Organization and expression analysis of 5S and 45S ribosomal DNA clusters in autotetraploid fish derived from Carassius auratus red Var . (♀) × Megalobrama amblycephala (♂)

48 Background: Autotetraploid fish (4n = 200, RRRR) (abbreviated as 4nRR) are derived from whole genome 49 duplication of red crucian carp (2n = 100, RR) (abbreviated as RCC). rDNA is often used to study molecular 50 evolution of repeated sequences because it has high copy rate and special conserved coding regions in 51 genomes. In this study, we determined the sequences (5S, ITS1-5.8S-ITS2 region), structure, methylation 52 level (NTS and IGS), and expression level (5S and 18S) of 5S and 45S rRNA genes in 4nRR and RCC in 53 order to elucidate the effects of autotetraploidization on ribosomal DNA (rDNA) in fish. 54 Results: Results showed that there was high sequence similarity of 5S, 5.8S and ITS1 region between 4nRR 55 and RCC. This study also identified two different types of ITS2 region in 4nRR and predicted the secondary 56 structure. It turns out that both secondary structures are functional. Compared with the diploid ancestor of 57 RCC, there was no significant difference in NTS (5S) methylation level, but the expression level of 5S 58 rRNA was lower in 4nRR, indicating that methylation had little effect on the expression level in 4nRR. IGS 59 (45S) was hypermethylated in 4nRR compared to RCC, but the expression of 18S gene were no significantly 60 different from that in RCC, indicating that methylation regulation affected gene expression in 4nRR. 61 Conclusion: These results demonstrate the effects of related structure and expression of 62 autotetraploidization on rDNA. In addition, this study provides reference for studying the effect of 63 autopolyploid on the evolution of species. Our the understanding of effects on ribosomal important significance for the evolutionary study of polyploid crucian carps. In addition, the information on the sequence and structure of the autotetraploid fish 45S rRNA) provides a reference for further studies on the evolution of rDNA in fish and other vertebrates.


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Background 70 Polyploidy studies have reported about different aspects of life such as genome duplication, gene expression, 71 and subsequent evolution (Adams et al., 2005;Hegarty et al., 2013). Polyploids can be classified into 72 autopolyploids and allopolyploids. The former presents two or more homologous chromosomes in a 73 homopolyploid which may contribute to the formation of polyvalent bodies during meiosis, whereas the 74 latter predominantly forms bivalent pairings (Comai, 2005;Qin et al., 2019a). It is worth noting that most 75 polyploidy associated studies mainly focus on plants and less on animals. In our previous studies, we 76 developed allotetraploid hybrids (4n = 148, RRBB) (abbreviated as 4nRB) from the first generation of  (Qin et al., 2014a). In subsequent studies, abnormal chromosomal behavior during meiosis in allotetraploid 79 hybrids (4nRB) led to the formation of autotetraploid sperm and autodiploid eggs, which eventually formed 80 autotetraploid fish (4nRR) (Qin et al., 2014b;Qin et al., 2015). Current research has mainly focused on 81 allopolyploids, with only few autopolyploid studies. As the first vertebrate to evolve, the genomes of fish 82 have been comprehensively studied, and thus they can be used to better understand the evolution of 83 vertebrate cell genome (Symonová and Howell, 2018). 84 Ribosomal DNA (rDNA) is commonly used to study the molecular evolution of multigene families. In 203bp; type II: 340bp; and type III: 477bp) are differentiated using NTS types (NTS I, NTS II, and NTS III) 91 (Long and Dawid, 1980;Korn and Brown, 1978;Qin et al., 2019a). IGS is a transcriptional regulatory 92 5 sequence of rDNA which modulates cellular processes (Ruffini et al., 2013;Ferná ndez et al., 2000).

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Previous analyses of rDNA repeats have mostly been carried out in invertebrates and plants. Therefore, 94 information on 5S and 45S rDNA in vertebrates is scarce. One study reported that rRNA molecules must 95 fold into secondary structures in order to function properly in ribosomes (Noller, 1984). ITS2 provides 96 useful biological information at a higher taxonomic level, even in all eukaryotes, because it has a conserved 97 secondary structure . Many gene promoter regions are rich in CpG, commonly known as 98 CpG islands. Studies have shown that cytosine methylation in CpG dinucleotide guanosine 5' plays an 99 important role in gene expression regulation (Bird, 1992;James et al., 1996). In this study, we analyzed the 100 sequence, structure, methylation, and expression changes in 5S and 45S rDNA clusters between 101 autotetraploid fish (4nRR) and its parental species (red crucian carp (RCC)). From an evolutionary 102 perspective, comparing the arrangement of synthetic autopolyploids with parents of the 5S and 45S rDNA 103 makes sense because of their similar genomic compositions. Our results will provide a new perspective for 104 the organization and evolution of multigene families.

