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  • Research article
  • Open Access

Identification of 74 cytochrome P450 genes and co-localized cytochrome P450 genes of the CYP2K, CYP5A, and CYP46A subfamilies in the mangrove killifish Kryptolebias marmoratus

Contributed equally
BMC Genomics201819:7

https://doi.org/10.1186/s12864-017-4410-2

  • Received: 10 August 2017
  • Accepted: 21 December 2017
  • Published:

Abstract

Background

The mangrove killifish Kryptolebias marmoratus is the only vertebrate that reproduces by self-fertilizing and is an important model species in genetics and marine ecotoxicology. Using whole-genome and transcriptome sequences, we identified all members of the cytochrome P450 (CYP) family in this model teleost and compared them with those of other teleosts.

Results

A total of 74 cytochrome P450 genes and one pseudogene were identified in K. marmoratus. Phylogenetic analysis indicated that the CYP genes in clan 2 were most expanded, while synteny analysis with other species showed orthologous relationships of CYP subfamilies among teleosts. In addition to the CYP2K expansions, five tandem duplicated gene copies of CYP5A were observed. These features were unique to K. marmoratus.

Conclusions

These results shed a light on CYP gene evolution, particularly the co-localized CYP2K, CYP5A, and CYP46A subfamilies in fish. Future studies of CYP expression could identify specific endogenous and exogenous environmental factors that triggered the evolution of tandem CYP duplication in K. marmoratus.

Keywords

  • Rivulus
  • Killifish
  • Model animal
  • Gene family expansion
  • Drug metabolism
  • Tandem duplication

Background

Cytochrome P450 (CYP) enzymes are heme-containing proteins that play critical roles in the metabolism of endogenous substrates (e.g., hormones and vitamins) and in the detoxification of xenobiotics (e.g., drugs and environmental pollutants) [15]. Together, the CYPs constitute one of the most diverse gene families. Different species, even closely related ones, can have different numbers of CYP genes [6, 7]. The CYP genes are hierarchically classified at three distinct levels into subfamilies, families, and clans based on their amino acid sequence similarity, phylogenetic relationships, and syntenic relationships [68]. Molecular phylogenetic studies have identified ten CYP clans and 19 families in vertebrates [6, 7, 9]. CYP genes in families 1 to 4 are mainly related to xenobiotic metabolism and are more diverse than the other CYPs, with less sequence conservation [10, 11]. In contrast, CYP genes in families 5 to 51 mainly have endogenous functions. Many studies of CYP genes in families 1 to 4 have focused on ecotoxicological model species, including teleosts [1, 3, 12]. Zebrafish and Japanese medaka are the teleosts most commonly used to study the mechanistic action of CYPs in response to chemical compounds. These model organisms have shown that CYPs alert the organism to the presence of carcinogenic and hormonal disruptive substances in aquatic ecosystems [13].

Over the past two decades, CYP genes have been intensively identified and characterized in fish. More than 130 CYP genes in 19 families have been identified in all fish species examined to date [3, 10]. For instance, Japanese pufferfish (Fugu rubripes) have 54 CYP genes (later updated to 61 CYP genes) [8], zebrafish (Danio rerio) have 94 CYP genes (without transcript variants, the number is closer to 86) [2, 12], marine medaka (Oryzias melastigma) have 65 CYP genes [14], and channel catfish (Ictalurus punctatus) have 61 CYP genes [15]. In addition, CYP genes with various functions have been studied in many other fish species [1, 3, 1619].

Kryptolebias marmoratus is the only vertebrate that reproduces by self-fertilization. K. marmoratus is a useful laboratory fish for studying molecular ecotoxicology because it is only 3–5 cm long, its life cycle is just 12–16 weeks, and it is easily maintained in aquaria [20]. As an ecotoxicological model species in which the entire genome has been sequenced [2123], it has provided a platform for assessing the impact of various chemicals on the marine environment. In a previous study, nine CYP genes co-localized on a scaffold were identified and their spatio-temporal expression patterns in response to various endocrine-disrupting chemicals (EDCs) were analyzed (e.g., benzo[α]pyrene, bisphenol A, octylphenol, and nonlyphenol) [24]. In this study, we identified and annotated the full complement of 74 CYP genes in K. marmoratus. We also analyzed the co-localized CYP2K, CYP5A, and CYP46A subfamilies and characterized their structural features.

