The 5S rDNA and their chromosomal loci in autotriploid Carassius auratus

Background Polyploidization can induces a flurry of genetic events and plays an important role in speciation. In this study, we comparatively analyzed the molecular organization and chromosomal locus of 5S rDNA in natural autotriploid (3nCC, 3n=150) and artificial autotriploid gynogenetic (3nG, 3n=150) Carassius auratus as well as the related diploid ancestors, diploid Carassius auratus (2nCC, 2n=100) and Carassius auratus red var. (RCC, 2n=100), which provide a excellent model to ascertain the genomic changes in autotriploidization process. Results The similarities of the 5S rDNA coding region (class I, class II and class III) between RCC and 3nG were 98.3%, 100% and 99.1%, respectively, and the similarities of the 5S rDNA coding region (class IV, class V and class VI) between 2nCC and 3nCC were 97.5%, 100% and 100%, respectively. Fluorescence in situ hybridization (FISH) with 5S rDNA as probe indicated that the expected numbers of maternal chromosomal loci were found in 3nG and 3nCC, respectively. Conclusions These observations indicate that autotriploidization has little influence on the divergence of 5S rDNA family in Carassius auratus.

Triploid C. auratus are considered of autotriploid origin [13] and have bisexual fertile and gynogenesis reproduction models [12]. These fertile autotriploids provide an attractive model for the study of the genomic changes.
The 5S rDNAs in higher eukaryotes are tandemly arrayed and involve a coding sequence (120bp) and a nontranscribed spacer (NTS) region containing some regulatory elements for coding sequence transcription [14,15]. Variation within the NTS region is mainly due to its length and molecular composition [16]. Because of its special structure, the 5S rDNA multigene, is an important maker to analyze and trace rapid evolutionary events. Many 5S rDNA studies have demonstrated the influence of polyploidy on intragenomic variation and gene expression [17,18]. The evolutionary dynamics of the 5S rDNA family likely reflect processes of chromosome repatterning that took place during speciation [19,20]. Therefore, the inheritance pattern and chromosome constitution of 5S rDNAs among polyploid hybrids and their parents can be used to explore the associated hereditary relationships [21]. This study performed a comparative analysis of the 5S rDNA sequence and chromosomal locus in 3nG and 3nCC as well as the related diploid ancestors, RCC and 2nCC.

Examination of chromosome number
In RCC individuals, 87.5% of the chromosomal metaphases had 100 chromosomes (Figure.

Fluorescence in situ Hybridization
The 5S rDNA probe (class I: 203 bp) hybridized with the metaphase chromosomes of RCC and 3nG. The 5S rDNA probe (class IV: 203 bp) hybridized with the metaphase chromosomes of 2nCC and 3nCC. The FISH results showed that eight 5S rDNA gene loci were present in 2nCC and RCC ( Figure. 6a, c, respectively; Table 2). Twelve 5S rDNA gene loci were present in 3nG and 3nCC ( Figure. 6b, d, respectively; Table 2).
The 5S rDNA probe (class II: 340 bp) hybridized with the metaphase chromosomes of RCC and 3nG. The 5S rDNA probe (class V: 340 bp) hybridized with the metaphase chromosomes of 2nCC and 3nCC. The FISH results showed that four 5S rDNA loci were present in 2nCC and RCC ( Figure. 7a, c, respectively; Table 2). Six 5S rDNA loci were found in 3nG and 3nCC ( Figure. 7b, d, respectively; Table 2).
The 5S rDNA probe (class III: 477 bp) hybridized with the metaphase chromosomes of RCC and 3nG. The 5S rDNA probe (class VI: 490 bp) hybridized with the metaphase chromosomes of 2nCC and 3nCC. The FISH results showed that eight 5S rDNA gene loci were identified in 2nCC and RCC ( Figure. 8a, c, respectively; Table 2). Twelve 5S rDNA gene loci were identified in 3nG and 3nCC ( Figure. 8b, d, respectively; Table 2). Therefore, it is reasonable to infer that autotriploidization has little influence on the divergence of the 5S rDNA family in C. auratus.

Discussion
Considering the general behavior of other polyploids, some changes following polyploidy were extremely probably to have occurred. Previous information has indicated that the           Examination of hybridizing signals by FISH (probes with 340 bp) in RCC, 3nG, 2nCC and 3nCC (a) There were two big (yellow arrows) and two small 5S gene loci (white arrows) in RCC; (b) There were three big (yellow arrows) and three small 5S gene loci (white arrows) in 3nG; (c) There were two big (yellow arrows) and two small 5S gene loci (white arrows) in 2nCC; (d) There were three big (yellow arrows) and three small 5S gene loci (white arrows) in 3nCC. Bar = 3 μm.