Histone H3 gene in the Pacific oyster, Crassostrea gigas Thunberg, 1793: molecular and cytogenetic characterisations

The Pacific oyster, Crassostrea gigas Thunberg, 1793 (2n = 20) is an economically important mollusc species cultured throughout the world. The most frequently used technique for molecular cytogenetic studies is fluorescence in situ hybridisation which offers new opportunities for the identification of oyster chromosomes. In oysters, it has been used to locate telomeric sequences, satellite DNA, simple sequence repeats, ribosomal RNA genes, and bacteriophage P1 clones. However, regarding chromosome identification, no study has been done with histone H3 gene. Histone H3 is among the most conserved eukaryotic proteins. Most histone H3 genes are repeatedly organised into clusters, which make them an ideal chromosomal marker. In bivalves, some data exist concerning sequence information but little knowledge is available concerning the physical mapping of histone genes. The histone H3 gene was sequenced in C. gigas and phylogenetic analysis revealed that C. gigas was more closely related to Ostrea edulis Linnaeus, 1758 and species of the genus Mytilus Linnaeus, 1758. In C. gigas, the histone H3 gene was mapped on two different pairs of chromosomes, one at an interstitial site on the long arm of chromosome pair 4, and the other on the telomeres of the smaller chromosome pair (pair 10). Polymorphism was detected on the telomeres of pair 10, once it was possible to observe single or double signals. Comparative chromosomal mapping should improve our understanding of bivalve genome organisation.


INTRODUCTION
The Pacifi c oyster, Crassostrea gigas Thunberg, 1793, is an economically important oyster species cultured throughout the world.This species has a diploid chromosome number of 2n = 20, and all chromosomes are metacentric.Several banding tehniques were already applied to oyster chromosomes.They are very useful for the identifi cation of individual chromosomes and also of particular regions of chromosomes."Classical" cytogenetic banding (G-, C-and NOR (nucleolus organiser regions) -banding techniques) was already performed in several species of the genus Crassostrea Sacco, 1897 Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics (review in Leitão, Chaves, 2008).Molecular cytogenetic banding was also applied to several species of the genus Crassostrea (review in Leitão, Chaves, 2008).RE (restriction endonucleases) -banding (Leitão et al., 2004(Leitão et al., , 2007;;Bouilly et al., 2005;Cross et al., 2005) and FISH (fl uorescence in situ hybridisation) technique with different kinds of labelled probes were already produced in different species of the genus Crassostrea.
Histone proteins are the major constituents of the eukaryotic chromatin.The family of histone proteins has been subdivided into the core histones (H2A, H2B, H3, and H4), which form the core particle of the nucleosome, and the linker histones (H1), which are involved in the generation of the higher-order chromatin structure (Thoma et al., 1979).Histone H3 is one of the most conserved eukaryotic proteins (Miller et al., 1993) and is an excellent probe for chromosome mapping as histone genes are usually organised in clusters (Maxson et al., 1983).Histone gene mapping has been achieved in a few species.In bivalves, little knowledge is available concerning the physical mapping of histone genes.Only fi ve species were studied until now: a mussel (Eirín-López et al., 2002, 2004) and four scallops (Zhang et al., 2007).In Mytilidae, FISH experiments on Mytilus galloprovincialis Lamarck, 1819 chromosomes revealed the presence of three pairs of signals in three chromosome pairs with the linker histone H1 probe (Eirín-López et al., 2002), and two pairs of signals in two chromosome pairs with a core histone gene probe (Eirín-López et al., 2004).In Pectinidae, histone H3 gene was clustered at two different loci in the genome of Patinopecten yessoensis Jay, 1857 or a single locus in the genome of Chlamys farreri Jones et Preston 1904, Chlamys nobilis Reeve, 1852, and Argopecten irradians Lamarck, 1819 (Zhang et al., 2007).Regarding mollusc molecular phylogenetic studies using histone genes, little information is available.No studies were realised in Ostreidae, but some studies have already been performed in Pectinidae (Puslednik, Serb, 2008), Mytilidae (Eirín-López et al., 2004), Veneridae (Kappner, Bieler, 2006) and in gastropods (Colgan et al., 2000).
In the present work, we partially sequenced the histone H3 gene in C. gigas and used that data to ascertain the phylogenetic relationships.We also applied FISH technique with histone H3 gene probe in order to determine its location and distribution in the genome of C. gigas.