Results

Identification of CYP genes

Using the available K. marmoratus genome and transcriptome assembly data, we identified 74 CYP genes and one CYP pseudogene that together mapped onto 36 scaffolds (Fig. 1; Table 1). Each scaffold contained one to ten CYP genes. The identified CYP genes were classified into ten clans (2, 3, 4, 7, 19, 20, 26, 46, 51, and mt) and 17 families (1, 2, 3, 4, 5, 7, 8, 11, 17, 19, 20, 21, 24, 26, 27, 46, and 51) (Table 1). Among the 18 teleost-specific subfamilies, K. marmoratus has 11 (CYP2K, CYP2N, CYP2P, CYP2V, CYP2X, CYP2Y, CYP2Z, CYP2AD, CYP3B, CYP7C, and CYP11C). Of the 74 CYP genes, four CYP genes (CYP2Z6, CYP3A176, CYP4T17, and CYP8A2) had alternatively spliced transcripts (CYP2Z6-like, CYP3A177, CYP4T18, and CYP8A2-like) (Table 1). During the CYP gene identification process, we obtained evidence of an additional CYP gene near the CYP2K38 gene, which turned out to be a pseudogene. This pseudogene (CYP2K38pseudo) was discovered by mapping the CYP2K38 gene onto the genome scaffolds. CYP2K38pseudo showed 98% sequence similarity in addition to structural similarity (nine exons) to CYP2K38, which is approximately 1 kb away on the complementary strand. However, CYP2K38pseudo has a stop codon at the end of the 4th exon. The corresponding transcript could not be identified from the RNA-seq data (Additional file 1: Figure S1).
Fig. 1
Fig. 1

Diagram of the cytochrome P450 genes and their genomic locations in K. marmoratus

Table 1

CYP genes identified in K. marmoratus

Clan

Family

CYP genes

ORF length (bp)

No. of Exons

Accession No.

Scaffold ID

Scaffold length (bp)

Start

End

Gene size (bp)

Strand

Clan 2

Family 1

CYP1A

1566

7

MF326082

NW_016094354

1,459,910

968,181

965,957

2224

  

CYP1B1

1617

2

MF326083

NW_016094495

528,655

243,436

240,336

3100

  

CYP1C1

1578

1

MF326084

NW_016094241

11,400,209

2,151,289

2,149,703

1586

  

CYP1C2

1575

1

MF326085

NW_016094241

11,400,209

2,147,560

2,145,986

1574

  

CYP1D1

1587

7

MF326086

NW_016094279

3,616,124

1,463,808

1,459,468

4340

 

Family2

CYP2AD12

1482

9

MF326087

NW_016094248

6,824,951

6,408,131

6,411,923

3792

+

  

CYP2AD12iso

1482

9

MF326088

NW_016094248

6,824,951

6,413,510

6,417,086

3576

+

  

CYP2K38

1521

9

MF326089

NW_016095595

18,832

6285

9742

3457

+

  

CYP2K39

1506

9

MF326090

NW_016094323

2,188,509

12,490

15,110

2620

+

  

CYP2K40

1506

9

MF326091

NW_016094323

2,188,509

17,216

20,474

3258

+

  

CYP2K41

1506

9

MF326092

NW_016094323

2,188,509

22,083

25,074

2991

+

  

CYP2K42

1503

9

MF326093

NW_016094323

2,188,509

27,002

31,421

4419

+

  

CYP2K43

1506

9

MF326094

NW_016094323

2,188,509

32,776

35,523

2747

+

  

CYP2K44

1503

9

MF326095

NW_016094323

2,188,509

39,265

42,491

3226

+

  

CYP2K45

1503

9

MF326096

NW_016094323

2,188,509

45,259

48,543

3284

+

  

CYP2K46

1488

9

MF326097

NW_016094323

2,188,509

57,299

59,861

2562

+

  

CYP2K47

1527

9

MF326098

NW_016094341

1,713,428

484,375

479,276

5099

  

CYP2K48

1419

9

MF326099

NW_016094341

1,713,428

477,446

472,119

5327

  

CYP2K49

1515

9

MF326100

NW_016094341

1,713,428

499,574

492,275

7299

  

CYP2K50

1521

9

MF326101

NW_016094341

1,713,428

466,024

461,828

4196

  

CYP2K51

1500

9

MF326102

NW_016094323

2,188,509

50,504

53,526

3022

+

  

