Hox genes reveal genomic DNA variation in tetraploid hybrids derived from Carassius auratus red var. (female) × Megalobrama amblycephala (male).

BACKGROUND: Allotetraploid F1 hybrids (4nF1) (AABB, 4n = 148) were generated from the distant hybridization of Carassius auratus red var. (RCC) (AA, 2n = 100) (♀) × Megalobrama amblycephala (BSB) (BB, 2n = 48) (♂). It has been reported that Hox gene clusters are highly conserved among plants and vertebrates. In this study, we investigated the genomic organization of Hox gene clusters in the allotetraploid F1 hybrids and their parents to investigate the polyploidization process.
RESULTS: There were three copies of Hox genes in the 4nF1 hybrids, two copies in RCC and one copy in BSB. In addition, obvious variation and pseudogenization were observed in some Hox genes from 4nF1.
CONCLUSION: Our results reveal the influence of polyploidization on the organization and evolution of Hox gene clusters in fish and also clarify some aspects of vertebrate genome evolution.

Background

Polyploidization is a widespread mechanism for speciation in eukaryotes, especially plants and vertebrates [1-5]. Polyploids with duplicated genomes may originate from a single species (autopolyploidy) or from different species through interspecific hybridization (allopolyploidy) [6]. Allopolyploids are prevalent in nature, suggesting there is an evolutionary advantage to obtaining multiple sets of genetic material for adaptation and development [7]. However, the molecular mechanisms underlying the processes and consequences of allopolyploidy remain unclear [8]. Polyploidy is relatively rare in animals compared with plants, and the influence of polyploidization on intragenomic variation in polyploid animals is poorly understood. In our earlier study, we successfully obtained fertile tetraploid hybrids from Carassius auratus red var. (RCC)(♀) × Megalobrama amblycephala (BSB)(♂) [9, 10]. RCC has 100 chromosomes and belongs to the Cyprinidae subfamily, while BSB has 48 chromosomes and belongs to the Cultrinae subfamily [11]. These new polyploid hybrids represent unique specimens for studying genomic changes in F1 hybrids and could significantly contribute to our understanding of evolution.

Hox genes, a set of important developmental regulatory genes, are highly conserved and typically organized cluster [12]. In vertebrates, Hox genes encode two exons, and the highly conserved homeodomain (60 aa) is encoded by the second exon [13]. Recent research has shown that gene duplication, sequence variation, and selective pressure played crucial roles in the origin and evolution of Hox genes [14]. The earliest indications of genome duplication came from the comparative analysis of Hox genes and clusters from different chordate lineages [15-18].

In general, polyploidization plays an important role in fish evolution [19]. The purpose of this research was to study the effects of allopolyploidization on Hox gene organization and evolution. In this article, three distinct Hox duplicates were observed in the 4nF1 genome, compared with two copies in RCC and one copy in BSB. Our data reveal the genetic variation and evolutionary characteristics of the Hox gene family in 4nF1 and provide new insights into their evolutionary patterns.

Results

Sequence information for RCC, BSB and 4nF1 clones

Using 11 pairs of degenerate primers (Additional file 1: Table S1), we obtained partial sequence information for eight putative Hox genes from RCC, four putative Hox genes from BSB, and 32 putative Hox genes from the 4nF1. All these fragments were between 700 and 1500 bp long and included the exon1-intron-exon2 region (Table 1). To avoid biased amplification of only one Hox gene copy, we selected 20 clones of each gene from 4nF1, 20 clones of each gene from RCC and 80 clones from BSB (20 clones for each Hox gene PCR fragment). All fragments from RCC, BSB and the 4nF1 were confirmed to be Hox gene sequences, and each included the homeobox. All Hox sequences have been submitted to GenBank; their accession numbers are listed in Table 1.

