Diversity of transposable elements and repeats in a 600 kb region of the fly Calliphora vicina

  • Bárbara Negre1, 2Email author and

    Affiliated with

    • Pat Simpson1

      Affiliated with

      Mobile DNA20134:13

      DOI: 10.1186/1759-8753-4-13

      Received: 19 December 2012

      Accepted: 5 March 2013

      Published: 3 April 2013

      Abstract

      Background

      Transposable elements (TEs) are a very dynamic component of eukaryotic genomes with important implications (e.g., in evolution) and applications (e.g., as transgenic tools). They also represent a major challenge for the assembly and annotation of genomic sequences. However, they are still largely unknown in non-model species.

      Results

      Here, we have annotated the repeats and transposable elements present in a 600 kb genomic region of the blowfly Calliphora vicina (Diptera: Calliphoridae) which contains most of the achaete-scute gene complex of this species. This is the largest genomic region to be sequenced and analyzed in higher flies outside the Drosophila genus. We find that the repeat content spans at least 24% of the sequence. It includes 318 insertions classified as 3 LTR retrotransposons, 21 LINEs, 14 cut-and-paste DNA transposons, 4 helitrons and 33 unclassified repeats.

      Conclusions

      This is the most detailed description of TEs and repeats in the Calliphoridae to date. This contribution not only adds to our knowledge about TE evolution but will also help in the annotation of repeats on Dipteran whole genome sequences.

      Keywords

      Calliphora vicina Diptera Helitrons Horizontal transfer Repeats Retrotransposons Transposable elements Transposons

      Background

      Transposable elements (TEs) are a common feature in eukaryotic genomes and constitute a major player in many of the processes that shape the genome and control gene expression [1, 2]. TEs can occupy a significant but highly variable portion of the genome. For example, at least 46% of the initial sequence of the human genome was recognized as TEs, and this percentage is probably higher than 50% when other repeats are considered [3]. Amongst species of Diptera sequenced to date the repeat content of euchromatic regions varies from only 6% in Drosophila melanogaster[4] to 16% in Anopheles gambiae[5], 28% in Culex quinquefasciatus[6] and 47% in Aedes aegypti[7]. TEs and other repeats pose a big challenge for the assembly and annotation of genomic sequences. Although many programs have been developed for the detection of TEs, most are difficult to use and their performance has not been properly tested [8]. They mostly rely on similarity to annotated elements or on the detection of known structures. The availability of well-annotated elements is thus of great help for their automatic detection and annotation.

      Detailed description of TEs is not only important for genome annotation but also essential for understanding genome structure, function and evolution. The presence of TEs can affect gene structure and gene expression in several ways: from local effects on the expression of adjacent genes, to global effects such as the generation of large chromosome rearrangements or transpositions [2, 9]. TEs are also important contributors to evolutionary adaptation [10]. Furthermore they contain historical information about the genome, and can be used as a sort of paleontological record. They provide a tool with which to solve evolutionary relationships and classification of species [1114]. Moreover, TEs have a direct application for transgenesis where they can be used as insertion vectors. Knowledge of the TE repertoire of a target species has important implications for vector choice, as it will influence the stability of the transgenes. These methods are not only valuable research tools but are also being developed for the control of pest species in the wild [15].

      TEs are divided into two main classes according to their structure and mechanism of transposition [16]. Class I elements, also called retrotransposons, transpose by reverse transcription of an RNA intermediate (DNA-RNA-DNA) mediated by a retrotranscriptase, whereas Class II elements transpose directly from DNA to DNA. Within each of these classes, TEs are further subdivided mainly on the basis of the structural features of their sequences [17, 18]. Class I elements are divided into two main types: with or without Long Terminal Repeats (LTR elements and non-LTR elements), such as LINEs and SINEs. Class II elements include cut-and-paste DNA transposons, rolling-circle DNA transposons (Helitrons) and self-synthesizing DNA transposons (Polintons). Cut-and-paste DNA transposons are characterized by the presence of Terminal Inverted Repeats (TIRs) flanking a transposase that catalyses the transposition reaction. Helitrons have been classified as Class II-DNA transposons that use a “rolling circle” (RC) mode of transposition [19].

      The Calliphoridae is a monophyletic family of calyptrate Muscomorpha (Diptera). These flies are of economic importance as a cause of myiasis in humans and animals, and as vectors of pathogens causing dysentery and other diseases. The larvae of most species are scavengers of carrion and dung, and fulfil an important ecological function in the decomposition of animal remains. They are among the first colonizers of cadavers, making them particularly useful for forensic entomology, predominantly to establish a minimum time since death, or minimum post-mortem interval [20]. This method usually relies on morphological identification of samples collected on corpses. Distinguishing between closely related taxa, such as Calliphora vicina and Calliphora vomitoria, can be a difficult process with major implications for post-mortem interval estimation. Mitochondrial sequences, like COI and COII, have been used for species identification but in some cases an overlap between intra- and inter-specific variability renders this method unreliable [20]. Measures to develop a TE-based simple and efficient marker system for the identification of forensically important carrion flies are currently being developed [21]. However, the retrotransposon landscape of carrion fly genomes remains largely unknown.

      Here we provide an inventory and classification of the TEs and other repeats found in 6 BAC clones covering most of the Achaete-Scute Complex of C. vicina. These sequences include the genes achaete (ac), scute (sc) and lethal of scute (l’sc) which are highly regulated and surrounded by large regulatory regions. It is a 600 kb euchromatic region of the 750 Mb C. vicina genome. We have identified 318 insertions classified as 75 different repeats; 42 of which are TEs and 33 are unclassified repeats. Elements which are complete or present at high copy number are described in some detail. We also discuss probable cases of horizontal transfer.