CYP2K52

1350

9

MF326103

NW_016094323

2,188,509

3986

9356

5371

+

  

CYP2N22

1488

9

MF326104

NW_016094248

6,824,951

6,424,268

6,427,747

3479

+

  

CYP2N23

1494

9

MF326105

NW_016094248

6,824,951

6,419,087

6,422,845

3758

+

  

CYP2P16

1497

9

MF326106

NW_016094248

6,824,951

6,406,727

6,401,086

5641

  

CYP2P17

1497

9

MF326107

NW_016094248

6,824,951

6,399,381

6,393,620

5761

  

CYP2P18

1497

9

MF326108

NW_016094248

6,824,951

6,392,341

6,385,519

6822

  

CYP2P19

1497

9

MF326109

NW_016094248

6,824,951

6,379,679

6,384,024

4345

+

  

CYP2P20

1506

9

MF326110

NW_016094248

6,824,951

6,373,446

6,378,689

5243

+

  

CYP2R1

1560

5

MF326111

NW_016094245

8,651,236

4,619,472

4,622,983

3511

+

  

CYP2U1

1602

5

MF326112

NW_016094240

11,911,191

4,154,835

4,159,576

4741

+

  

CYP2X24

1461

11

MF326113

NW_016094701

214,116

47,337

53,345

6008

+

  

CYP2X25

1461

11

MF326114

NW_016096522

9746

N/A

5537

N/A

+

  

CYP2X26

1479

11

MF326115

NW_016094701

214,116

56,638

71,729

≈ 15,091

+

  

CYP2X27

1458

11

MF326116

NW_016094701

214,116

35,726

45,002

≈ 9276

+

  

CYP2Y9

1476

9

MF326117

NW_016094386

1,104,698

13,051

9566

3485

  

CYP2Z6

1515

9

MF326118

NW_016094248

6,824,951

3,433,325

3,429,696

3629

  

CYP2Z6-like*

1500

9

MF326119

NW_016094248

6,824,951

3,433,325

3,429,696

3629

 

Family17

CYP17A1

1548

8

MF326142

NW_016094300

2,667,381

1,094,110

1,104,539

10,429

+

  

CYP17A2

1539

9

MF326143

NW_016095167

43,372

7511

12,189

4678

+

 

Family21

CYP21A1

1572

12

MF326147

NW_016094332

1,971,969

1,370,794

1,374,238

3444

+

Clan3

Family3

CYP3A176

1530

13

MF326120

NW_016094240

11,911,191

8,795,434

8,800,292

4858

+

  

CYP3A177*

1548

12

MF326121

NW_016094240

11,911,191

8,795,434

8,799,969

4535

+

  

CYP3B10

1485

13

MF326122

NW_016094243

9,347,475

2,955,181

2,949,946

5235

 

Family5

CYP5A1

1701

13

MF326127

NW_016094285

3,446,830

2,031,496

2,035,382

3886

+

  

CYP5A2

1662

13

MF326128

NW_016094285

3,446,830

2,037,833

2,044,122

≈ 6289

  

CYP5A3

1656

13

MF326129

NW_016094285

3,446,830

2,046,601

2,051,404

4803

+

  

CYP5A4

1722

13

MF326130

NW_016094285

3,446,830

2,052,641

2,057,508

4867

+

  

CYP5A6

1668

13

MF326131

NW_016094285

3,446,830

2,068,924

2,077,955

9031

+

Clan4

Family4

CYP4F128

1617

13

MF326123

NW_016094474

596,310

272,969

277,073

4104

+

  

CYP4T17

1539

12

MF326124

NW_016094273

4,016,886

3,420,192

3,425,137

4945

+

  

CYP4T18*

1575

10

MF326125

NW_016094273

4,016,886

3,420,192

3,425,137

4945

+

  

CYP4V2

1623

11

MF326126

NW_016094240

11,911,191

7,707,782

7,701,568

6214

Clan7

Family7

CYP7A1

1539

8

MF326132

NW_016094845

138,999

31,992

26,587

5405

  

CYP7C1

1563

5

MF326133

NW_016096556

9473

9282

6496

2786

 

Family8

CYP8A1

1446

10

MF326134

NW_016094274

3,987,237

3,283,074

3,288,823

5749

+

  