MATERIAL AND METHODS
Animal material and genomic DNA extraction.DNA was extracted from muscle tissue preserved in 70% ethanol using the Quickgene DNA tissue kit S (Fujifi lm Life Science).Tissue was sampled from adult oysters bred at the IFREMER (Institut Français de Recherche pour l'Exploitation de Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics la Mer) hatchery in La Tremblade (Charente-Maritime, France).For chromosome preparations, embryos were used.Gametes were collected by stripspawning sexually mature animals.Fertilized gametes were cultured at 23°C in 150-L fi breglass larval tanks of seawater.
PCR.PCR amplifi cation of the histone H3 gene was realised using a pair of primers (forward: 5'-CGTAAATCCACTGGAGGCAAGG-3'; reverse: 5'-GGATGGCGCACAGGTTGGT GTC-3') designed from the H3 nucleotide sequences of Pecten jacobaeus Linnaeus, 1758 and Mimachlamys varia Linnaeus, 1758 retrieved from GenBank (AY070153 and AY070154 respectively).The PCR reactions used were the standard ones, except that in the labelling PCR we also used digoxigenin-11-dUTP (Roche Molecular Biochemicals).PCR was performed under the following conditions: 5 min denaturation at 94°C, 30 cycles of 1 min at 94°C, 40 s at 66°C and 1 min at 72°C, and a fi nal extension of 5 min at 72°C.PCR amplifi cation products were subjected to 1.5% agarose gel electrophoresis and stained with ethidium bromide.
Sequencing of histone H3 gene in Crassostrea gigas, sequence and phylogenetic analyses.The PCR amplifi cation product was excised from a 1.5% agarose gel and purifi ed using the Geneclean II Kit (QBioGene MP Biomedicals).A band of ~ 300 bp was obtained.The purifi ed PCR product was sequenced in both directions using the primers H3 forward (5'-CGT AAATCCACTGGAGGCAAGG-3') and H3 reverse (5'-GGATGGCGCACAGGTTGG TGTC-3').The DNA sequence of the histone H3 gene from C. gigas was deposited in the GenBank Database with the following accession number: HQ009488.The histone H3 gene sequence in C. gigas was analysed for similarity with other known sequences using the National Centre for Biotechnology Information Blast database tool (http://www.ncbi.nlm.gov/Blast/).CLC Sequence Viewer version 6.2 (http://www.clcbio.com) was used to aligne the sequence with other histone H3 gene sequences and for phylogenetic reconstruction.A total of 42 histone H3 gene sequences were used for phylogenetic analysis (Table 1).Distance-tree was constructed with the Neighbour-Joining (NJ) algorithm (Saitou, Nei, 1987), 100 bootstrap replicates.Bootstrap values lower than 50% were not shown.
Chromosome preparations.Chromosome preparations were made as described in Bouilly et al. (2008).Briefl y, 6 hours old embryos were treated with 0.005% colchicine in seawater for 25 min.A hypotonic shock was applied for 10 min in 0.9% sodium citrate.Then, embryos were fi xed in 3:1 absolute ethanol-acetic acid solution.Embryos cells were dissociated in 50% acetic acid and the suspension obtained was dropped onto slides heated at 42°C and air-dried.
Fluorescence in situ hybridisation (FISH).Chromosome spreads were pretreated with 0.005% pepsin in 10 mM HCl at 37°C for 5 min and air-dried.Slides were then fi xed with formaldehyde as described in Chaves et al. (2002).Briefl y, slides were rinsed twice in phosphate-buffered saline (PBS) for 5 min, and after incubation in 1% formaldehyde in PBS, for 20 min at room temperature, they were washed twice in PBS for 5 min.The slides were then dehydrated in a chilled ethanol series (70%, 90%, and 100%; 5 min each) and air-dried.Slides were aged overnight at 65°C, and then, dehydrated in 100% ethanol at -20°C for 3 min.Chromosomes preparations were denaturated at 72°C in 70% formamide in 2 × SSC for 2 min, followed by dehydratation in 70%, 90%, and 100% chilled ethanol, for 5 min each.

Comp. Cytogenet., 2010 4(2)
Comparative Cytogenetics 10 min and cooled immediately.Hybridisation solution containing the denaturated probe was dropped onto the denaturated spreads, the slides were covered and kept overnight in a humid chamber at 37°C.After hybridisation, the slides were washed at 37°C once in 2 × SSC for 5 min, then twice in 50% formamide in 2 × SSC for 5 min, and once in 2 × SSC for 5 min.The blocking agent was 3% BSA, for 10 min at room temperature.The digoxigenin-labelled-probe was detected with anti-digoxigenin-rhodamine fab fragments (Roche Molecular Biochemicals).The slides were counterstained with DAPI and mounted in Vectashield (Vector Laboratories).Chromosome observation.Chromosomes were observed with a Zeiss Imager.Z1 microscope coupled with an Axiocam digital camera and a Zeiss LSM Image Browser software.Digitised photos were prepared for printing in Adobe Photoshop (Version 9.0); contrast and color optimisation were the functions used and all affected the whole of the image equally.

