Organization and variation analysis of 5S rDNA in gynogenetic offspring of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂)
© Qin et al.; licensee BioMed Central. 2015
Received: 1 December 2014
Accepted: 2 March 2015
Published: 13 March 2015
The offspring with 100 chromosomes (abbreviated as GRCC) have been obtained in the first generation of Carassius auratus red var. (abbreviated as RCC, 2n = 100) (♀) × Megalobrama amblycephala (abbreviated as BSB, 2n = 48) (♂), in which the females and unexpected males both are found. Chromosomal and karyotypic analysis has been reported in GRCC which gynogenesis origin has been suggested, but lack genetic evidence.
Fluorescence in situ hybridization with species-specific centromere probes directly proves that GRCC possess two sets of RCC-derived chromosomes. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (abbreviated as NTS) reveals that three types of 5S rDNA class (class I; class II and class III) in GRCC are completely inherited from their female parent (RCC), and show obvious base variations and insertions-deletions. Fluorescence in situ hybridization with the entire 5S rDNA probe reveals obvious chromosomal loci (class I and class II) variation in GRCC.
This paper provides directly genetic evidence that GRCC is gynogenesis origin. In addition, our result is also reveals that distant hybridization inducing gynogenesis can lead to sequence and partial chromosomal loci of 5S rDNA gene obvious variation.
Gynogenesis is an important reproductive mode in fish. The Amazon Molly, Poecilia formosa, is one such fish that reproduces by gynogenesis and produces all-female offspring. Natural gynogenetic males of this species are, however, also found and several studies have documented that they are of hybrid origin . UV-treated sterile sperm of Megalobrama amblycephala (BSB, 2n = 48) and cold shock (0–4°C) for 30 min to double the eggs’ chromosomes have been used to activate Carassius auratus red var. (RCC, 2n = 100) eggs, resulting in all-female gynogenetic progeny . Interestingly, we obtained both male and female offspring with 100 chromosomes (GRCC) in the first generation of Carassius auratus red var. (RCC, 2n = 100) (♀) × Megalobrama amblycephala (BSB, 2n = 48) (♂). Chromosomal and karyotypic analysis has been reported in GRCC which gynogenesis origin has been suggested referring to it’s the female parent , but lack sufficient genetic evidence.
In vertebrate, 5S rDNA consists of a conserved coding region of 120 bp (5S) with a variable intergenic spacer usually referred to as the nontranscribed spacer (NTS) [4-7]. The coding region is highly conserved, even among nonrelated taxa, whereas the spacer region is more variable. Many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA in distantly related taxa [8-11]. Thus, 5S rDNA sequences have been used as genetic and cytogenetic markers in evolutionary studies and for studying intraspecific variation, genome evolution, and phylogenetic reconstruction [12-15]. Indeed, little is known about the nucleotide sequences and chromosomal location of 5S rRNAs in teleosts in comparison with other vertebrate classes. Our previous study revealed the influence of polyploidy on the organization and evolution of the 5S rDNA multigene family in teleosts . In an attempt to further understand the genetic variation in gynogenetic fish, and to extend the knowledge of 5S rDNA organization in teleosts, the 5S rDNA sequences and chromosomal loci of RCC and GRCC were isolated and characterized. Our data reveal the genetic variation and evolutionary characteristics of the 5S rDNA multigene family in fish, and provide new insights into the evolutionary patterns of this multigene family in vertebrates.
All samples were cultured in ponds at the Protection Station of Polyploidy Fish, Hunan Normal University, and fed with artificial feed. 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.
During the reproductive seasons (from April to June) in 2006 and 2007, each 15 mature females and 15 mature males of both RCC and BSB were chosen as the maternal fish and paternal fish, respectively. The crossings were performed by two groups. In the first group, RCC was used as the maternal parent, and BSB was used as the paternal fish. In the second one, the maternal fish and paternal fish were reversed. In the reverse cross-BSB(♀) × RCC(♂), there was no living progeny, while in the cross-RCC(♀) × BSB(♂), there existed the living offspring including diploid gynogenetic red crucian carp (GRCC), triploid and tetraploid hybrids .