CYP8A2

1467

10

MF326135

NW_016094250

6,612,770

5,812,657

5,818,781

6124

+

  

CYP8A2-like*

1521

10

MF326136

NW_016094250

6,612,770

5,810,209

5,818,781

8572

+

  

CYP8B1

1530

1

MF326137

NW_016094328

2,090,648

988,883

990,412

1529

+

  

CYP8B14

1530

1

MF326138

NW_016094328

2,090,648

995,773

997,302

1529

+

Clan19

Family19

CYP19A1

1551

9

MF326144

NW_016094822

150,677

61,672

N/A

N/A

+

  

CYP19A2

1518

10

MF326145

NW_016094246

8,378,829

5,605,491

5,608,355

2864

+

Clan20

Family20

CYP20A1

1389

13

MF326146

NW_016094638

271,067

153,258

157,717

4459

+

Clan26

Family26

CYP26A1

1467

7

MF326149

NW_016094402

996,503

529,169

537,817

8648

+

  

CYP26B1

1539

7

MF326150

NW_016094716

201,440

39,690

10,153

≈ 29,537

  

CYP26C1

1608

7

MF326151

NW_016094465

632,235

493,674

502,741

9067

+

Clan46

Family46

CYP46A1

1512

15

MF326156

NW_016094252

6,456,249

3,898,087

3,893,214

4873

  

CYP46A2

1515

15

MF326157

NW_016094252

6,456,249

3,614,329

3,621,112

6783

+

  

CYP46A4

1527

15

MF326158

NW_016094252

6,456,249

3,606,320

3,612,983

6663

+

  

CYP46A5

1515

15

MF326159

NW_016094252

6,456,249

3,589,333

3,605,213

15,880

+

Clan51

Family51

CYP51A1

1497

10

MF326160

NW_016094242

10,095,097

8,228,424

8,223,720

4704

Clanmt

Family11

CYP11A1

1572

9

MF326140

NW_016094246

8,378,829

4,196,019

4,193,094

2925

  

CYP11C1V1

1632

9

MF326141

NW_016094273

4,016,886

9463

16,380

6917

+

 

Family24

CYP24A1

1542

11

MF326148

NW_016094251

6,557,273

3,641,436

3,635,899

5537

 

Family27

CYP27A1

1593

9

MF326152

NW_016094376

1,154,157

145,481

140,290

5191

  

CYP27A3

1614

11

MF326153

NW_016094297

2,892,702

2,218,077

2,237,829

≈ 19,752

+

  

CYP27B1

1566

9

MF326154

NW_016094274

3,987,237

1,230,560

1,227,105

3455

  

CYP27C1

1623

9

MF326155

NW_016094245

8,651,236

41,377

52,309

≈ 10,932

+

*Alternatively spliced transcript of the gene directly above

N/A, the exact location of the gene could not be determined because the gene was mapped to the end of the scaffold

