Molecular and FISH analysis of 45S rDNA on BAC molecule of Saccharina Japonica

IGS is abundant in polymorphism, which is widely used in the analysis of intraspecic genetic diversity and phylogenetic relationships among geographical populations. In this study, the 45S rDNA repeat unit of Saccharina japonica was obtained for the rst time by BAC clone sequencing. The total length of 45S rDNA repeat unit of S. japonica was 8995 bp, including 5420 bp of 18s-5.8s-25s rDNA and 3575 bp of IGS (Intergenic Spacer), with the GC content of 51.4%. IGS was composed of 465 bp 3’-outer transcribed spacer (ETS), 874 bp 5’-ETS, and 2236 bp non transcribed spacer (NTS), with the GC content of 50.1%.Fiber-FISH (ber-uorescence in situ hybridization, ber-FISH) analysis of 45S rDNA on the BAC molecule of female gametophytes of S. japonica illustrated that each ber had at least ve continuous moniliform hybridization signal points, indicating the distribution of 45S rDNA repeat unit on the bacterial articial chromosome. This study provided a new candidate molecular marker for detecting intraspecic polymorphisms of S. japonica, and the successful ber-FISH analysis of 45S rDNA on BAC molecule would contribute to the construction of the physical map and Map-based cloning of this kelp.


Introduction
In eukaryotes, ribosomal 45S rRNA genes (rDNA) are located in the nucleolar organizer region (NOR) of chromosomes in the form of tandem repeats [1][2][3]. Each 45S rDNA repeat unit is composed of ribosomal gene coding regions (25S, 5.8S and 18S rDNA), two internal transcribed spacer (ITS) and one intergenic spacer (IGS) [4]. Due to different natural selection pressures, 45s rRNA demonstrated different genetic development among different species and even high intraspeci c genetic diversity. IGS is located between the 3-terminal of 25s rRNA gene and the 5-terminal of 18S rRNA gene. IGS contains a 3' external transcribed spacer (ETS), a 5'-ETS, a non transcribed spacer (NTS), on which there is a transcription initiation site (TIS), a transcription termination site (TTS) and a series of regulatory sequences. In comparision with the conservation of rRNA gene, IGS is highly polymorphic and has been widely used in species classi cation and identi cation [3,5].
Due to the complicated structure of IGS, including the high GC content, self-complementary structure and the repeated sequence, it is di cult to obtain the full-length sequence of IGS by PCR [6]. Till now, only few IGS full-length sequenceshad been reported from macroalgae including Pyropia yezoensis [7,8], Pyropia haitanensis [9,10], Bangia [11], Ulva prolifera and Blidingia sp [12]. Bacterial arti cial chromosome (BAC) is an engineered DNA molecule in bacterial cells. Large segments ranging from 100 kb to about 300 kb of an organism could be inserted into BACs for whole genome sequencing [13], physical map construction [14], and map based cloning [15,16] due to its high genetic stability.
Saccharina japonica is an economically and ecologically important brown alga. So far, no 45S rDNA complete repeat unit had been reported. On the basis of BAC library of female gametophyte of S. japonica, the BAC clone containing 45S rDNA repeat unit was screened and sequenced by using the third generation of PacBio sequencing technology. In addition, with the 18S rDNA sequence of S. japonica as a probe, the 45S rDNA repeat unit was successfully located in bacterial arti cial chromosome by using ber-FISH. This was the rst report about uorescent in situ hybridization of molecular marker on DNA bers in macroalgae. The results would contribute to the construction of physical map and map-based cloning of S. japonica and other macroalgae.
1 Materials And Methods

Materials
The female gametophyte BAC library of S. japonica was constructed and preserved in our laboratory. It contains 31872 BAC monoclones with an average length of 115 kb, covering 6.57 times of the whole genome of S. japonica gametophyte. The high molecular weight BAC plasmid DNA of the screened clones was extracted witha DNA Extraction Kit (Qiagen, Germany).Pulsed eld gel electrophoresis was performed for measure the size of the inserted DNA fragment after NotI digestion of the extracted plasmid. The inserted fragments were sequenced using the third generation of PacBio sequencing technology (Wuhan Eight Star Bio-tech Co., Ltd., Wuhan, China).

DNA Sequence analysis of BAC clone
The obtained sequence was pre-processed to remove cloning vector sequences from the reads using Vec Screen(https://www.ncbi.nlm.nih.gov/tools/vecscreen/) of NCBI. Sequence similarity analysis was conducted by using Blastn https://blast.ncbi.nlm.nih.gov/Blast.cgi . The Plant-CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was employed to predict the conserved cis-element motifs presented in IGS. CpG islands in the IGS were predicted by using CpGPlot [17]. The physical map of the inserted fragment of the positive BAC clone (625-E18) was constructed according to the genome sequenceof S. japonicaby using IBS1.0 [18] and MapChart2.32 [19].