Molecular characterisation.
A histone H3 gene sequence in C. gigas with a size of approximately 300 base pairs was isolated and sequenced.A contig was produced using Vector NTI Advance 10.The length of this contig was 326 bp.Primers were aligned with the contig and unalignable regions were excluded from phylogenetic analysis.These included positions 1 to 3 and 320 to 326.The sequence, after analysis, was 316 base pairs long (Fig. 1).The analysis of C. gigas H3 gene sequence using NCBI Blast database tool showed a high similarity with H3 gene sequence in different mollusc species.For example, C. gigas H3 gene had 87% similarity with O. edulis H3 gene (AY070151).The GC content (56.3%) was higher than the AT content (43.7%).The histone H3 gene sequence from C. gigas and other histone H3 gene sequences were reduced to 305 bp for phylogenetic analysis to have a good alignment with the 41 histone H3 gene sequences selected from GenBank Database.The NJ tree separated the species into two clades (Fig. 2).The fi rst clade was subdivided into two groups, one constituted of 20 Bivalvia (12 Pectinidae, 5 Mytilidae, 2 Ostreidae and 1 Limidae) all from the subclass Pteriomorphia, and the other one of 5 Gastropoda (corresponding to the superorder of Vetigastropoda), and 7 Bivalvia (all Heteroconchia: 4 Veneroida, 2 Myoida, except one which was Pteriomorphia: 1 Limoida).
In bivalves, phylogenetic relationships with histone H3 gene sequences have already been studied in Pectinidae (Puslednik, Serb,  According to the phylogenetic tree some closely related species were not clustered together.The partial sequence of histone H3 gene seems to be too short to give a good resolution in phylogenetic reconstructions.Indeed, some groups were not supported by a signifi cative bootstrap value, and thus, H3 gene did not allow reconstructing an accurate phylogeny as the different clusters not always correspond to the taxonomical (family level or class level) groups.

Cytogenetic characterisation.
We examined 80 good chromosome spreads from oyster embryos.Our results showed that the histone H3 gene was mapped at two different loci in the genome of C. gigas (Fig. 3).The histone H3 gene was located at an interstitial site (about half-way from the centromere to the telomeres) on the long arm of chromosome pair 4, and on the telomeres of the smaller chromosome pair (pair 10).Polymorphism was detected on the telomeres of pair 10.On the 80 metaphases that were examined, 16 presented single signals in both chromosomes (20%) (Fig. 3, A-B), 29 showed double signals (36%) (Fig. 3, C-D), and 35 had a single signal on one chromosome and a double signal on the homologue (44%) (Fig. 3, E-F).The histone H3 gene is a qualifi ed chromosomal marker.
In other bivalve species, histone genes were also localised in one, two or three loci.In Mytilidae, FISH results on Mytilus galloprovincialis chromosomes located core histone genes at two loci in two different chromosome pairs (Eirín-López et al., 2004), and the linker histone H1 probe at three loci in three chromosome pairs (Eirín-López et al., 2002).In both experiments, the hybridisation signals mapped the histone genes at telomeric chromosomal positions or interstitial positions.In Pectinidae, histone H3 gene was mapped at two loci in the genome of Patinopecten yessoensis or a single locus in the genome of Chlamys farreri, Chlamys nobilis, and Argopecten irradians (Zhang et al., 2007).Positions were also interstitial or telomeric as in our experiment.In Mytilidae and Pectinidae studies, no polymorphism was reported.Histone gene clusters were rather conservative in chromosome location in most cases where closed species have been analysed (Hankeln et al., 1993;Ranz et al., 2003;Cabrero et al., 2009).Chromosomal localisation of histone H3 gene should be determined in other Crassostrea species for oyster comparative studies.Histone H3 gene in O. edulis was characterised molecularly (Giribet, Distel, 2003), but no cytogenetic studies were realised with this sequence.
Major ribosomal DNA genes (Xu et al., 2001;Wang et al., 2004), NORs (Thiriot-Quiévreux, Insua, 1992) and C-bands (Bouilly et al., 2008) were also localised on the telomeres of long arm of pair 10 in C. gigas.During mitosis, the major ribosomal genes are the nucleotidic component of the NORs (Goessens, 1984), therefore, the chromosomal sites of major ribosomal gene clusters correspond to NOR sites.NOR sites were already detected to colocalise with C-banded Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics heterochromatic region in an insect Bradysia hygida Sauaia et Alves, 1968 (Gaspar et al., 2002).In salmonids, the chromosomal location of the major histone cluster was adjacent or close to regions with C+ heterochromatin (Pendás et al., 1994).Histone genes were already found to be associated with 5S rDNA in two Crustacean species, Artemia salina Linnaeus, 1758 (Andrews et al., 1987) and Asellus aquaticus Linnaeus, 1758 (Barzotti et al., 2000).Multicolour-FISH will be of great interest to check if the major and minor rDNA genes are colocalised or not with the histone H3 genes in C. gigas.
In conclusion, histone H3 gene is a useful karyotypic marker which can be used in oyster cytotaxonomy.Our data support that molecular and cytogenetic characterisations of histone H3 gene in other oyster and bivalve species will be useful for comparative studies, evolutionary and phylogenetic studies, and for understanding genome organisation in oysters.

Fig. 3 ,
Fig. 3, A-F.FISH with histone H3 gene probe applied on chromosomes of Crassostrea gigas (in red).The chromosomes were contrasted with DAPI (blue).A -a metaphase cell with single/single signals on telomeres of pair 10, and B -its corresponding karyotype.C -a metaphase cell with double/double signals on telomeres of pair 10, and D -its corresponding karyotype.E -a metaphase cell with single/double signals on telomeres of pair 10, and F -its corresponding karyotype.Bars = 10 μm.

Table 1 .
The probe was denaturated at 80°C for List of histone H3 gene sequences in different molluscan species used for the phylogenetic analysis with GenBank accession numbers.