Table 1

PCR amplification bands in RCC, BSB and 4nF1

Species	Locus	Size (bp)	Exon1 (bp)	Intron (bp)	Exon 2 (bp)	GenBank accession no.
RCC	HoxA4ai	1181	89-500	501-974	975-1181	JX282274
	HoxA4aii	1184	89-500	501-977	978-1184	JX282275
	HoxA9ai	867	1-381	382-670	671-867	JX282276
	HoxA9aii	819	1-381	382-622	623-819	JX282277
	HoxA2bi	1486	1-314	315-901	902-1486	JX282278
	HoxA2bii	1448	1-314	315-863	864-1448	JX282279
	HoxD4ai	960	1-315	316-735	736-960	JX282280
	HoxD4aii	952	1-315	316-719	728-952	JX282281
BSB	HoxA4a	1188	89-500	501-981	982-1188	JX282282
	HoxA9a	879	1-381	382-682	683-879	JX282283
	HoxA2b	1479	1-311	312-894	895-1479	JX282284
	HoxD4a	911	1-306	307-686	687-911	JX282285
4nF1	HoxA4ai	1183	89-500	501-976	977-1183	JQ901468
	HoxA4aii	1169	89-500	501-962	963-1169	JX282286
	HoxA4aiii	1177	89-500	501-970	971-1177	JX282287
	HoxA9ai	867	1-381	382-670	671-867	JX282288
	HoxA9aii	817	1-381	382-620	621-817	JX282289
	HoxA9aiii	863	1-381	382-666	667-863	JX282290
	HoxA2bi	1486	1-314	315-901	902-1486	JX282291
	HoxA2bii	1448	1-314	315-863	864-1448	JX282292
	HoxA2biii	1475	1-314	315-890	891-1475	JX282293
	HoxA11bi	1251	3-590	591-1153	1154-1251	JX282294
	HoxA11bii	1411	3-590	591-1313	1314-1411	JX282295
	HoxA11biii	1437	3-590	591-1339	1340-1437	JX282296
	HoxB1bi	733	1-477	478-567	568-733	JX282297
	HoxB1bii	734	1-477	478-568	569-734	JX282298
	HoxB1biii	731	1-477	478-565	566-731	JX282299
	HoxB5bi	1196	1-561	562-990	991-1196	JX282300
	HoxB5bii	1195	1-561	562-989	990-1196	JX282301
	HoxB5biii	1190	1-561	562-984	985-1190	JX282302
	HoxB6bi	807	1-169	170-667	668-807	JX282303
	HoxB6bii	819	1-169	170-679	680-819	JX282304
	HoxB6biii	812	1-169	170-672	673-819	JX282304
	HoxC4ai	1176	1-410	411-935	936-1176	JX282306
	HoxC4aii	1173	1-410	411-932	933-1173	JX282307
	HoxC4aiii	1169	1-410	411-928	929-1169	JX282308
	HoxC4a-1	1179	1-410	411-938	939-1179	JX282309
	HoxD4ai	960	1-315	316-735	736-960	JX282310
	HoxD4aii	952	1-315	316-719	728-952	JX282311
	HoxD4aiii Ψ	942	-	-	-	JX282312
	HoxD9a Ψ	897	-	-	-	JX282313
	HoxD10a Ψ	1481	-	-	-	JX282314
	HoxD10aii	1554	1-589	590-1324	1325-1554	JX282315
	HoxD10aiii	1495	1-592	593-1265	1266-1495	JX282316