FISH on BAC clone molecule
The positive BAC clones were cultured in LB liquid medium containing 12.5 µg/mL chloramphenicol. The BAC plasmid DNA was extracted with plasmid DNA Extraction Kit (Qiagen, Germany). 5 µL BAC-DNA (about 50 ng) was diluted in 5 µL sterilized deionized water and then pipette onto a poly-L-lysine glass slide. A 18 mm⋅18 mm coverslip was used to spread the molecular to prevent molecular broken [20]. Slide was air-dried for 30 min at room temperature, and then the coverslip was washed off in water. After air dry, slide was xed in the Carnot's xative (Ethanol / glacial acetic, 3:1, v/v) [21] for 2 min, and dried at 60℃ for 30 min.
The 18S rDNA probes were labeled by nick-translation with Alex ow green-5-dUTP (PerkinElmer, Boston, USA). FISH was performed according to the procedure described by Liu et al. [22].Slide with BAC-DNA was cross-linked for 2-3 times in a ultraviolet cross linker, and then 8 µL of hybridization solution, including 6 µL of mixture (2×SSC + 1×TE buffer) and 2 µL of 18S rDNA probe, was pipette on the slide.
After covering with a coverslip, the slide was water bath at 100℃ for 5 min. Then put it in an oven at 55℃ for hybridization overnight. After hybridization, the slide was washed with 2 × SSC at 42 ℃ for 10 min and then dried at 55 ℃ for 15 min. At last, 5 µL DAPI staining solution was added. After incubation for 10 min, the results were observed under a uorescence microscope. LeicaDM4000 uorescence microscope (Germany) and Orca ER camera were used to take pictures, and Adobe PhotoShop CS6.0 was used to process the pictures.
2 Results And Analysis 2.1 Characterization of the BAC clone harbouring 45S rDNA With 18S rRNA gene as probe, one BAC clone (625-E18) containing 18S rRNA gene was successfully screened from the BAC library of female gametophyte of S. japonica by three-dimensional PCR (Fig. S1). The result of PFGE showed that the inserted fragment of this positive clone was about 95 kb in length ( Fig. 1).
In total, 181233 reads were derived from the third generation of PacBio sequencing results, with an average length of 3990 bp (Fig. 2). After splicing, an inserted DNA fragment with 24437 bp was obtained.
On the basis of Blastn alignment results, three DNA fragments between 6074 bp-7895 bp, 15069 bp-16890 bp and 21510 bp-23331 bp on the sequenced region of BAC clone 625-E18 were matched with 18S rRNA genes of S. japonica gametophytes. These results implied that the sequenced region of BAC clone 625-E18 harbouredthree 45S rDNA repeat units, and each repeat unit was 8995 bp in length, which included 5420 bp of 18S-5.8S-25S rDNA sequence and 3575 bp of IGS.

Sequence characterization of IGS ofS. japonica
The size of IGS of S. japonica was 3575 bp in length, which was constituted by 465 bp of 3'-outer transcribed spacer (ETS), 874 bp of 5'-ETS and 2236 bp of non transcribed spacer (NTS) in turn (Fig. 3). The GC content of the IGS sequence was 50.1%. A transcription termination site (TTS) was predicted in the NTS, which was rich in cytosine (CCCCCCCCCCCCCA), and was highly consistent with the reported 25s rRNA TTS of higher plants [23,24]. It was speculated to participate in the termination of S. japonica rRNA gene transcription. At 2702-2713 bp of 5'-ETS, a transcription initiation site (TIS,TTTTTAGGGGGG) was predicted, of which the base A at 2707 bp might be the RNA transcription initiation site. In addition, three CpG islands were predicted in the IGS.
On the basis of the basic local alignment search results, apart from the two fragments of 6756 bp-6827 bp and 9366 bp-9435 bp in Scaffold4565, the sequenced region of BAC clone (625-E18) matched well with the sequence between 785 bp and 10030 bp of Scaffold4565 of S. japonica genome (Fig. 3), which accounted for 75% of the total length of Scaffold4565 [25]. In the physical map of the sequenced region of BAC clone 625-E18, it was noted thatthe IGS and 25S rDNA in the third 45S rDNA unit were slightly shorter than the other two. In addition, the region between 9435 bp-10030 bp in Scaffold4565 could not be matched with the corresponding 25S rRNA gene sequence of S. japonica (Fig. 3). These results illustrated that BAC clone sequencing and the physical map construction were e cient way of the genome sequence error diagnosis and thus improving the accuracy of genome sequences.

