelRed™ Nucleic Acid Gel Stain (Biotium, Inc.) and visualized under UV light. The amplicons were then cloned into the plasmid, pSC-B-amp/kan using the StrataClone Blunt PCR Cloning Kit (Agilent Technologies) as per the manufacturer’s protocol with a few modifications. The modifications were as follows: 2.5 μL of PCR product and 0.5 μL of StrataClone Blunt Vector Mix were used in the ligation; 25-50 μL of StrataClone SoloPack cells was used for the transformation; an incubation of 30–45 min was used because the insert was large (3.3 kb); 500 μL of Terrific Broth was used instead of 250 μL of LB medium; and 50 μL and 400 μL were plated instead of 5 μL and 100 μL.
Positive colonies (white) were triple-streaked onto new plates and grown overnight. One of the three streaks was added with a toothpick to 10 μL of water and incubated at 99.9 °C for 3 min. One microliter was amplified in a 25 μL reaction with two sets of primers: Pok-2904 F with Pok-3811R (5’ ccg tgt tac ttc acc atc gg) to generate a 910 bp fragment; and Pok-3720 F (5’ cag ttc aaa gag tgg ctc c) with Pok-4488R (5’ gaa tcg ctc gcg agt cat gg) to generate a 770 bp fragment. Reactions contained 1X GenScript buffer (GenScript USA Inc.), 0.04 mM dNTPs, 0.04 μM of each primer, and 0.5 units of GenScript DNA polymerase (GenScript USA Inc.). Reactions were run on either a PTC-100 Thermocycler (MJ Research) or a T100 Thermocycler (Bio-Rad Laboratories, Inc.) using the following protocol: 2 min initial denaturation at 94 °C; 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 55 °C, and 1 min extension at 72 °C; followed by a final elongation at 72 °C for 5 min. Amplicons from 12 clones per isolate were sequenced with the primers Pok-2904 F and Pok-3720 F in 12 μL reactions. The reactions contained 0.3 μL BigDye® Terminator v3.1 Ready Reaction Mix (Applied Biosystems), 0.4X Sequencing Buffer (Applied Biosystems), 0.83 μM of primer, and 2 μL of amplicon. Sequencing reactions were run for 1 min at 96 °C followed by 30 cycles of a 20 s denaturation at 96 °C, a 20 s annealing at 55 °C, and a 4 min extension at 60 °C. Reactions were resolved on an ABI 3730 DNA Analyzer (Applied Biosystems) by the Genomics Facility at the University of Guelph. Sequences were analyzed using CLC Main Workbench software (CLC Bio). We used MEGA 5.0  to generate a 1608 bp alignment containing 48 sequences from the four NA D. pulicaria isolates. The maximum composite likelihood model was used to estimate a matrix of pairwise sequence divergence from which a Neighbor-Joining dendrogram was generated. Gaps were excluded from this analysis using the pairwise deletion option. In addition, a consensus sequence was generated for each isolate from the 12 sequences. The number of differences between each sequence and its consensus was determined in MEGA. Gaps were included in this analysis, but consecutive gaps were coded as single nucleotide changes. ANOVA was used to test for heterogeneity among the mean number of differences in each of the four isolates. A post hoc Tukey’s HSD test was used to compare the means in the average and high isolates from the same population.
18S, 18S rRNA gene; 28S, 28S rRNA gene; bp, base pair; CT, threshold cycle; ETS, external transcribed spacer; EU, European; gmPok1, mPok1 found outside rDNA; gmPok2, mPok2 found outside rDNA; gPokey, Pokey elements found outside rDNA; gPokeyA, mPok1 found outside rDNA; gPokeyB, mPok2 found outside rDNA; IGS, intergenic spacer; ITS, internal transcribed spacer; kb, kilo base pairs; LTR, long terminal repeats; MITE, Miniature Inverted-repeat Transposable Element; NA, North American; PAE, percent amplification efficiency; qPCR, quantitative Polymerase Chain Reaction; rDNA, ribosomal DNA; rmPok1, mPok1 in the 28S gene; rmPok2, mPok2 in the 28S gene; rPokey, Pokey elements found in the 28S gene; rPokeyA, mPok1 in the 28S rRNA gene; rPokeyB, mPok2 in the 28S rRNA gene; rRNA, ribosomal RNA; TE, transposable element; TIR, terminal inverted repeat; tmPok1, mPok1 in the genome; tmPok2, mPok2 in the genome; tPokey, Pokey elements found in the genome; tPokeyA, PokeyA in the genome; tPokeyB, PokeyB in the genome; TSD, target site duplication.
We thank Andrew Beckerman, Melania Cristescu, France Dufresne, Michael Lynch, Seanna McTaggart and Julia Reger for providing some of the Daphnia samples, and the Genomics Facility at the University of Guelph for assistance with the qPCR and sequencing of plasmid clones. Comments from Tyler Elliott and two anonymous reviewers greatly improved the manuscript.
This research was funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to TJC. SHCE was partially supported by an Ontario Graduate Studies Science and Technology Scholarship. The funding agencies had no involvement in the design of the study, the collection, analysis, and interpretation of data, or writing the manuscript.
Availability of data and material
The sequence dataset supporting the conclusions of this article is available in GenBank, with accession numbers KX354776 to KX354823. All other data supporting the conclusions of this article are included within the article and its additional files.
SHCE participated in the design of the study, carried out the molecular genetic analyses, analyzed the data, and drafted the manuscript. TJC conceived of the study and participated in its design, and helped to draft the manuscript. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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