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Expression sequence analysis of 5S rRNA coding region and ITS1-5.8S-ITS2 sequence 108 A total of 40 copies of the gene sequences were analyzed from 4nRR and RCC. Amplification of the 5S 109 rRNA coding region in 4nRR and RCC produced a 120 bp band. BLASTn alignment of the sequences 110 detected a few base substitution changes when the autotetraploids (4nRR) were compared with the 111 corresponding parents (RCC) (Fig. 1). Moreover, the coding region sequences of 4nRR had high sequence 112 identity (average similarity of 97.5%) with corresponding sequences from RCC. Therefore, our preliminary 113 analysis showed that the 5S rRNA coding region of 4nRR had high similarity with the corresponding 114 parental species sequence (GenBank Accession Nos. MZ041022 and MZ041023). It is well known that two specific sequences (called internal transcription spacers) separate the mature rRNA 116 sequences: ITS1 (between 18S rRNA and 5.8S rRNA) and ITS2 (between 5.8S and 28S rRNA). We cloned 117 and sequenced PCR products in order to compare the internal transcription region (ITS1-5.8S-ITS2) of 118 4nRR and RCC. We divided the ITS region into ITS1, 5.8S, and ITS2 regions for better comparison.

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BLASTn sequence alignments showed that the ITS1 and 5.8S rRNA of 4nRR had 100% similarity (Fig. 2) 120 to RCC (ITS1: GenBank Accession Nos. MZ041015 and MZ041016; 5.8S: GenBank Accession Nos. 121 MZ041020 and MZ041021). Nevertheless, we found two different types of ITS2 in 4nRR: type I (inherited 122 from the parental species (RCC)) and type II (a newly formed type which was only expressed in tetraploid 123 species) (Fig. 3) (GenBank Accession Nos. MZ041017-MZ041019). Figure 3 showed intraspecific variation 124 of these sequences. Results indicated that type II ITS2 had obvious insertion, deletion and base substitution. 125 These findings suggest that the ITS1 sequence is much more conserved than ITS2. that only helix II and helix III are recognizable and are essentially common in all organisms (Coleman,130 2007). In this study, we predicted the secondary structure of ITS2 according to the two different sequences 131 of type I and type II (Fig. 4). It turned out that both secondary structures were functional. The results showed 132 that helix II (pyrimidine-pyrimidine) and helix III had high conservation in type I and type II of ITS2, 133 especially the 5' side of helix III (CCGGTGG).

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Expression analysis of 5S and 18S rRNA 136 We compared the expression of 5S and 18S rRNA genes in 4nRR using quantitative real-time PCR with 137 RCC acting as the control group (Fig. 4). Results showed that the amount of 5S transcriptional products in 1995; Allaby and Brown, 2001). In addition, hybridization is accompanied by genome changes in order to 169 overcome threats to its survival (Mcclintock, 1984).

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The ITS2 secondary structure presented in this study is consistent with other ITS2 structure predictions.

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ITS2 usually has four helices, with helix II and helix III being recognizable in almost all organisms. Helix II 172 is very short, does not have any branches, and has a pyrimidine-pyrimidine mismatch. On the other hand, 173 helix III is usually longer than helix II and often has branches. Previous studies reported that the largest 174 absolute sequence conserved region in the entire ITS2 is located on the 5' side (YCGGTGGR) of helix III 175 close to the tip Joseph, 1999;Coleman and Vacquier, 2002). Moreover, these conservative 176 characteristics are preserved in type I and type II helices. The ITS2 conserved structural motifs is necessary 177 for all aspects of ribosomal processing (Keller et al., 2009). The helix of region I is highly similar in both 178 types. Traditionally, helix IV is the most variable region in ITS2, thus, it is normal for the two types of 4nRR 179 to be different, both secondary structures are functional (Young and Coleman, 2004;. These 180 differences may also reflect differences in the formation of mature functional ribosomes because there are 181 many steps involved in the production of a mature rRNA gene (Johansen et al., 2006).     We thank many researchers for maintaining and nursing autotetraploid fish for many years. Liu and Qin 341 contributed to the conception and designed the study. All authors read and approved the final manuscript.