Preparation of chromosome spreads
To determine ploidy, chromosome counts were performed using kidney tissue from 20 individuals each of BSB, RCC, and GRCC at 1 year of age. After culture for 1–3 d at a water temperature of 18–22°C, the samples were injected with concanavalin one to three times at a dose of 2–8 mg/g body weight. The interval between injections was 12–24 h. Six hours prior to dissection each sample was injected with colchicine at a dose of 2–4 mg/g body weight. The kidney tissue was ground in 0.9% NaCl, followed by hypotonic treatment with 0.075 m KCl at 37°C for 40–60 min and then fixed in 3:1 methanol–acetic acid with three changes. The cells were dropped onto cold, wet slides and stained for 30 min in 4% Giemsa. The shape and number of chromosomes were analyzed under a microscope. For each type of fish, 400 metaphase spreads (20 metaphase spreads from each sample) of chromosomes were analyzed. The preparations were examined under an oil lens at a magnification of 3330 ×.
PCR amplification and sequencing of 5S rDNA sequences
Total genomic DNA was isolated from peripheral blood cells according to the standard phenol: chloroform extraction procedure described Sambrook et al. . One pair of primers (5SP1, 5′-GCTATGCCCGATCTCGTCTGA-3′: 5SP2R, 5′-CAGGTTGGTATGGCCGTAAGC-3′) was designed and synthesized to PCR amplify 5S rDNA repeats directly from genomic DNA. The PCR reaction was performed in a volume of 25 μl with approximately 20 ng of genomic DNA, 1.5 mM of MgCl2, 200 μM of each dNTP, 0.4 μM of each primer, and 1.25 U of Taq polymerase (Takara). The temperature profile was: initial denaturation step at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 10 min. Amplification products were separated on a 3.0% agarose gel using TBE buffer. The DNA fragments were purified using a gel extraction kit (Sangon) and ligated into pMD-18 T. Plasmids were transformed into Escherichia coli DH5a, propagated and then purified. The cloned DNA fragments were sequenced by an automated DNA sequencer (ABI PRISM 3730). Sequence homology and variation among the fragments amplified from RCC and GRCC were analyzed using ClustalW software (http://www.ebi.ac.uk/clustalw/ intex.html).
Fluorescence in situ hybridization
The probes of fluorescence in situ hybridization (FISH) for 5S gene and Species-specific centromere were constructed for RCC and amplified by PCR using the 5SP1 and 5SP2R primers, the primers 5′-TTCGAAAAGAGAGAATAATCTA-3′ and 5′-AACTCGTCTAAACCCGAACTA-3′, respectively. The PCR reaction of Species-specific centromere primers was performed in a volume of 25 μl with approximately 20 ng of genomic DNA, 1.5 mM of MgCl2, 200 μM of each dNTP, 0.4 μM of each primer, and 1.25 U of Taq polymerase (Takara). The temperature profile was: initial denaturation step at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, with a final extension step at 72°C for 10 min. The FISH probes were produced by Dig-11-dUTP labeling (using a nick translation kit, Roche, Germany) of purified PCR products. FISH was performed according to the method described by Caradonna et al. . For each type of fish, 200 metaphase spreads with 100 chromosomes (20 metaphase spreads in each sample) were analyzed.
Chromosome number and composition
Examination of chromosome number of RCC and GRCC
No. of fish
No. of metaphase
Distribution of chromosome number
5S rDNA sequence characterization and molecular analysis
GenBank accession numbers of the 5S rDNA sequences in RCC and GRCC
GenBank accession no. of the sequences
338 bp;340 bp
477 bp;487 bp
Chromosomal loci of 5S rDNA
Examination of chromosomal locus in RCC and GRCC
No. of chromosome
The probe with 203 bp
The probe with 477 bp
The probe with 340 bp
No. of locus
No. of locus
No. of large locus
No. of small locus
Gynogenesis is an especial reproductive mode, in which genetic material of the female parent controls individual development. The main differences between natural gynogenesis and artificial gynogenesis are the UV treatment of sperm and the cold shock to eggs. BSB sperm without UV treatment were able to enter RCC eggs, but BSB genetic material is excluded during embryonic development. Thus, GRCC possess two sets of RCC-derived chromosome and BSB-derived microchromosomes . This study also provides directly genetic evidence that GRCC is gynogenesis origin.