Ψ denotes a pseudogene

Molecular organization of the Hox gene sequence

We comparatively analysed the inferred amino acid sequences of the Hox genes in 4nF1 with those in zebrafish, fugu, medaka, and BSB (Additional file 1: Table S2), which indicated that the 4nF1 sequences were similar to those of the other species. The organization of the Hox clusters in 4nF1 is shown in Fig. 1. The clusters can be summarized as HoxAai, HoxAaii, HoxAaiii, HoxAbi, HoxAbii, HoxAbiii, HoxBai, HoxBaii, HoxBaiii, HoxBbi, HoxBbii, HoxBbiii, HoxCai, HoxCaii, HoxCaiii, HoxCbi, HoxCbii, HoxCbiii, HoxDai, HoxDaii, and HoxDaiii (Table 1). Among these copies, we found that HoxD4aiiiΨ, HoxD9aΨ, and HoxD10aΨ in 4nF1 were pseudogenes (Fig. 2). Two deletions at codons 316 and 317 in the coding region of HoxD4aiiiΨ suggested that it was a pseudogene. The alignment of the putative HoxD4a sequences is shown in Fig. 2a. HoxD9aΨ has become a pseudogene because a stop codon prematurely terminates expression of the full-length functional product (Fig. 2b). An insertion was observed at codon 593 in the HoxD10aΨ coding region; alignment of the putative HoxD10a duplicated sequences is shown in Fig. 2c. HoxD10aΨ had an inserted G nucleotide compared with HoxD10aiii, whereas a T in HoxD4aΨ was replaced by a G compared with HoxD4ai. Thus, non-functionalization is a possible fate for some duplicated Hox genes. The GC levels of the pseudogenes tended to be lower than that of their counterpart genes (Additional file 1: Table S3). For instance, in 4nF1, the exons of the pseudogene HoxD4aiiiΨ exhibited a GC content of 50.1%, which was lower than that of its functional counterparts HoxD4ai and HoxD4aii (51.3%, 52.1%). As shown in Additional file 1: Table S3, the exon GC content of the pseudogene HoxD10aiΨ was 49.4%, which was lower than those of its putative functional counterparts HoxD10aii and HoxD10aiii (49.6% and 49.9%, respectively) in 4nF1. Similarly, the exon GC content of the pseudogene HoxD9aΨ (43.3%) was slightly lower than that of its putative functional HoxB1b paralogues (50.1%, 50.2%, and 50.2%). During duplication, one copy typically remains functional, whereas the other copy may lose its function, which generally leads to a decreased GC level for the non-functional gene.

Fig. 1

Hox cluster architecture in 4nF1 compared with zebrafish. We identified a total of 32 Hox genes. Nine Hox genes were present in three copies, one Hox gene was present in four copies, and one was present as a single copy in 4nF1. Copies of the HoxD9a, HoxD4a, and HoxD10a genes were pseudogenes. Black boxes represent Hox genes from Danio rerio, and “E” refers to EVX (even-skipped related gene). Aa, Ab, Ba, Bb, Ca, Cb, Da and Db refer to classes of genes

Fig. 2

The pseudogenes HoxD9a, HoxD4aiii, and HoxD10ai in 4nF1. a Two deletions occurred in codons 316 and 317 in the coding region of HoxD4aiiiΨ. b HoxD9aΨ became a pseudogene because a stop codon prematurely terminated expression of the full-length functional product. c One insertion occurred in codon 593 in the coding region of HoxD10aiΨ

Phylogenetic relationships

For most genes, such as HoxA4a, HoxB1b, and HoxD10a, three distinct orthologues of the zebrafish genes were identified in 4nF1. These duplicated genes shared a high identity percentage for the deduced amino acid sequences (Additional file 1: Tables S2 and S3). An identity analysis of the putative amino acid sequences suggested that the duplicated sequences were more closely related to each other than to the reported zebrafish orthologues except for the HoxC4aiii sequences. For instance, the percentage nucleotide identity between the HoxA11bi, HoxA11bii, and HoxA11biii orthologues from 4nF1 and HoxA11b from zebrafish was only 89.9%, 89.9%, and 92.4%, respectively. Conversely, the identity between the paralogues HoxA11bi and HoxA11bii, HoxA11bi and HoxA11biii, and HoxA11bii and HoxA11biii in 4nF1 was 98.6%, 96.4%, and 96.0%, respectively (Additional file 1: Table S2 and Fig. 2a). The identity between HoxB1bi and HoxB1bii, HoxB1bi and HoxB1biii, and HoxB1bii and HoxB1biii was 99.5%, 95.7% and 96.2%, whereas the similarity to their zebrafish orthologues was 91.0%, 90.6% and 91.5% (Additional file 1: Table S2 and Fig. 3b). These results showed that HoxA11bi, HoxA11bii, and HoxA11biii as well as HoxB1bi, HoxB1bii and HoxB1biii all share a mostly closed ancestral cluster and are true orthologues of the zebrafish genes HoxA11b and HoxB1b. Analysis of the sequences obtained for HoxC4a suggested that four distinct copies of this gene exist in 4nF1, which were named HoxC4ai, HoxC4aii, HoxC4aiii and HoxC4a-1. The putative amino acid sequence of HoxC4a-1 shares approximately 100%, 100% and 99% similarity to those of HoxC4ai, HoxC4aii, and HoxC4aiii, respectively. However, the nucleotide similarity to all three sequences is 100%, which suggests the mutation was synonymous.