      Results

      We have analysed a 613,063 bp genomic region within which we have identified a total of 318 TE insertions and repeats (Table 1, Table 2, Figure 1, Additional file 1, Additional file 2). The repeats have been classified and are described below.
      Table 1

      Transposable elements and other repeats identified in C. vicina

       

      Family

      Name

      Copies

      Total size

      Average

      Longest

      Class I (retrotransposons)

      LTR/Gypsy-CsRn1

      CsRn1_Cv1

      1

      4294

      4294

      4294

      LTR/Gypsy-Osvaldo

      Cv_Isis-like

      1

      10995

      10995

      10995

      LTR/Pao

      Pao_Cv1

      1

      6420

      6420

      6420

      Total LTR

      3

      3

      21709

      7236

      10995

         

      3.54%

        

      LINE/CR1

      CR1-1_CV

      3

      2703

      901

      1461

      LINE/CR1

      CR1-2_CV

      2

      309

      155

      309

      LINE/CR1

      CR1-3_CV

      1

      188

      188

      188

      LINE/Jockey

      Jockey_Cv1

      1

      553

      553

      553

      LINE/Jockey

      Jockey_Cv2

      1

      180

      180

      180

      LINE/LOA

      LOA-1_Cv

      1

      536

      536

      536

      LINE/LOA

      LOA-2_Cv

      1

      1636

      1636

      1636

      LINE/LOA

      LOA-3_Cv

      1

      157

      157

      157

      LINE/LOA

      LOA-4_Cv

      1

      294

      294

      294

      LINE/LOA

      LOA-5_Cv

      1

      447

      447

      447

      LINE/LOA

      LOA-6_Cv

      3

      2414

      805

      1157

      LINE/LOA

      LOA-7_Cv

      1

      472

      472

      472

      LINE/LOA

      LOA-8_Cv

      1

      129

      129

      129

      LINE/LOA

      LOA-9_Cv

      1

      83

      83

      83

      LINE/LOA

      LOA-10_Cv

      2

      890

      445

      660

      LINE/L2

      L2-1_Cv

      1

      315

      315

      315

      LINE/RTE

      RTE-1_Cv

      1

      1111

      1111

      1111

      LINE/RTE

      RTE-2_Cv

      1

      726

      726

      726

      LINE

      LINE1_Cv

      1

      276

      276

      276

      LINE

      LINE2_Cv

      3

      2541

      847

      2125

      LINE

      LINE3_Cv

      1

      2116

      2116

      2116

      Total LINE

      21

      29

      18076

      623

      2125

         

      2.95%

        

      Class II (DNA transposons)

      DNA/ITm-mariner

      Cv-mar1

      40

      30549

      764

      1296

      DNA/ITm-mariner

      Cv-mar2

      14

      6154

      440

      989

      DNA/ITm-mariner

      Cv-mar3

      5

      950

      190

      311

      DNA/ITm-mariner

      Cv-mar5

      1

      427

      427

      427

      DNA/ITm-DD37E

      DD37E_Cv1

      4

      3073

      768

      1304

      DNA/ITm-Tc1

      AMARI_Cv1

      5

      1863

      373

      657

      DNA/ITm-Tc1

      SMAR_CV1

      1

      357

      357

      357

      DNA/ITm-Tc1

      CRMAR_CV1

      1

      305

      305

      305

      DNA/ITm-Tc3

      Tc3_CV1

      1

      356

      356

      356

      DNA/ITm-Tc3

      Tc3_CV2

      1

      414

      414

      414

      DNA/MITE

      MITE_Cv1

      8

      1524

      191

      245

      DNA/Chapaev-Chapaev3

      Chapaev3-1_CV

      2

      1658

      829

      1075

      DNA/Chapaev-Chapaev3

      Chapaev3-2_CV

      1

      302

      302

      302

      DNA/hAT

      hAT_CV1

      2

      265

      133

      163

      Total DNA

      14

      86

      48197

      560

      1304

         

      7.86%

        

      RC/Helitron

      Helitron1-Cv

      3

      1431

      477

      1269

      RC/Helitron

      Helitron2-Cv

      41

      19310

      471

      767

      RC/Helitron

      Helitron3-Cv

      40

      9901

      248

      821

      RC/Helitron

      Helitron4-Cv

      1

      85

      85

      85

      Total RC

      4

      85

      30727

      361

      1269

         

      5.01%

        

      Unclassified

      Unknown

      unknown1

      4

      2637

      659

      1010

      Unknown

      unknown2

      5

      542

      108

      127

      Unknown

      unknown3

      4

      1459

      365

      521

      Unknown

      unknown4

      1

      535

      535

      535

      Unknown

      unknown5

      20

      4144

      207

      269

      Unknown

      unknown6

      12

      3283

      273

      482

      Unknown

      unknown7

      4

      3088

      772

      1009

      Unknown

      unknown8

      6

      2051

      342

      600

      Unknown

      unknown9

      1

      160

      160

      160

      Unknown

      unknown10

      5

      598

      120

      130

      Unknown

      unknown12

      2

      228

      114

      228

      Unknown

      unknown13

      7

      3069

      438

      570

      Unknown

      unknown14

      2

      246

      123

      129

      Unknown

      unknown15

      2

      212

      106

      127

      Unknown

      unknown16

      1

      151

      151

      151

      Unknown

      unknown17

      2

      173

      87

      115

      Unknown

      unknown18

      3

      658

      219

      247

      Unknown

      unknown19

      1

      60

      60

      60

      Unknown

      unknown20

      10

      1057

      106

      138

      Unknown

      unknown21

      1

      55

      55

      55

      Unknown

      unknown22

      1

      146

      146

      146

      Unknown

      unknown23

      8

      3012

      377

      902

      Unknown

      unknown24

      1

      138

      138

      138

      Unknown

      unknown25

      1

      182

      182

      182

      Unknown

      unknown26

      1

      242

      242

      242

      Unknown

      unknown27

      1

      82

      82

      82

      Unknown

      unknown28

      1

      137

      137

      137

      Unknown

      unknown29

      1

      105

      105

      105

      Unknown

      unknown30

      1

      108

      108

      108

      Unknown

      unknown31

      1

      103

      103

      103

      Unknown

      unknown32

      1

      110

      110

      110

      Unknown

      unknown33

      1

      1429

      1429

      1429

      Unknown

      unknown34

      8

      1913

      239

      359

      Total unknown

      33

      120

      32113

      268

      1429

         

      5.24%

        
       

      Total repeats

      75

      323

      150822

        
          

      24.60%

        
      Table 2

      Total repeat content in C. vicina BAC sequences

        

      Repeats

        

      BAC

      Length

      Number

      Total bp

      % of sequence

      113H10

      96426

      61

      23570

      24.44

      99 M22

      102758

      78

      28879

      28.10

      97 L04

      111044

      38

      38432

      34.61

      62B24

      90178

      44

      28013

      31.06

      16B10

      135393

      66

      27144

      20.05

      104 L14

      115595

      68

      19608

      16.96

      Total BACs

      651394

      355

      165646

      25.43

      Total region*

      613063

      317

      149226

      24.34

      (*without repeated alleles).

      http://static-content.springer.com/image/art%3A10.1186%2F1759-8753-4-13/MediaObjects/13100_2012_Article_75_Fig1_HTML.jpg
      Figure 1

      Distribution of transposable elements and repeats in sequenced BACs. TEs and repeats are represented as rectangles: Class I-LTR elements are shown in dark green, Class I-LINEs in light green, Class II-cut and paste DNA-transposons in blue, Class II-rolling circle transposons in red and unclassified repeats in yellow; dark blue arrows represent the C. vicina genes found in this region: achaete (ac), scute (sc) and lethal of scute (l'sc).