Our previous study revealed the influence of polyploidy on 5S rDNA in fish, including parental genome-specific loss, formation of new types of 5S rDNA class, substitutions and insertion-deletions in the NTS sequence . The evolution of 5S rDNA is driven by birth-and-death processes with strong purifying selection [8-11], which can lead to the existence of the different types of NTS . In this paper, GRCC have three types of 5S rDNA class that completely derived from their female parent (RCC), but no new types of 5S rDNA class is found. Although obvious base variation and insertions-deletions of NTS of 5S rDNA were also observed in GRCC, all sequences analyzed here were likely to correspond to functional genes, because they exhibit all the necessary features for the correct gene expression: three ICRs (box A, internal element, and box C), a TATA control element, and a T-rich tail. It was possible for the genome of the BSB sperm to recombine with the genome of RCC, which could lead to genetic variation via recombination, for example, sox gene shows obvirous variation in GRCC . In this article, our results revealed that GRCC had three types of NTS sequences, in which NTS-III showed an insertion-deletion and obvious nucleotide variations. Thus, we confirmed that the formation of bisexual gynogenetic fish by hybridization can induce obvious base substitutions and insertion-deletions in the NTS sequence, and that these variations in the NTS sequence can serve as an effective molecular marker to distinguish RCC and GRCC.
Fluorescence in-situ hybridization (FISH) is effective way to determine the chromosomal location of the 5S rDNA. Using specific NTS sequences as probes is able to check the exact chromosome location of the different 5S rDNA classes. In this paper, in addition to the 5S ribosomal gene characterization among RCC and GRCC, the chromosomal loci of 5S rDNA performed here allows for a more thorough cytogenetic comparison. FISH, using the 5S rDNA sequence with 340 bp (class II) as probe, showed that a pair of large 5S gene loci were located on a homologous submetacentric chromosome, and a pair of small 5S gene loci were located on a homologous subtelocentric chromosome. Two large and two small 5S gene loci were expected in GRCC, however, only one large and two small 5S gene loci were detected. A large 5S gene locus on the submetacentric chromosome was deleted, suggesting that the chromosomal location of the 340 bp 5S rDNA was changed in GRCC. In addition, hybridization of 5S rDNA sequence with 203 bp (class I) to RCC metaphase chromosomes showed eight 5S gene loci. Eight 5S loci were expected in GRCC, but only six 5S loci were found. It is also reveals chromosomal loci variation in GRCC.
Several studies have documented that such changes to the genome are prerequisites for the successful establishment of a newly formed hybrid or allopolyploid species, because the purging of genetic incompatibility is essential for high fertility in a hybrid or allopolyploid that has diverged from the parents [19-22]. For example, a positive correlation between the amount and rapidity of sequence elimination and the fertility of the various newly synthesized allopolyploids has been observed . GRCC metaphase spreads possess two sets of RCC-derived chromosome and BSB-derived microchromosome . We hypothesized that, to improve fertility, GRCC are required for genetic recombination, mutation, and removal of DNA to reduce incompatible parental genetic material (RCC and BSB). Consequently, recombination and elimination of 5S rDNA sequences and Chromosomal loci variation were revealed in GRCC.
We obtained both male and female offspring with 100 chromosomes (GRCC) in the first generation of Carassius auratus red var. (RCC, 2n = 100) (♀) × Megalobrama amblycephala (BSB, 2n = 48) (♂) . This paper provides directly genetic evidence that GRCC is gynogenesis origin. In addition, our results also prove that distant hybridization inducing gynogenesis can lead to sequence and partial chromosomal loci of 5S rDNA gene obvious variation.
This research was supported by the National Natural Science Foundation of China (Grant No. 31201987, 31430088, and 31210103918), the Doctoral Fund of Ministry of Education of China (Grant No. 20124306120006), the Natural Science Foundation of Hunan Province (Grant No. 14JJ6008), Training Program of the Major Research Plan of the National Natural Science Foundation of China (Grant No. 91331105),the National Key Basic Research Program of China (Grant No. 2012CB722305), the National High Technology Research and Development Program of China (Grant No. 2011AA100403), Cooperative innovation center of engineering and new products for developmental biology (20134486), and the construct program of the key discipline in Hunan province and China.
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