≈ Approximate genome size because the scaffold contains ‘Ns’ in the gene area

Homology of CYP genes in other fish

Molecular phylogenetic analysis based on the inferred amino acid sequences was used to characterize the relationship of K. marmoratus CYP genes with CYP genes in other intensively studied fish species such as zebrafish (D. rerio), Japanese medaka (Oryzias latipes), and fugu (F. rubripes) (Fig. 2). The phylogenetic tree indicated that the clan structure was robust among these fish species with the CYP genes in clan 2 showing the most expanded pattern in K. marmoratus (Fig. 2). Compared with the zebrafish CYP genes, the K. marmoratus CYP genes were arranged into similar subfamilies, with the exception that CYP39, CYP2AA, and CYP2AE were lost in K. marmoratus (Fig. 3). For the CYP1, CYP17, CYP19, CYP20, CYP21, and CYP46 families, the gene members and their structures in K. marmoratus were similar to those in zebrafish but with different degrees of sequence similarity. Each CYP2R1 and CYP2U1 subfamily has a single CYP gene consisting of five exons. These genes can be considered to be orthologs of CYP2R1 and CYP2U1 in humans and in other fish [12, 15, 25]. CYP1A, CYP1B, CYP2U, and CYP2R appear to be evolutionarily conserved across species. In K. marmoratus, the CYP26 family consists of CYP26A1, CYP26B1, and CYP26C1, as shown in zebrafish. In both species, CYP26A1 and CYP26C1 showed similar gene structures. While zebrafish CYP26B1 has six exons, K. marmoratus CYP26B1 has seven exons. This difference is because the 3rd exon in zebrafish is split into two exons, thus forming the 3rd and 4th exons in K. marmoratus. The CYP2 family is largest in K. marmoratus and consists of 32 genes in nine subfamilies. The nine genes (CYP2N22, CYP2N23, CYP2AD12, CYP2AD-iso, CYP2P16, CYP2P17, CYP2P18, CYP2P19, and CYP2P20) in the three CYP2 families are homologous to human CYP2J2 because phylogenetic analysis grouped them together into a clade with the zebrafish CYP2 subfamilies (CYP2N, CYP2P, CYP2V, CYP2AD, and CYP2AE) (Additional file 2: Figure S2). All nine genes have been reported to be located in tandem on a scaffold (NW_016094248) and to share synteny with 11 zebrafish genes [24]. Four CYP2X genes are present in two separate scaffolds. The CYP2X subfamily showed a different gene structure from other members in the CYP2 family in this species with the exceptions of CYP2R1 and CYP2U1. Gene members in CYP2X have 11 exons instead of 9 (Table 1), because the 5th and 7th exons are split into two exons each. CYP2X25 is located on scaffold NW_016096522, while the other three CYP2Xs (CYP2X27, CYP2X24, and CYP2X26) are located in tandem on scaffold NW_016094701 (Fig. 1). Based on their sequence identity and the phylogenetic analysis results, we predicted that these four genes would be on the same scaffold. While the best mapping position of CYP2X25 was on scaffold NW_016096522, the 2nd best location was the same area of CYP2X26. This finding is likely because the two proteins share 86% amino acid sequence similarity and the genes share 90% nucleotide sequence identity. Considering that the gaps in the area spanning the CYPX26 gene on scaffolds NW_016094701 and NW_016096522 were relatively short, we suspected that an assembly error had occurred in the region. In order to confirm whether this was assembly errors or not, we mapped four CYP2X genes onto the published genome scaffolds of another killifish strain with the higher number of contigs [22]. Unfortunately, only two CYP2X genes (CYP2X24 and CYP2X25) were mapped onto one scaffold. However, CYP2X25, which was isolated in this study, was mapped to one scaffold with one of four genes together and the scaffold was mapped back onto the CYP2Xs-containing scaffold (NW_016094701) of this study. Based on this analysis, this isolation of CYP2X25 is more likely due to the assembly error, instead of the translocation.
Fig. 2
Fig. 2

Phylogenetic tree of cytochrome P450 genes in K. marmoratus and other teleosts. Km, Kryptolebias marmoratus; Ol, Oryzia latipes; Dr., Danio rerio; Tr, Takifugu rubripes

Fig. 3
Fig. 3

Comparison of cytochrome P450 subfamily member homologies among humans, zebrafish, and K. marmoratus. Image is modified from Nelson (2003)

