Organization and Variation Analysis of 5S rDNA in Different Ploidy-level Hybrids of Red Crucian Carp × Topmouth Culter

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

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

<p>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.</p

Examination of chromosome number in RCC, TC, 2nRT, 3nRT and 4nRT hybrids.

a<p>The chromosome number is less than what they should be, owning to the loss of chromosomes in the procedure of chromosome preparation.</p

Mean DNA content of RCC, TC, 2nRT, 3nRT and 4nRT hybrids.

a<p>The intensity of fluorescence (unit, channel).</p>b<p>The observed ratio was not significantly different (<i>P</i>>0.05) from the expected ratio.</p

The results of sequences.

<p>The results of sequences.</p

Cytometric histograms of RCC, TC and their hybrid offspring.

<p>(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).</p

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

<p>Dots indicate the identical nucleotides and ICRs are included in the boxes.</p

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

<p>(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.</p

Appearance of RCC, TC and their hybrid offspring.

<p>(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.</p