Fig. 3

Maximum likelihood tree inferred from the alignment of amino acid sequences. This tree illustrates the phylogenetic relationships among putative Hox sequences in 4nF1 and reported orthologues from blunt snout bream (Megalobrama amblycephala), zebrafish (Danio rerio), fugu (Fugu rubripes), and medaka (Oryzias latipes). The numbers at the branch nodes indicate percentage bootstrap support for each node based on 1000 replicates. a–d HoxA11b, HoxB1b, HoxB5b, and HoxB6b

To evaluate the speciation of 4nF1, the nucleotide identity percentages among all known representatives of the HoxA4a, HoxA9a, HoxA2b, and HoxD4a gene groups in RCC, BSB, and the 4nF1 were examined (Table 2, Fig. 4). The identities of orthologous ‘i’ or ‘ii’ genes between 4nF1 and RCC were much higher than those between 4nF1 and BSB. For example, the nucleotide identity percentages of the orthologous HoxA4ai, HoxA9ai, HoxA2bi, and HoxD4ai genes between 4nF1 and RCC were 99.5%, 99.4%, 99.6% and 99.6%, respectively. Conversely, the similarity of these genes between 4nF1 and BSB was 97.0%, 92.3.0%, 97.2%, and 93.7%, respectively. Although similarly high identity was observed, the ‘iii’ gene in 4nF1 did not exhibit higher similarity to the gene in RCC or BSB for all four Hox sequence groups, suggesting no obvious orthologous relationship between the two species. Thus, we speculated that the ‘iii’ genes were variants of RCC or BSB genes. For example, the HoxA4aiii, HoxA9aiii, HoxA2biii, and HoxD4aiii genes from 4nF1 and the HoxA4a, HoxA9a, HoxA2b, and HoxD4a genes from BSB shared 98.0%, 92.2%, 97.7%, and 94.0% identity (Table 2).

Table 2

Percentage nucleotide identity (on the left) and percentage amino acid identity (on the right) between duplicated Hox coding regions in 4nF1, RCC, and BSB

	HoxA4a (%)	HoxA9a (%)	HoxA2b (%)	HoxD4a (%)
4nF1 i:4nF1 ii	97.4/97.5	91.8/89.5	96.9/96.9	98.3/97.7
:4nF1 iii	98.5/98.0	98.9/98.9	97.5/97.6	96.2/61.6
:RCC i	99.5/99.5	99.4/99.4	99.6/100.0	99.6/100.0
:RCC ii	97.2/96.6	92.3/90.1	96.5/96.6	98.3/97.7
:BSB	97.0/96.6	92.3/91.6	97.2/97.9	93.7/94.9
4nF1 ii:4nF1 iii	98.3/98.5	91.6/88.5	96.9/95.9	95.3/60.5
:RCC i	97.5/98.0	92.0/89.0	96.8/96.9	98.3/97.7
:RCC ii	98.8/98.5	94.9/92.1	98.3/97.6	100.0/100.0
:BSB	96.7/97.0	91.0/86.4	96.4/96.3	93.3/92.7
4nF1 iii:RCC i	98.7/98.5	99.1/98.4	97.4/97.6	95.9/61.6
:RCC ii	98.2/98.5	92.2/89.0	96.5/95.9	95.3/60.5
:BSB	98.0/98.5	92.2/90.6	97.7/97.3	94.0/59.4
RCC i:RCCii	97.4/97.0	92.5/89.5	96.6/96.6	98.3/97.7
:BSB	97.2/97.0	92.5/91.1	97.1/97.9	94.0/94.9
RCC ii:BSB	96.9/97.0	91.5/88.0	96.2/96.6	93.3/92.7