      Class I – RNA-mediated TEs

      LTR retroposons

      LTR elements are characterized by the presence of direct long terminal repeats (LTRs) that range from a few hundred base pairs to more than five kilobases long [17]. Between the LTRs there are generally only one or two open reading frames (ORFs) that encode a polymerase (pol) protein and a protein related to the retroviral group-associated antigen (gag) protein. The pol protein contains reverse transcriptase (RT), ribonuclease H (RNaseH), protease (PR) and integrase (IN) domains that are important for the process of retrotransposition. The gag protein binds nucleic acids or forms a nucleocapside shell. Some LTR retrotransposons also have an env (envelope)-like domain that encodes a transmembrane receptor-binding protein that allows the transmission of retroviruses.

      We have identified three LTR retrotransposon elements, each with one insertion. These elements are recent insertions; all three are full length, have identical or almost identical LTRs and at least two of the three insertions are polymorphic (see below).

      Isis-like
      This is the largest identified repeat with 10,995 bp (Figure 2, Additional file 3: Figure S1). It is closely related to the Isis TE recently described in Drosophila buzzatii[22]. It belongs to the Osvaldo lineage of the Gypsy family. The LTRs of Isis-like are 2577 and 2574 bp long and there are 4 bp Target Site Duplications (TSD: CGTG) and two ORFs. The first ORF encodes a 531-amino acid (aa) gag protein with a 40% identity (and 70% similarity) with Isis. It contains a RING finger domain which is absent in Isis but present in Osvaldo (also from the same family). The second ORF encodes a 1,137-aa pol protein, which has 60% identity (and 85% similarity) with the Isis pol protein. However, Isis-like lacks the env domain and the LTR of both elements are very different (742 vs. 2574 bp long). This is a recent insertion, less than 25,000 years old, and is polymorphic as it is present in only one of the two sequenced alleles covering this region.
      http://static-content.springer.com/image/art%3A10.1186%2F1759-8753-4-13/MediaObjects/13100_2012_Article_75_Fig2_HTML.jpg
      Figure 2

      Structure of the LTR elements. Diagram showing the structural features of the three LTR elements identified in this study. All features are drawn to scale (except PBS and PPT). See legend for colour code. Full sequences of these elements can be found in Additional file 3: Figure S1, Additional file 4: Figure S2, and Additional file 5: Figure S3, respectively.

      CsRn1_Cv1

      This element is 4294 bp long. It comprises 179 and 180 bp LTRs and two ORFs 267 and 1,036-aa long (Figure 2, Additional file 4: Figure S2). It belongs to the CsRn1 lineage of the Gypsy family [23]. This lineage is characterized by the presence of a PBS complementary to tRNA-Trp, a CHCC gag motif and the GPY motif in the 3 of the Integrase protein, all of which are present in this element. However, it seems to present a 6 bp TSD (CAAGTG) instead of the 4 bp TSD typical of the group. We have estimated this insertion to be 350,000 years old, which makes it the oldest of the three LTR elements.

      Pao_Cv1

      The last LTR element identified belongs to the Pao family, and is related to the Ninja-I element. Pao_Cv1 is 6420 bp long, has 355 bp long LTRs, and one ORF coding for a 1881 aa protein (Figure 2, Additional file 5: Figure S3). It has 5 bp TSDs (GCGGG). It is inserted inside a mariner element. This insertion is polymorphic and furthermore the two LTRs are completely identical which indicates that it is very young (less than 88,000 years old).

      Non-LTR retroposons (LINEs)

      A total of 29 insertions have been classified as 21 different LINE elements, most of which are short and degraded fragments. The insertions average 745 bp in size and ten of them are smaller than 500 bp, whereas size typically ranges from 1 to 7 kb for this group [24]. The absence of canonical sequences for comparison makes it difficult to classify them properly. This is particularly acute for the LAO elements, from which we have found many very short fragments (for eight out of ten putative elements the longest fragment is smaller than 1 kb, the smallest being 83 bp only) (Table 1). We cannot exclude the possibility that some of the insertions we have defined as separate elements are in reality different regions of the same element. The size and degraded nature of these elements suggests they are all old insertions. Overall the identified LINEs span 18 kb of the sequenced region (2.9%).

      Class II – DNA transposons

      Cut-and-paste DNA transposons

      Cut-and-paste DNA transposons are characterized by 10 to 200 bp terminal inverted repeats (TIRs) flanking one or more ORFs encoding a transposase. We have identified 14 different cut-and-paste DNA elements with a total of 89 insertions spanning 7.86% of the sequenced region. One element belongs to the MITE family, two to the Chapaenov family, one to the hAT family, and the remaining 10 to the IS630-Tc1-mariner (ITm) superfamily. The most common elements belong to the Mariner family of the ITm superfamily.

