Organization and variation analysis of 5S rDNA in different ploidy-level hybrids of red crucian carp × topmouth culter

Abstract

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I-N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

Figure 1. Appearance of RCC, TC and their hybrid offspring.

(A) RCC. (B) TC. (C) 2nRT hybrids of RCC♀×TC♂. (D) 3nRT hybrids of RCC♀×TC♂. (E) 4nRT hybrids of RCC♀×TC♂. Scale bar in A–E, 1 cm.

Figure 2. Chromosome spreads at metaphase and corresponding karyotypes of RCC, TC, and their hybrid offspring.

(A) The 100 chromosomes of RCC, with no large submetacentric chromosome. (B) The 48 chromosomes of TC, with a pair of the largest submetacentric chromosomes indicated (solid arrows). (C) The 74 chromosomes of 2nRT hybrids, with a piece of the largest submetacentric chromosome indicated (solid arrow). (D) The 124 chromosomes of 3nRT hybrids, with a piece of the largest submetacentric chromosome indicated (solid arrow). (E) The 148 chromosomes of 4nRT hybrids, with a pair of the largest submetacentric chromosomes indicated (solid arrows). (F) The karyotype of RCC, in which no large submetacentric chromosome is detected. (G) The karyotype of TC, which includes a pair of the largest submetacentric chromosomes (solid arrow). (H) The karyotype of 2nRT hybrids, comprising one set of chromosomes from RCC and one set from TC. The solid arrow indicates a piece of the largest submetacentric chromosome, which is similar to that of TC. (I) The karyotype of 3nRT hybrids, consisting of two sets of chromosomes from RCC and one set from TC. The solid arrow indicates a piece of the largest submetacentric chromosome, which is similar to that of TC. (J) The karyotype of 4nRT hybrids, consisting of two sets of chromosomes from RCC and two sets from TC. The solid arrow indicates a pair of the largest submetacentric chromosomes similar to those of TC. Scale bar in A–J, 3 µm.

Figure 3. Cytometric histograms of RCC, TC and their hybrid offspring.

(A) The mean DNA content of RCC (peak 1∶101.29). (B) The mean DNA content of TC (peak 1∶67.40). (C) The mean DNA content of 2nRT hybrids (peak 1∶84.19). (D) The mean DNA content of 3nRT hybrids (peak 2∶136.17). (E) The mean DNA content of 4nRT hybrids (peak 1∶163.01).

Figure 4. DNA bands amplified from RCC, TC and their hybrid offspring.

M: DNA ladder markers (50 bp increments); lane 1: two DNA bands (∼200 and 300 bp) from TC; lane 2: three DNA bands (∼200, 350 and 500 bp) from RCC; lane 3: three DNA bands (∼200, 350 and 500 bp) from 2nRT hybrids; lane 4: three DNA bands (∼200, 350 and 500 bp) from 3nRT hybrids; lane 5: four DNA bands (∼200, 350, 400 and 500 bp) from 4nRT hybrids.

Figure 5. Representative sequences of monomeric 5S rDNA and dimeric 5S rDNA.

(A) Arrangement of eukaryotic 5S rDNA and the illustration of the PCR amplification with 5S primers P₁ and P₂R; (B) Representative sequences of 5S rDNA Class IV and Class V from TC; (C) The dimeric 5S rDNA tandem arrays (NTS–I–N) of 4nRT hybrids. The gene sequences of 5S rDNA are underlined and the shaded regions show the 5S primers. Dashes indicate alignment gaps; asterisks represent variable sites; TATA element (TAAA) is included in box.

Figure 6. Complete 5S rRNA genes of RCC, TC and their hybrid offspring.

Dots indicate the identical nucleotides and ICRs are included in the boxes.

Figure 7. Alignment results of the NTS sequences from CC, RCC, TC and their hybrid offspring.

(A) The 59 bp and 68 bp NTS sequences (NTS–IV) of TC, 2nRT and 3nRT hybrids; (B) NTS–I from RCC, CC, 2nRT and 4nRT hybrids; (C) NTS–II from RCC, 2nRT, 3nRT and 4nRT hybrids; (D) NTS–III from RCC, 2nRT, 3nRT and 4nRT hybrids. The NTS upstream TATA-like sequences are included in boxes. Dots indicate sequence identity and hyphens represent insertions/deletions.

Figure 8. Fluorescence photomicrographs of mitotic metaphase chromosomes of RCC, TC and their hybrid offspring.

Signals were detected with fluorescein isothiocyanate (FITC)-conjugated avidin and all the metaphase chromosomes were stained with DAPI. A–E (mitotic metaphase chromosomes of RCC, TC, 2nRT, 3nRT and 4nRT hybrids respectively) show the single-label FISH results hybridized with a probe from the cloned repeated fragments of class IV; F–J (mitotic metaphase chromosomes of RCC, TC, 2nRT, 3nRT and 4nRT hybrids respectively) demonstrate the hybridization results after single-label FISH with a probe from the cloned repeated fragments of class V. The white arrowheads indicate the fluorescent signals (green) of 5S rDNA. Scale bar in A–J, 3 µm.