Tandem duplicated CYP genes

Similar to the CYP evolution patterns in other animals, tandem duplication of a number of CYP genes was observed in the K. marmoratus genome. Of 74 CYP genes from K. marmoratus, we examined the region of tandem duplicated CYP genes to investigate the duplicated pattern in the genome. Eight scaffolds contained more than two copies of tandem duplicated CYP genes, five of which had CYP genes with more than four copies (Fig. 1). Of CYP2K subfamily, ten CYP2K genes (CYP2K39, CYP2K40, CYP2K41, CYP2K42, CYP2K43, CYP2K44, CYP2K45, CYP2K46 CYP2K51, and CYP2K52) were clustered in the 48-kb region of scaffold NW_016094323 and four CYP2K genes (CYP2K47, CYP2K48, CYP2K49, and CYP2K50) were in the 40 kb region of scaffold NW_016094341 (Figs. 1 and 4). Synteny analysis revealed that zebrafish have eight CYP2K genes clustered in a homologous region (116 kb), whereas T. rubripes and O. latipes have only two copies of CYP2K genes in the 9-kb and 10-kb regions, respectively (Fig. 4a). Four CYP2K genes comprise another cluster on scaffold NW_016094341 (Fig. 4). Phylogenetic analysis of CYP2K genes in fish (with human genes as the outgroup) showed that the four CYP2K genes are similar to medaka-CYP2KP29 and medaka-CYP2K30, which are located on chromosome 24 (Fig. 5). Synteny analysis of this region did not identify homologous genes outside the clusters for any species (Fig. 4). In addition, the CYP5A tandem genes and the CYP46A tandem genes were clustered in scaffolds NW_016094285 and NW_016094252, respectively (Fig. 1). While zebrafish has only one gene in the CYP5A subfamily, K. marmoratus has five copies of CYP5A genes (5A1, 5A2, 5A3, 5A4, and 5A6). These copies were also arrayed in tandem on scaffold NW_016094285 (Figs. 1 and 6a). Synteny analysis showed homology with zebrafish chromosome 18 (Fig. 4b). In the CYP46A subfamily, CYP46A1, CYP46A2, CYP46A4, and CYP46A5 also showed tandem duplication on scaffold NW_016094252 in the K. marmoratus genome (Fig. 6b). This region seemed to share synteny with D. rerio chromosome 20, Japanese medaka chromosome 24, and Fugu chromosome 16 (Fig. 6b), although some gene order mismatches in both K. marmoratus and D. rerio were observed, compared with pufferfish and Japanese medaka. Considering the presence of a big gap (~170 kb) between bcl-11 and CYP46A1 in K. marmoratus, we also suspected the assembly error in this region. However, comparing with the genome assembly by Kelley et al. [22], the gene order in K. marmoratus in both assemblies was consistent. In pufferfish and Japanese medaka, two copies of CYP46A-like tandem genes were surrounded by the genes, ccdc85cb and ism2b, in the synteny region. It seemed that CYP46As and neighboring genes, including ccdc85cb, CCNK, and bcl-11, were inverted in the area with an additional duplication of CYP46A copies, which was uncertain if the tandem duplication occurred before or after the inversion. Thus, based on the synteny analysis of the zebrafish, gene duplication probably has occurred prior to the inversion, although zebrafish seems to have small difference in the evolutionary repertoires in this region.
Fig. 4
Fig. 4

Synteny analysis of CYP2K genes of K. marmoratus and other teleosts. a) Synteny of the CYP2K39–46, 2 K51, and 2 K52 genes. b) Synteny of the CYP2K47–50 genes

Fig. 5
Fig. 5

Phylogenetic tree of the CYP2K subfamily in K. marmoratus and other fish species with an outgroup (CYP2W1) from human. Colored bars at right side of the tree stand for grouping gene copies on particular chromosomes or scaffolds. Km, Kryptolebias marmoratus; Ol, Oryzias latipes; Dr., Danio rerio; Tr, Takifugu rubripes; Hs, Homo sapiens

Fig. 6
Fig. 6

Synteny analysis of tandemly duplicated CYP5A (a) and CYP46A genes (b)

Discussion

Comparison of CYP subfamilies in teleosts

Using whole genome sequences and RNA-seq data, we identified a full complement of CYP genes in the K. marmoratus genome. K. marmoratus has a total of 74 CYP genes in 17 families within 10 clans. Ten clans and 19 families have been reported in vertebrate animals [6, 7, 9]. Among the 19 CYP families of vertebrates, we did not identify the CYP39 or CYP16 family in K. marmoratus. CYP39 families have recently been identified in teleost fish. Before this discovery, the CYP39 family was thought to be unique to mammals or to have arisen in the tetrapod lineage after it diverged from fish [8]. Goldstone et al. [12] reported the presence of CYP39 genes in zebrafish. However, CYP39 genes were not found in other published fish genomes, including Fugu. K. marmoratus does not have the CYP16 family. This family was lost in mammals and is also absent from zebrafish. Out of all published fish genomes, CYP16 was reported only in Fugu [15].