      Cv-mar1
      The most frequent transposon is Cv-mar1 with 41 different insertions that span overall more than 30 kb. All insertions are partially degraded and range from 320 to 1296 bp, the consensus sequence is 1,275 bp long (Figure 3, Additional file 6: Figure S4). This element shows 78% identity at the nucleotide level with the Desmar1 mariner element from the Hessian fly Mayetiola destructor[2527] (Additional file 7: Figure S5). Its TIRs have been identified by similarity to those of Desmar1 [25], with which they show 3 nucleotide (nt) substitutions and 1 nt insertion. However, the 5TIR of Cv-mar1 is incomplete and the 3TIR is present in only a single copy of the element (the fragment of the consensus sequence derived from a single element is delimited by a blue dash in Additional file 6: Figure S4). Although none of the annotated elements displays a complete transposase, we were able to derive a “complete” copy from the consensus sequence. In position 993 (shown in red) the consensus sequence has a T that results in a stop codon in the transposase, however a third of the sequences have an A at this position, which would result in an arginine (R) residue. The next stop codon is in the same position as that of the Desmar1 element (Additional file 8: Figure S6). If we consider this longer transposase it is 345 aa long.
      http://static-content.springer.com/image/art%3A10.1186%2F1759-8753-4-13/MediaObjects/13100_2012_Article_75_Fig3_HTML.jpg
      Figure 3

      Structure of the DNA-transposons. Diagram showing the structural features of the cut-and-paste and rolling circle transposons for which we obtained consensus sequences. All features are drawn to scale. See legend for colour code. Full sequences of these elements can be found in Additional file 6: Figure S4, Additional file 9: Figure S7, Additional file 12: Figure S10, Additional file 13: Figure S11, and Additional file 14: Figure S12.

      Cv-mar2

      In the region analysed there are 14 copies of Cv-mar2 which span a total of 6 kb. The average insertion is 440 bp long, with the longest being 989 bp. Although none of the insertions is full length we were able to derive a consensus full length sequence which is 1299 bp long (Figure 3, Additional file 9: Figure S7), individual copies are 77% to 91% identical to the consensus. It has 35 bp TIRs and a 344 aa transposase. However, this consensus element would be non-functional as the TIRs have five mismatches and the transposase has four stop codons and commences with a leucine instead of a methionine. This element is very similar to the Mariner1_DYa from Drosophila yakuba[28]. The consensus obtained has a 78% identity at the nucleotide level with Mariner1_DYa and the two transposases show 73% identity at the amino acid level (Additional file 10: Figure S8 and Additional file 11: Figure S9).

      DD37E_Cv1

      The DD37E_Cv1 element belongs to the ITm-DD37E family [26]. This family was first discovered in mosquitos and is characterized by a unique DD37E catalytic domain. The full-length copy of this element is 1298 bp long with a 354 aa ORF and 27 bp ITRs (Figure 3, Additional file 12: Figure S10). At both ends of the insertion we find the TA sequence, the canonical dinucleotide target site duplication of the family [29]. Three additional copies are fragmented, highly degraded and in two cases enclose other nested repeats. This element has been present in the C. vicina genome for a long time (presence of degraded insertions). The identification of a full-length copy suggests this element has also been active recently in Calliphora.

      Rolling circle (RC) transposons - Helitrons

      Helitrons have been classified as class II-DNA transposons that use a “rolling circle” mode of transposition [19]. They encode proteins similar to helicases, ssDNA-binding proteins and replication initiation proteins [4, 19]. Helitrons lack inverted repeats but are characterized by much-conserved termini and hairpin structures close to the 3 end. As with other TEs, the Helitrons present both autonomous and non-autonomous elements. DINE-1 and mini-me elements from Drosophila, which show some unique characteristics, are now classified as non-autonomous Helitrons [30, 31]. They lack coding capacity, do not have these characteristic termini, but have subterminal inverted repeats and the hairpin structures at the 3 region [30]. Four different elements of the Helitron family are present in our sample. Two of them show a high copy number, with 40 and 41 insertions, respectively. Helitrons cover 5.01% of the analysed sequence.

      Helitron2_Cv

      Was identified by similarity to the 5region of the Arylphorin subunit from C. vicina (X63340). RepeatMasker indicated it is related to Helitron-1N1_Dvir and mini-me elements [32]. We have annotated 41 copies of this element, from 136 to 767 bp long. The consensus sequence is 750 bp long (Figure 3, Additional file 13: Figure S11). Eight copies are full length and show a 95% to 97% identity with the consensus. Helitron2_Cv shows the structural features of non-autonomous DINE1-like Helitrons: 11 bp subTIRs, partial inverted repeats next to the 5 subTIRs, GTCY-rich protosatellites and short hairpin stem-loops (with 9 bp stems) next to the 3end of the element. It is closely related to the autonomous and non-autonomous elements Helitron-1-Dvir and Helitron-1N1_Dvir of D. virilis[32]. Helitron2_Cv shows a 65% and 70% identity in the 5region (up to protosatellite repeat) and 3end (last 100 bp), respectively, with the D. virilis elements. Copies of this element represent 3% of the sequenced region. Given the level of divergence of the full length insertion, autonomous copies of this element probably exist in the C. vicina genome.

      Helitron3_Cv

      This is also a DINE1-like Helitron. We have identified 40 copies that range from 71 to 821 bp. They can be divided into two subtypes, whose consensus sequences are 395 and 396 bp long. The consensus of the two subtypes differs in one nucleotide indel and 54 nucleotide substitutions, half of which are located in the region just after the protosatellite repeat. All features typical of DINE1-like Helitrons are present except the 3 subTIR (Figure 3, Additional file 14: Figure S12). The protosatellite repeat (GTCT)2 is expanded in 3 of the insertions: one has 4 repeats, another 5 repeats and the third 108 repeats.

      Unclassified repeats

      These repeats have been mainly identified by similarity within and between BAC sequences and with other published Calliphora sequences (blastn – non-redundant nucleotide NCBI database). They are mostly short and with no obvious structure or similarity with known elements. Overall these repeats span 5.24% of the analysed region.

      Unknown 5

      This repeat was first identified by blastn to the non-redundant NCBI database, as it is present in intergenic or intronic regions of two different alleles of the Xdh gene of C. vicina (M30316, M30488). We have annotated 20 insertions of this element in the region we analysed. The consensus sequence is 275 bp long (Additional file 15: Figure S13). The 5region of the element is rich in polyA and polyT tracts, whereas the 3region of the element is highly conserved between copies (red region in Additional file 15: Figure S13). However, no structural features or internal repeats could be recognized.

      Unknown 6

      A short fragment of this element was first identified by RepeatMasker as a fragment of a Helitron. However, in this sequence, which is present 12 times in the C. vicina sequences, we could not identify any of the features of a Helitron and thus it remains unclassified. The consensus sequence of this element is 488 bp long (Additional file 16: Figure S14). From nucleotide 1 to 465 the sequence is palindromic (with 92% identity).