FISH on BAC molecule
With 18S rDNA as probe, 45S rDNA repeat units were successfully visualized on the BAC 625-E18 molecular ber by FISH. FISH results illustrated that the hybridization signal points representing repeat units were lined like continuous moniliform on the molecular ber (Fig. 4A). On the selected three molecular bers, ve, seven and seven signal points were visualized respectively as shown in Fig. 4B, Fig. 4C and Fig. 4D. These results implied that BAC clone 625-E18 harboured at least seven 45s rDNA repeat units.

IGS sequence of S. japonica
In this study, the full-length IGS sequence of S. japonica was 3575 bp, which was the rst report of IGS in brown algae. IGSs were more variable than protein coding genes such as nr SSU, 5.8S rDNA, and nr LSU in 45S rDNA [26]. The length of IGS varied greatly among different organisms, which was from about 1 kb in yeast to 12 kb in plant [27]. In macroalgae, only few IGS full length sequences had been documented. Generally, the reported sizes of IGSs of red algae were longer than that of S. japonica, and they were 5984 bp, 6969 bp and 4613 bp in Pyropia yezoensis [8], P. haitanensis [9], and Bangia [11], respectively. In contrast, the IGSs of green algae were slightly shorter than that of S. japonica, such as the IGSs of Ulva proliferaand Blidingia sp, which were 3388 bp and 3059 bp in length [12], respectively. According to that IGS was more suitable for phylogenetic analysis at the level of genus, species and subspecies [24], the full length sequence of IGS reported in this paper would provide useful molecular marker for phylogenetic analysis at the level of subspecies of this kelp.
In contrast to the high variability of the sequence length of IGS, its structure and function were relatively conserved. All the IGS contained a TTS, a TIS and some transcriptional regulatory elements [5]. In the IGS of S. japonica, the sequence of TTS was composed of CCCCCCCCCCCCCA, of which the characteristic of pyrimidine enrichment was also common in the higher plants [23,24]. The TIS of S. japonica was composed of TTTTTAGGGGGG.It had been reported that the sequences from − 5 to + 6 at both ends of TIS sequence had important functions and were highly invariant [28]. In most plants, TATA-box and GGGG-box were common elements at the 5 'end of rRNA precursor. There was a GGGG-box downstream of the transcription initiation site of S. japonica TIS, but the TATA-box was instead of TTTTT upstream. This phenomenon of TATA-box variation was also described in the higher plants, such as Punica granatum L and Quercus palustris Münchh.,in which TATA-box was instead of TCTTT [24,29]. In addition, the IGS of S. japonica had some methylation sites (three CpG islands). In plants, cytosine in CpG CpNpG and CpHp was prone to methylation, which leaded to gene silencing. The expression of rRNA gene was regulated by ribosome requirement. In actively growing cells, the transcription of rRNA accounted for the majority of the total RNA, while in the cells with inactive growth, the transcription of rRNA gene was greatly reduced, indicating that cells mainly regulated the synthesis of rRNA on the level of transcription.
DNA methylation and post transcriptional modi cation of histidine were considered to be involved in gene silencing. There were plenty of repeats in ribosomal genes, and the methylation of CpG in IGS region was considered to be enough to inhibit the transcription of rRNA gene [30]. Three CpG islands in the IGS sequence of S. japonica were identi ed in this study, which also might participate in the regulating the transcription of rRNA gene through DNA methylation.

Distribution of 45S rDNAsitesin BACmolecule
In this study, at least seven 45S rDNA repeat units of S. japonica were localized in BAC molecule. This is the rst report about the cytological localization of DNA molecular of macroalgae by ber-FISH. Ribosomes play an important role in protein synthesis and cell growth. rRNA accounts for 50%-80% of total RNA of cells [31,32]. Correspondingly, there are tandem repeats of rDNA in the nucleolar organizer region of one or more chromosomes [33].The nuclear genome of eukaryotes may contain hundreds to thousands of rRNA gene repeats. For macroalgae, there was no report on the number of 45S rDNA repeat units yet. In this study, three 45S rDNA repeat units were screened on the basis of BAC contig sequencing, and at least seven 45S rDNA sites were localized in BAC molecule by ber-FISH. Although the number of 45S rDNA repeat units deduced from this study might be lower than actual number, the molecular and cytological analysis of 45S rDNA of S. japonica in this study would provide new candidate molecular markers for the study of the genetic diversity of S. japonica, and the successful conduction of ber-FISH on BAC molecule would contribute to the construction of the physical map and Map-based cloning of S. japonica and other macroalgae.

Availability of data and material
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.    Physical map of BAC clone 625-E18 sequence (lower) and its location in the assembled scaffold of S. japonica (upper).