Gene expansion by lineage-specific duplication

While CYP genes are commonly expanded by tandem duplication [6, 15, 2628], the basic mechanisms by which a certain gene is selected for such duplication remain unclear. We predominantly focused on comparing the K. marmoratus CYP genes with the zebrafish CYP genes because the two species have similar total numbers of CYP genes and the homology of their CYP genes with all human CYP genes is known (Fig. 3). Phylogenetic and synteny analyses revealed lineage-specific duplication of many CYP genes, which was apparent in some tandem duplications of CYP genes. Among the eight genomic regions where tandemly duplicated CYP genes were located in the K. marmoratus genome, five subfamilies (CYP2P, CYP2AD, CYP2K, CYP5A, CYP8B, and CYP46A) in the four regions showed lineage-specific duplication (Figs. 1 and 2). Although the gene members in the subfamilies were duplicated in a lineage-specific manner with different copy numbers, the syntenies (including the tandem duplicated genes) were the same between the two species (Fig. 3) [24]. Specifically, CYP46As in K. marmoratus and zebrafish showed strong homology within gene members and gene structures, albeit with different degrees of sequence similarity, compared to other subfamilies with the same syntenies. However, we note that gene order in the K. marmoratus CYP46As synteny is different, suggesting that both species appear to have undergone evolutionary events independently after the tandem duplication of CYP46A. CYP46A1 has been identified in many species, including teleosts, and plays an important role in cholesterol turnover in the central nervous system in vertebrates [29]. In humans, CYP46A1 functions as a cholesterol 24(S)-hydroxylase and a 24-hydroxy-cholesterol-hydroxylase [2931]. Although mutations in CYP46A1 have been associated with neurodegenerative diseases such as Alzheimer’s and Huntington’s disease in humans [3235], the function of CYP46A1 in teleosts has not been studied. Ten CYP2Ks on scaffold NW_016094323 belong to the subfamily that shows the highest level of lineage-specific tandem duplication in K. marmoratus, while four CYP2Ks on another scaffold do not seem to be duplicated in a lineage-specific manner and share synteny with those of zebrafish (Figs. 1 and 4).

Kryptolebias marmoratus-specific gene expansion

Cytochrome P450 enzymes have two main functions: metabolism of endogenous molecules and detoxification of xenobiotic compounds. Phylogenetic studies have suggested that CYP genes, which are responsible for the endogenous functions, are stable across animal species and that copy expansion is rare [11]. In contrast, CYP genes related to xenobiotic metabolism have been shown to be phylogenetically unstable with a relatively high rate of birth-death evolution [11, 36, 37]. Within this context, the most apparent gene expansion due to lineage-specific tandem duplication in K. marmoratus occurred in two CYP subfamilies, CYP2K and CYP5A. Similar to what has been observed in other teleost species, CYP2K was the most expanded subfamily in K. marmoratus (Fig. 4). Since CYP2Ks are highly expanded in teleosts and the members in CYP2K vary across species, the functions of CYP2K genes have received comparatively little attention. CYP2Ks share synteny with human CYP2W1, a tumor-specific CYP that oxidizes indole and chlorzoxazone [3840]. Rainbow trout CYP2K1 and zebrafish CYP2K6 show an orthologous relationship and both metabolize aflatoxin B1 (AFB1) to exo-8,9-AFB1 epoxide, which is carcinogenic. However, their metabolic features differ somewhat, as only rainbow trout CYP2K1 can metabolize lauric acid [13, 41]. Based on the clan identity of CYP2K, the expansion by high level tandem duplication may have resulted from the diversity of exogenous xenobiotic substrates. Thus, rapid evolutionary selection could have favored tandem duplication as a means of coping with xenobiotic stress.

Kryptolebias marmoratus have five copies (CYP5A1, CYP5A2, CYP5A3, CYP5A4, and CYP5A6) of CYP5A subfamily members, while other teleosts including zebrafish, pufferfish, and channel catfish maintain the subfamily with a single gene copy [8, 12, 15]. CYP5A1 (thromboxane A2 synthase) catalyzes the conversion of prostaglandin H2 into thromboxane A2 and has been associated with human cardiovascular disease related to platelet aggregation [42]. Rather than metabolizing xenobiotics, CYP5A1 seems to be primarily involved in endogenous functions. Considering that genes involved in conserved endogenous functions are rarely expanded, the K. marmoratus-specific expansion of CYP5A is an interesting finding. Gene duplication and subsequent divergence of the duplicated copies are basic mechanisms by which gene subfamilies are formed and are considered essential sources of genetic complexity and evolutionary change [4345]. Gene expansion by tandem duplication leading to gene clusters appears to be an important mechanism by which these needs are met for cytochrome P450 in various species. Analysis of the expression profiles of the CYP genes expanded specifically in K. marmoratus could generate insight into the endogenous and exogenous environmental factors driving CYP evolution.