      Unknown 20

      This element was first identified by blastn with similarity to a Lucilia cuprina intronic sequence (M89990). There are 10 insertions of this sequence present in the region of C. vicina that was analysed. The consensus sequence is 140 bp long (Additional file 17: Figure S15). No structural features or internal repeats were identified which could help classify this repeat.

      Candidates of horizontal transfer

      Four of the analysed repeats show a remarkable similarity with elements from other species. To assess the possibility of horizontal transfer we have taken a closer look at these elements and checked their distribution on available sequences (NCBI and Insect genome sequences – see Methods). These elements are the LTR element Isis, the DNA cut-and-paste elements Cv-mar1 and Cv-mar2, and the Helitron Helitron2_Cv.

      The elements Isis from D. buzzatii and Isis-like from C. vicina have 40% and 60% identity in their ORFs, however they differ in the presence of the RING (present only in Isis-like) and env (present only in Isis) domains. The sequence (and length) of their LTRs is also very different. Of the sequenced genomes, only D. mojavensis presents an Isis element. We have found no evidence of Isis-like. The limited distribution of these elements suggests that they arrived by horizontal transfer to the D. buzzatii-D. mojavensis ancestor (after the split of D. virilis) and to C. vicina (or its ancestors).

      The Cv-mar1 element shows 70% to 80% identity with multiple Mariner elements described in different insect species [3335] besides Desmar1 [25]. The whole genome sequences of Mayetiola, Rhodnius prolixus (Hemiptera), Solenopsis invicta (Hymenoptera) and Anopheles gambiae (Nematocera) include fragments of this element (500 to 800 bp long) with 80% identity. The broad distribution of this element suggests it is mainly vertically transmitted.

      The Mariner element Cv-mar2 is present in D. yakuba (Mariner1_Ya) with which it shows 78% identity over its whole length. We have also found several hits with 80% identity in the ants Camponeatus floridanus and Harpegnathos saltator (Hymenoptera), covering 80% and 60% of the length of the element, respectively. We found no evidence of this element in other species. Its high similarity and limited distribution suggest its transmission by horizontal transfer between Diptera and Hymenoptera which diverged approx. 300 Myr ago.

      The Helitron2_Cv is similar to Helitron-1N1_Dvir from D. virilis. They have 50% identity over the whole element, and 65% to 70% identity at the 5 and 3end, respectively. Multiple hits with 60% to 90% identity around sequenced genes of Lucilia, Musca and other species show that this element is very common within the Muscomorpha. No hits were found in the whole genome sequences with Helitron2_Cv. Using Helitron-1N1_Dvir as query, we find multiple hits in Drosophila species but nothing outside the Drosophila genus. This suggests that this element is vertically transmitted, the absence of hits in other insect is probably due to evolution of the sequence of this element.

      Discussion

      We have analysed a small (600 kb) region of the Calliphora genome. It contains most of the Achaete-Scute complex: with the genes ac, sc and l’sc. The low gene density in this region is due to the presence of large regulatory regions (Negre and Simpson, submitted). It is euchromatic in nature although we do not know its position in the chromosome or whether it is representative of the genome in terms of TE content and diversity but there are no reasons that would indicate otherwise. The discussion that follows is only a first approximation to the repeat landscape of this fly species, C. vicina, which has a big genome with 750 Mb (Spencer Johnston personal communication).

      Fraction of genomic DNA occupied by repeats

      Repeats span 24% of the region analysed (600 kb). This percentage is relatively high but not unusual for fly genomes. Larger genomes usually show a higher proportion of repeats; however, repeat content is not proportional to genome size and is highly variable between dipteran genomes (Table 3). For example, there are several species whose genome is around 200 Mb with a repeat content ranging from 3% to 25%.
      Table 3

      Repeat content in dipteran genomes

        

      % Genome

      Class I

       

      Class II

         

      Species

      Genome size

      All TEs

      LTR

      Non-LTR (LINE)

      DNA (TIR)

      Helitron

      Other

      Unclassified

      Drosophila melanogaster

      180 Mb

      6%1

      4.20%1

      1.38%1

      0.30%1

      -

      0.12%1

      -

      Drosophila ananassae

      231 Mb

      25%1

      15.5%1

      7.00%1

      1.25%1

      -

      1.25%1

      -

      Drosophila virilis

      206 Mb

      14%1

      9.94%1

      3.36%1

      0.28%1

      -

      0.42%1

      -

      Drosophila grimshawii

      200 Mb

      3%1

      1.53%1

      0.66%1

      0.57%1

      -

      0.24%1

      -

      Calliphora vicina (600 kb region)

      750 Mb

      24% *

      3.54%

      2.95%

      7.86%

      5.01%

      -

      5.24%

      Anopheles gambiae

      278 Mb

      16%2

      2.64%4

      3.75%4

      4.54%4

      0.11%4

      -

      -

      Aedes aegypty

      1,376 Mb

      47%3

      12.41%4

      12.67%4

      13.97%4

      1.26%4

      -

      -

      Culex quinquefasciatus

      579 Mb

      28%4

      3.89%4

      4.45%4

      19.40%4

      0.49%4

      -

      -

      *% of TEs based on the 600 kb region analysed in this study, data from other species comes from whole genome sequences. References: 1Drosophila 12 genomes consortium 2007; [37]2Holt, et al.[5]; 3Nene, et al.[7]; 4Arensburger, et al.[6].

      Repeat content is also variable within genomes, being most abundant in heterochromatin and pericentromeric regions. Unfortunately, we have no information about the position within the chromosome of the region we analysed. In D. melanogaster it is close to the tip of the X chromosome, however chromosomes are very dynamic in terms of gene order, so we do not expect the position to be necessarily conserved.

      Abundance of the different classes of repeats

      If we look at the distribution of repeats in Dipterans, the abundance of the different classes appears to be constant within lineages independently of total repeat content, but very divergent between lineages (Table 3). In D. melanogaster LTRs are the most abundant TEs, followed by non-LTR and then TIR elements [36] (there is no information about Helitrons). The same pattern is observed in the other 11 Drosophila species that have been sequenced [37]. The pattern changes in mosquitos where TIR elements are the most abundant, followed by non-LTR, LTRs and finally Helitrons with less than 1% (Table 3). As in Drosophilidae, all mosquitos show the same pattern, although in Anopheles and Aedes the quantity of TIR, non-LTR and LTR elements is very similar, whereas in Culex TIR elements represent more than half of the repeat content. In Calliphora we see again a completely different pattern. As in mosquitoes TIR elements are the most frequent but they are now followed by Helitrons. LTR and non-LTR elements (in this order) are the least frequent in C. vicina (Table 3). It is noteworthy that if we consider the unclassified repeats in Calliphora this would be the second most frequent class of repeats.