Values before slashes (/) denote nucleotide identity, values after slashes denote amino acid identity

Fig. 4

Maximum likelihood tree inferred from the alignment of amino acid sequences. This tree illustrates the phylogenetic relationships among putative Hox sequences in 4nF1, BSB, and RCC and the reported orthologues from blunt snout bream (Megalobrama amblycephala), zebrafish (Danio rerio), and medaka (Oryzias latipes). The number at each branch node indicates the percentage bootstrap support for that node based on 1000 replicates. a–c HoxA4a, HoxA9a and HoxA2b.

Discussion

The structure of cloned Hox gene sequences

Prior PCR surveys and genomic library screening have identified interesting variability in Hox gene content among teleosts [12, 15, 16, 20, 21]. Luo et al. [22] estimated there were 14–16 Hox gene clusters in goldfish. Our data suggested 18–21 Hox gene clusters were present in 4nF1, with each was located on a different acrocentric chromosome. The Hox gene clusters in 4nF1 were approximately the sum of the clusters in RCC and BSB, except that some clusters were lost. The topology of the Hox gene maximum likelihood tree (Fig. 3) further suggested that some of the Hox genes orthologous to zebrafish genes were present in two copies in RCC, one copy in BSB, and three copies in 4nF1. However, the third copy did not exhibit notably higher similarity to the gene in RCC or BSB. We speculated that variation and reorganization of the genome likely occurred during polyploidization, resulting in new copies in 4nF1. This might be evidence that allopolyploidization induces a variety of rapid genomic changes in a 4nF1 population [23, 24]. Using sequence alignment in 4nF1, we isolated 32 fragments that can be characterized as HoxA, HoxB, HoxC, and HoxD family genes. However, amplified RCC and BSB DNA were only characterized as the HoxA and HoxD genes. We speculated that the increase in the number of 4nF1 genes might be related to polyploidization. This situation was also observed in our previous study [25, 26]; the number of 4nF1 fragments increased, and some genes from RCC and BSB were lost. At present, although we have no precise data explaining this outcome, we speculate that allotetraploidization might lead to rapid changes in 4nF1 genome diversity. Our study is the first to evaluate the organization of Hox clusters in a 4nF1 population. This theory is also strongly supported by other studies examining Hox genes [22], other gene families [27], and DNA content [28].