Methods

Fish rearing

Kryptolebias marmoratus mangrove killifish were reared at the aquarium facility of Sungkwunkwan University (Suwon, South Korea). The fish were maintained in an automated flow-through system with constant water quality (pH 8.0 and 15 practical salinity units [psu]) at 25 °C under a 12/12-h light/dark cycle. The fish were maintained in glass aquaria (20 L capacity). Each aquarium accommodated 40 fish larvae (length ≈ 1.0 ± 0.2 cm, approximately 7 days post-hatching [dph]). Fish were fed with Artemia spp. brine shrimp (<24 h after hatching) once per day.

Genome-wide identification of CYP genes

The assembled K. marmoratus whole genome (ASM164957v1) and transcriptome (SRX1765072) sequences have been published [23]. Using CYP gene sequences in other teleosts including zebrafish (D. rerio), Japanese medaka (O. latipes), and pufferfish (F. rubripes) (Additional file 3: Table S1), we searched for putative CYP sequences in the K. marmoratus genome. BLAST analysis of coding sequences was performed to confirm the sequence similarities. All CYP gene sequences were obtained by performing BLASTp searches of the fully assembled transcripts against the nonredundant (NR) NCBI database. A significant hit was defined as a hit with an E-value ≤10−5. The putative CYP coding sequences from K. marmoratus were translated into amino acids; further annotation was carried out by Prof. David R. Nelson (University of Tennessee Health Science Center) and Dr. Gared V. Goldstone (Woods Hole Oceanographic Institution). Gene structure was identified by comparing sequences between the genome scaffolds and transcriptomes. Synteny analysis was carried out by comparing the CYP gene clusters in K. marmoratus with those of Japanese medaka (O. latipes), pufferfish (T. rubripes), and zebrafish (D. rerio). Data were collected from the published chromosome assembly information at Ensemble (https://www.ensembl.org/index.html) with further identification.

Phylogenetic analysis

The entire amino acid sequences encoded by the CYP genes of zebrafish (D. rerio) (Dr-CYPs) and Japanese medaka (O. latipes) (Ol-CYPs) were retrieved from GenBank (Additional file 3: Table S1). Multiple alignments of amino acid sequences from K. mamoratus, Japanese medaka, and zebrafish were performed with Clustal algorithm [46]. To establish a best-fit substitution model for phylogenetic analysis, the model showing the lowest score according to the Bayesian information criterion (BIC) [47] and the Akaike information criterion (AICc) [48, 49] was determined by maximum likelihood (ML) analysis. According to the results of the model test, the LG + γ + I model was chosen to generate a phylogenetic tree using MEGA6 software (Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA) [50]. For phylogenetic analysis, full-length protein sequences were aligned and a phylogenetic tree was obtained as described above with an additional bootstrapping test (1000 replicates) [51]. Phylogeny data were deposited in the Treebase repository with the accession number 22004.

Conclusions

In this study, we identified and annotated the full complement of 74 CYP genes in K. marmoratus. We also analyzed the co-localized CYP2K, CYP5A, and CYP46A subfamilies and characterized their structural features.

Abbreviations

CYP: 

Cytochrome P450

Declarations

Acknowledgements

We thank two anonymous reviewers for their valuable comments on the manuscript.

Funding

This work was supported by a grant to Jae-Seong Lee from the Collaborative Genome Program (PJT200620), which is funded by the Korean Ministry of Oceans and Fisheries and also supported by a grant to Bo-Young Lee from the National Research Foundation (NRF-2017R1D1A1B03036026).

Availability of data and materials

Sequencing data is available via NCBI by the accession number below.

K. marmoratus whole genome sequence: ASM164957v1.

K. mamoratus transcriptome sequences: SRX1765072.

K. mamoratus CYP sequences: MF326082-MF326155.

Phylogy tree data of Figs. 2 and 5: http://purl.org/phylo/treebase/phylows/study/TB2:S22052

Authors’ contributions

BYL, DHK, HSK and BMK analyzed data and wrote the paper. JH and JSL discussed all the issues during experiment and finally confirmed the manuscript. All authors have read and approved the manuscript.

Ethics approval

All animal handling and experimental procedures were approved by the Animal Welfare Ethical Committee and the Animal Experimental Ethics Committee of the Sungkyunkwan University (Suwon, South Korea). Experiments were carried out in accordance with the approved guidelines of the Animal Experimental Ethics Committee of the Sungkyunkwan University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Authors’ Affiliations

(1)
Department of Biological Science, College of Science, Sungkyunkwan University, Suwon, 16419, South Korea

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