      Age of TE insertions

      Nested elements

      Of the 322 identified repeats 11 (3.4%) are nested within other elements. Two of the three LTR elements are nested within other repeats, whereas none of the LTR elements themselves show insertions of other elements. This is consistent with the fact that they are recent insertions. At the other extreme, the unclassified (unknown) elements, in spite of being the most numerous (37%), show the smallest proportion of nested elements: only one copy is nested and two include insertions of other elements. The fact that one copy of unknown 20 is nested within another TE suggests that this element is mobile although no structural features have been identified (see results). On the other hand, the fact that only one of the 119 unknown repeats is nested suggests that some of them might not be mobile. For the other types of elements (LINE, DNA and RC) the frequency of nested copies is proportional to the number of insertions. However, LINEs show a high number of copies serving as landing sites. This, together with the small size and degraded nature of most copies, indicates that most LINE insertions are very old. Of the RC elements, all three nested insertions belong to Helitron2, two of which are full length. Two of the three are nested inside fragmented copies of the DNA element DDE37E_Cv1.

      New vs. old insertions

      All LTR insertions found in this sample are recent in origin. All three insertions are full length and at least two of them are polymorphic. We have found no fragments or degraded copies. This is a very different picture to that found in all other TE classes where none (non-LTR elements) or only a few (DNA and RC elements) insertions are full-length. In all these classes most insertions are fragmented and highly degraded. A similar trend was found in D. melanogaster. LTR families appear to be transposing in the D. melanogaster genome at higher rates than TEs from other orders leading to the observation that LTR elements, as a group, tend to be younger [38]. Recent analyses suggest that this trend is due to a higher intrinsic rate of transposition of LTR elements and not to a recent increase of transposition [39].

      Role of horizontal transfer

      The mobile nature of TEs makes them prone to horizontal transfer. It is thought to be an essential step in TE life cycle, which allows them to escape vertical extinction [40, 41].

      Four TEs showed a remarkable similarity with elements from other species. Although we could not compare the rates of synonymous mutations between the TEs and orthologous genes, we have checked the distribution of these elements in sequenced insect species to detect possible instances of horizontal transfer.

      The broad distribution of the Mariner element Cv-mar1 and the Helitron Helitron2_Cv shows they are vertically transmitted. We cannot rule out completely horizontal transfer in Cv-mar1, but its detection would require a much thorough analysis (which is out of the scope of this study).

      The elements Isis and Cv-mar2 do seem to have undergone horizontal transfer. Isis moved between Calliphoridae and Drosophilidae which diverged approximately 100 Myr ago, and Cv-mar2 between Diptera and Hymenoptera which diverged approximately 300 Myr ago.

      Overall two of the 43 identified TEs show evidence of horizontal transfer. One is an LTR and the other a DNA transposon, the two classes more often involved in transfer events [40].

      Conclusions

      This is the first detailed description of TEs in carrion flies. Although the analysis includes only a small region of the genome it gives an overview of the classes of TEs present and their abundance. Moreover, the description of these TEs and repeats can help in the annotation of repeat sequences in other Dipteran genomes, e.g., those currently being sequenced.

      Methods

      Sequences analysed

      We have analysed the sequences of six overlapping BAC clones, in a region which contains most of the Achaete-Scute Complex (AS-C) of Calliphora vicina (cloning and sequencing of this region is described in Negre and Simpson, submitted). The clones comprise a total of 651,394 base pairs (bp), of which 38,331 bp correspond to identical alleles in two overlapping clones (see Table 2). Thus we have analysed 613,063 bp of unique sequence.

      Identification of repetitive elements

      Several tools were used for the identification and classification of repeats: RepeatMasker was run against the Drosophila database and all hits were considered, for protein-based RepeatMasker (A.F.A. Smit, R. Hubley and P. Green, RepeatMasker at http://​repeatmasker.​org) all hits were also considered; blastn and blastp were run against NCBI non-redundant databases [42] and hits longer than 100 bp with identities over 60% were further analysed. LTR-Finder [43] was used to identify LTR elements and some of their structural features such as PBS and PPT sequences. The online program Palindromes (http://​mobyle.​pasteur.​fr) was used to aid in the identification of TIRs. All hits were compared between methods and manually inspected. Most repeats are identified by more than one method. Non-overlapping hits smaller than 50 bp were discarded. The best match was used for repeat classification. Annotated repeats were added to a local database to help in the identification of further copies of the same repeats. Comparison between Calliphora sequences (with blast2sequences-blastn) allowed the identification of many short unclassified repeats which are found recurrently in the Calliphora genome. Some of the elements we have annotated are also present in GeneBank sequences (in intronic and intergenic regions), but these were all unannotated. Consensus sequences were obtained by ClustalW [44, 45] or Tcoffee [46, 47] alignment and manually corrected with the aid of Bioedit.

      Divergence time of TE insertions

      The age of TE insertions (t) has been calculated as in [4]; t = K/v, where K is the average divergence of TE copies from the consensus and v the neutral substitution rate. We have used the neutral substitution rate for Drosophila (v=0.016 substitutions/Myr) [48]. For LTR elements we have used t = K/2v, where K stands for the divergence between the two LTRs of one insertion [4].

      Identification of similar elements in other species

      Distribution of similar elements in other species was assessed by similarity searches (blastn) against: (1) the non-redundant NCBI database and (2) insect whole genome sequences (flybase) [42, 49]. Only hits with >60% identity over half the length of the query sequence were considered.

      Abbreviations

      Env: 

      Envelope

      IN: 

      Integrase

      IR: 

      Inverted repeat

      ITm: 

      IS360-Tc1-mariner superfamily

      LTR: 

      Long terminal repeat

      ORF: 

      Open reading frame

      PBS: 

      Primer binding site

      PR: 

      Protease

      PPT: 

      Polypurine tract

      RC: 

      Rolling circle

      RNaseH: 

      Ribonuclease H

      RT: 

      Reverse transcriptase

      TE: 

      Transposable element

      TIRs: 

      Terminal inverted repeats

      TSD: 

      Target site duplication

      Declarations

      Acknowledgements

      We would like to thank Pr. Spencer Johnston for the estimate of the Calliphora vicina genome size, Sung Ly and Carol McKimmie for technical assistance and Josefa González and two anonymous reviewers for comments on the manuscript. This work was supported by the Wellcome Trust grant 29156.