The significance of polyploidization

Polyploidization likely increases genomic variation rates, which can result in the formation of new polyploid species [29]. First, the process of polyploidization can itself generate species that are reproductively isolated from their diploid progenitors, increasing the number of species as a by product. For example, several studies have indicated that a polyploidization event occurred in an ancestor of teleost fish shortly after this lineage diverged from the lineage leading to tetrapods [30-32]. Second, an entirely different trait can result in increased rates of polyploidization [6]. Synonymous mutations increase genomic variation. For example, the putative amino acid sequence of HoxC4a-1 shares approximately 100%, 100%, and 99% similarity with those of HoxC4ai, HoxC4aii, and HoxC4aiii, respectively. The identity of their nucleotide sequences is 100%. In the polyploidization process, genome duplication produces abundant genomic DNA, so the organism maintains the dosage balance or rapidly stabilizes the duplicated genomes via retention/exclusion of redundancy. Lynch et al. [33] suggested there are three outcomes in the evolution of duplicate genes: non-functionalization, neo-functionalization and sub-functionalization. Interestingly, we found some pseudogenes in 4nF1, such as HoxD4aiiiΨ, HoxD9aΨ and HoxD10aΨ. Pseudogenes are formed either by random mutations that create stop codons and prematurely terminate full-length functional product expression or by insertions/deletions that shift the reading frame, rendering the translated protein non-functional. We speculate that dosage effects generated selection pressure from the loss of Hox genes or the formation of pseudogenes after whole genome duplication. This pressure is consistent with the expectation that there are Hox clusters in the 4nF1 genome that have lost functional Hox genes due to the reduction of redundancy following the polyploidization event. However, 4nF1 required genetic recombination, mutation, and pseudogenization to reduce the amount of incompatible genetic material and improve fertility [34]. Thus, we unexpectedly obtained autotetraploids with greater fertility among the 4nF1 progeny, and we successfully established an autotetraploid fish line [35]. Our characterization of the Hox gene clusters in tetraploid hybrids improves our understanding of the evolutionary processes occurring after Hox gene duplication in vertebrates.

Conclusions

We identified three copies of Hox genes in 4nF1, two copies in RCC and one copy in BSB. In addition, obvious variation and pseudogene generation were observed in some 4nF1 Hox genes. These results reveal the effects of polyploidization on the organization and evolution of Hox gene clusters in fish and also help to clarify aspects of vertebrate genome evolution.

Methods

DNA extraction

Specimens of 4nF1 (4n = 148), RCC (2n = 100), and BSB (2n = 48) were obtained from the Engineering Research Center of Polyploid Fish Breeding and Reproduction of the State Education Ministry at Hunan Normal University. Fish treatments were carried out according to the regulations for protected wildlife and the Administration of Affairs Concerning Animal Experimentation, and approved by the Science and Technology Bureau of China. Approval from the Department of Wildlife Administration was not required for the experiments conducted in this paper. The fish were deeply anesthetized with 100 mg/L MS-222 (Sigma-Aldrich, St Louis, MO, USA) before dissection. Narcotic drugs was fed before blood sampling. Total genomic DNA was isolated from peripheral blood cells using the standard phenol chloroform extraction procedures described by Sambrook et al. [36].

Cloning and sequencing of Hox genes

We amplified fragments of Hox genes from genomic DNA by PCR amplification using several combinations of degenerate primers (Table 1). PCR was performed in 50-μL reaction volumes using Taq DNA polymerase (TaKaRa, Dalian, China). A typical PCR programme consisted of a denaturation step at 94°C for 5 min; 35 cycles of 98°C for 15 s, 55°C for 45 s and 72°C for 1 min; and a final elongation step at 72°C for 10 min. PCR products were cloned into a T vector and sequenced with an automated ABI 3700 DNA sequencer. The sequences were BLAST searched against the non-redundant protein database maintained at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) to determine their identity.

Sequence comparison and analysis

Sequence homology and variation among the fragments amplified from RCC, BSB and the 4nF1 were analysed in BioEdit [37, 38]. Partial DNA sequences for each gene were verified using a BLASTx search. To increase the probability of detecting duplicated paralogues and circumventing PCR errors, we sequenced 20 clones for each gene from 4nF1, RCC and BSB. The obtained sequences were screened for Hox gene fragments using different BLAST searches (BLASTn, BLASTp, and BLASTx) against GenBank (http://www.ncbi.nlm.gov/Blast.cgi). Then, we evaluated the organization of the 4nF1 Hox clusters compared to RCC and BSB to characterize the Hox genes.

Phylogenetic analysis

Using Clustal X 1.81, the derived amino acid sequences of these fragments were aligned with the Hox genes from BSB, zebrafish, fugu, medaka and other teleosts retrieved from GenBank [38]. Regions of sequences that were difficult to align were removed from the alignment. Gaps were also removed from the alignment. The maximum likelihood method implemented in the online software RAxML was used to construct a phylogenetic tree [39].