      Authors’ Affiliations

      (1)
      Department of Zoology, University of Cambridge
      (2)
      EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), and Universitat Pompeu Fabra (UPF)

      References

      1. Kidwell MG, Lisch DR: Transposable elements and host genome evolution. Trends Ecol Evol. 2000, 15: 95-99. 10.1016/S0169-5347(99)01817-0.View ArticlePubMed
      2. Biemont C, Vieira C: Genetics: junk DNA as an evolutionary force. Nature. 2006, 443: 521-524. 10.1038/443521a.View ArticlePubMed
      3. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C: Initial sequencing and analysis of the human genome. Nature. 2001, 409: 860-921. 10.1038/35057062.View ArticlePubMed
      4. Kapitonov VV, Jurka J: Molecular paleontology of transposable elements in the Drosophila melanogaster genome. Proc Natl Acad Sci U S A. 2003, 100: 6569-6574. 10.1073/pnas.0732024100.PubMed CentralView ArticlePubMed
      5. Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark AG, Ribeiro JM, Wides R, Salzberg SL, Loftus B, Yandell M, Majoros WH, Rusch DB, Lai Z, Kraft CL, Abril JF, Anthouard V, Arensburger P, Atkinson PW, Baden H, de Berardinis V, Baldwin D, Benes V, Biedler J, Blass C, Bolanos R, Boscus D, Barnstead M: The genome sequence of the malaria mosquito Anopheles gambiae. Science. 2002, 298: 129-149. 10.1126/science.1076181.View ArticlePubMed
      6. Arensburger P, Megy K, Waterhouse RM, Abrudan J, Amedeo P, Antelo B, Bartholomay L, Bidwell S, Caler E, Camara F, Campbell CL, Campbell KS, Casola C, Castro MT, Chandramouliswaran I, Chapman SB, Christley S, Costas J, Eisenstadt E, Feschotte C, Fraser-Liggett C, Guigo R, Haas B, Hammond M, Hansson BS, Hemingway J, Hill SR, Howarth C, Ignell R, Kennedy RC: Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics. Science. 2010, 330: 86-88. 10.1126/science.1191864.PubMed CentralView ArticlePubMed
      7. Nene V, Wortman JR, Lawson D, Haas B, Kodira C, Tu ZJ, Loftus B, Xi Z, Megy K, Grabherr M, Ren Q, Zdobnov EM, Lobo NF, Campbell KS, Brown SE, Bonaldo MF, Zhu J, Sinkins SP, Hogenkamp DG, Amedeo P, Arensburger P, Atkinson PW, Bidwell S, Biedler J, Birney E, Bruggner RV, Costas J, Coy MR, Crabtree J, Crawford M: Genome sequence of Aedes aegypti, a major arbovirus vector. Science. 2007, 316: 1718-1723. 10.1126/science.1138878.View ArticlePubMed
      8. Lerat E: Identifying repeats and transposable elements in sequenced genomes: how to find your way through the dense forest of programs. Heredity. 2010, 104: 520-533. 10.1038/hdy.2009.165.View ArticlePubMed
      9. Oliver KR, Greene WK: Transposable elements: powerful facilitators of evolution. BioEssays. 2009, 31: 703-714. 10.1002/bies.200800219.View ArticlePubMed
      10. Gonzalez J, Petrov DA: The adaptive role of transposable elements in the Drosophila genome. Gene. 2009, 448: 124-133. 10.1016/j.gene.2009.06.008.PubMed CentralView ArticlePubMed
      11. Kumar A, Hirochika H: Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci. 2001, 6: 127-134. 10.1016/S1360-1385(00)01860-4.View ArticlePubMed
      12. Hamon P, Duroy PO, Dubreuil-Tranchant C, Mafra D’Almeida Costa P, Duret C, Razafinarivo NJ, Couturon E, Hamon S, de Kochko A, Poncet V, Guyot R: Two novel Ty1-copia retrotransposons isolated from coffee trees can effectively reveal evolutionary relationships in the Coffea genus (Rubiaceae). Mol Genet Genomics. 2011, 285: 447-460. 10.1007/s00438-011-0617-0.View ArticlePubMed
      13. D’Onofrio C, Lorenzis G, Giordani T, Natali L, Cavallini A, Scalabrelli G: Retrotransposon-based molecular markers for grapevine species and cultivars identification. Tree Genetics & Genomes. 2010, 6: 451-466. 10.1007/s11295-009-0263-4.View Article
      14. Mansour A: Utilization of genomic retrotransposons as cladistic markers. J Cell Molec Biol. 2008, 7: 17-28.
      15. Scolari F, Siciliano P, Gabrieli P, Gomulski LM, Bonomi A, Gasperi G, Malacrida AR: Safe and fit genetically modified insects for pest control: from lab to field applications. Genetica. 2011, 139: 41-52. 10.1007/s10709-010-9483-7.View ArticlePubMed
      16. Finnegan DJ: Transposable elements. Curr Opin Genet Dev. 1992, 2: 861-867. 10.1016/S0959-437X(05)80108-X.View ArticlePubMed
      17. Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, Flavell A, Leroy P, Morgante M, Panaud O, Paux E, SanMiguel P, Schulman AH: A unified classification system for eukaryotic transposable elements. Nat Rev Genet. 2007, 8: 973-982. 10.1038/nrg2165.View ArticlePubMed
      18. Kapitonov VV, Jurka J: A universal classification of eukaryotic transposable elements implemented in Repbase. Nat Rev Genet. 2008, 9: 411-412. Author reply 414View ArticlePubMed
      19. Kapitonov VV, Jurka J: Rolling-circle transposons in eukaryotes. Proc Natl Acad Sci U S A. 2001, 98: 8714-8719. 10.1073/pnas.151269298.PubMed CentralView ArticlePubMed
      20. Amendt J, Krettek R, Zehner R: Forensic entomology. Die Naturwissenschaften. 2004, 91: 51-65. 10.1007/s00114-003-0493-5.View ArticlePubMed
      21. Thompson ML, Gauna AE, Williams ML, Ray DA: Multiple chicken repeat 1 lineages in the genomes of oestroid flies. Gene. 2009, 448: 40-45. 10.1016/j.gene.2009.08.010.View ArticlePubMed
      22. Garcia Guerreiro MP, Fontdevila A: Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii. Mol Genet Genomics. 2007, 277: 83-95.View ArticlePubMed
      23. Tubio JM, Naveira H, Costas J: Structural and evolutionary analyses of the Ty3/gypsy group of LTR retrotransposons in the genome of Anopheles gambiae. Mol Biol Evol. 2005, 22: 29-39.View ArticlePubMed
      24. Kidwell MG: Transposable elements and the evolution of genome size in eukaryotes. Genetica. 2002, 115: 49-63. 10.1023/A:1016072014259.View ArticlePubMed
      25. Russell VW, Shukle RH: Molecular and cytological analysis of a mariner transposon from Hessian fly. J Hered. 1997, 88: 72-76. 10.1093/oxfordjournals.jhered.a023062.View ArticlePubMed
      26. Shao H, Tu Z: Expanding the diversity of the IS630-Tc1-mariner superfamily: discovery of a unique DD37E transposon and reclassification of the DD37D and DD39D transposons. Genetics. 2001, 159: 1103-1115.PubMed CentralPubMed
      27. Behura SK, Shukle RH, Stuart JJ: Assessment of structural variation and molecular mapping of insertion sites of Desmar-like elements in the Hessian fly genome. Insect Mol Biol. 2010, 19: 707-715. 10.1111/j.1365-2583.2010.01028.x.View ArticlePubMed
      28. Jurka J: Mariner-type families from fruit fly. Repbase Reports. 2009, 9: 477-
      29. Biedler JK, Shao H, Tu Z: Evolution and horizontal transfer of a DD37E DNA transposon in mosquitoes. Genetics. 2007, 177: 2553-2558. 10.1534/genetics.107.081109.PubMed CentralView ArticlePubMed
      30. Yang HP, Barbash DA: Abundant and species-specific DINE-1 transposable elements in 12 Drosophila genomes. Genome Biol. 2008, 9: R39-10.1186/gb-2008-9-2-r39.PubMed CentralView ArticlePubMed
      31. Kapitonov VV, Jurka J: Helitrons on a roll: eukaryotic rolling-circle transposons. Trends in genetics: TIG. 2007, 23: 521-529. 10.1016/j.tig.2007.08.004.View ArticlePubMed
      32. Kapitonov VV, Jurka J: Helitrons in fruitflies. Repbase Reports. 2007, 7: 127-132.
      33. Rezende-Teixeira P, Siviero F, Andrade A, Santelli RV, Machado-Santelli GM: Mariner-like elements in Rhynchosciara americana (Sciaridae) genome: molecular and cytological aspects. Genetica. 2008, 133: 137-145. 10.1007/s10709-007-9193-y.View ArticlePubMed
      34. Rezende-Teixeira P, Lauand C, Siviero F, Machado-Santelli GM: Normal and defective mariner-like elements in Rhynchosciara species (Sciaridae, Diptera). Genet Mol Res. 2010, 9 (2): 849-857. 10.4238/vol9-2gmr796.View ArticlePubMed
      35. Haine ER, Kabat P, Cook JM: Diverse Mariner-like elements in fig wasps. Insect Mol Biol. 2007, 16 (6): 743-752. 10.1111/j.1365-2583.2007.00767.x.View ArticlePubMed
      36. Bergman CM, Quesneville H, Anxolabehere D, Ashburner M: Recurrent insertion and duplication generate networks of transposable element sequences in the Drosophila melanogaster genome. Genome Biol. 2006, 7: R112-10.1186/gb-2006-7-11-r112.PubMed CentralView ArticlePubMed
      37. Drosophila 12 genomes Consortium: Evolution of genes and genomes on the Drosophila phylogeny. Nature. 2007, 450: 203-218. 10.1038/nature06341.View Article
      38. Bergman CM, Bensasson D: Recent LTR retrotransposon insertion contrasts with waves of non-LTR insertion since speciation in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2007, 104: 11340-11345. 10.1073/pnas.0702552104.PubMed CentralView ArticlePubMed
      39. Petrov DA, Fiston-Lavier AS, Lipatov M, Lenkov K, Gonzalez J: Population genomics of transposable elements in Drosophila melanogaster. Mol Biol Evol. 2011, 28: 1633-1644. 10.1093/molbev/msq337.PubMed CentralView ArticlePubMed
      40. Loreto ELS, Carareto CMA, Capy P: Revisiting horizontal transfer of transposable elements in Drosophila. Heredity. 2008, 100: 545-554. 10.1038/sj.hdy.6801094.View ArticlePubMed
      41. Schaack S, Gilbert C, Feschotte C: Promiscuous DNA: horizontal transfer of tranposable elements and why it matters for eukaryotic evolution. TREE. 2010, 25: 537-546.PubMed CentralPubMed
      42. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.View ArticlePubMed
      43. Xu Z, Wang H: LTR_FINDER: an efficient tool for the prediction of full-length LTR retrotransposons. Nucleic Acids Res. 2007, 35: W265-W268. 10.1093/nar/gkm286.PubMed CentralView ArticlePubMed
      44. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23: 2947-2948. 10.1093/bioinformatics/btm404.View ArticlePubMed
      45. Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R: A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 2010, 38: W695-W699. 10.1093/nar/gkq313.PubMed CentralView ArticlePubMed
      46. Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang JM, Taly JF, Notredame C: T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011, 39: W13-W17. 10.1093/nar/gkr245.PubMed CentralView ArticlePubMed
      47. Notredame C, Higgins DG, Heringa J: T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000, 302: 205-217. 10.1006/jmbi.2000.4042.View ArticlePubMed
      48. Li W-H: Molecular Evolution. 1997, Sunderland, MA: Sinauer
      49. McQuilton P, St Pierre SE, Thurmond J, The FlyBase Consortium: FlyBase 101 – the basics of navigating FlyBase. Nucleic Acids Res. 2012, 40 (Database issue): D706-D714.PubMed CentralView ArticlePubMed

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      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.