A Novel Allotriploid Hybrid Derived From Female Goldfish × Male Bleeker's Yellow Tail

Abstract

Hybridization is a traditional and effective strategy to alter the genotypes and phenotypes of the offspring, and distant hybridization is a useful strategy to generate polyploids in fish. In this study, goldfish (Carassius auratus, GF, 2n = 100) and Bleeker's yellow tail (Xenocypris davidi Bleeker, YT, 2n = 48), which belong to different subfamilies, were crossed with each other. The cross of female GF × male YT successfully obtained hybrid offspring (GFYT hybrids), while the cross of female YT × male GF was lethal, and all the fertilized eggs stopped developing before the neurula stage of embryogenesis. All GFYT hybrids possessed 124 chromosomes (3n = 124) with two sets from GF and one set from YT. The measurable and countable traits of GFYT hybrids were identified, and the genetic characteristics of 5S rDNA between GFYT hybrids and their parents were also revealed. There were, respectively, four and three different 5S rDNA types in GF (assigned as GF-Ⅰ∼Ⅳ) and YT (assigned as YT-Ⅰ∼Ⅲ), and GFYT hybrids specifically inherited YT-Ⅰ and YT-Ⅱ 5S rDNA types from YT and GF-Ⅲ and GF-Ⅳ from GF. In addition, there were only testis-like and fat-like gonads been found in GFYT hybrids. Interestingly, there were pyknotic and heteromorphous chromatin and invaginated cell membrane observed in the spermatids of testis-like gonads, but no mature sperm were found. Furthermore, TUNEL assays indicated that, compared with control, apparent apoptotic signals, which were mainly distributed around spermatid regions, were detected in the testis-like gonads, and the expression of apoptosis pathway-related genes including p53, bcl-2, bax, and caspase9 was significantly upregulated. Moreover, the expression of meiosis-related genes including spo11, dmc1, and rad51 showed an abnormally high expression, but mns1 and meig1, two key genes involved in the maturation of spermatid, were extremely downregulated. In brief, this is the first report of allotriploid via distant hybridization between GF and YT that possessing different chromosome numbers in vertebrates. The obtainment of GFYT hybrids not only harbors potential benefits and application in aquaculture but also further extends the understanding of the influence of hybridization and polyploidization on the genomic constitution of the hybrid offspring. Furthermore, they can be used as a model to test the origin and consequences of polyploidization and served as a proper resource to study the underlying mechanisms of spermatogenesis dysfunctions.

Keywords: allotriploid; distant hybridization; inheritance; recombination; sterility.

FIGURE 1

Reciprocal crosses, main stages of embryonic development, and comparisons of morphological traits between GFYT hybrids and their parents. (A) Cross of female GF × male YT and their hybrid offspring. (B) Lethal cross of female YT × male GF. (C) Main stages of embryonic development of reciprocal crosses. The upper lane shows the cross of female GF × male YT, and the lower presents the cross of female YT × male GF. (D) Comparisons of the measurable traits between GFYT and their parents. (E) Comparisons of the countable traits between GFYT hybrids and their parents. For each comparison, different symbols (# or *) mean significant difference (p < 0.05). TL, total length; BL, body length; BH, body height; HL, head length; HH, head height; CPL, caudal peduncle length; CPH, caudal peduncle height; LS, lateral line scales; ALS, scale rows above the lateral line; BLS, scale rows below the lateral line; DFR, dorsal fin rays; PFR, pelvic fin rays; AFR, anal fin rays; ST, single tail; TT, twin tail.

FIGURE 2

Flow cytometric histograms and chromosomal constitution of GFYT hybrids and their parents. (A–C) Mean DNA content. (A) GF (peak 1: 98.39). (B) YT (peak 1: 60.71). (C) GFYT hybrids (peak 1: 126.12). (D–F) Chromosome spreads at metaphase. (D) 100 chromosomes of GF. (E) 48 chromosomes of YT. (F) 124 chromosomes of GFYT hybrids. (G–I) Fluorescence in situ hybridization of mitotic metaphase chromosomes. (G) GF, about 100 signals. (H) YT, no signal. (I) GFYT, about 100 signals.

FIGURE 3

Hereditary characteristics of 5S rDNA units in GFYT hybrids and their parents. (A) DNA bands amplified from GF, YT, and GFYT hybrids. Marker: DNA ladder markers with 100 bp increments. (B) Alignment of the NTS sequences. (C) Ideogram presenting the genetic constitution of 5S rDNA units in GFYT hybrids.

FIGURE 4

Histological structure of gonads in GFYT hybrids and their parents. (A–H) HE-stained gonads and (I–L) ultrastructure of testis-like gonads of GFYT hybrids. (A) Ovary of GF. (B) Testis of GF. (C) Ovary of YT. (D) Testis of YT. (E,F) Testis-like gonad of GFYT hybrids. (G,H) Fat-like gonad of GFYT hybrids. (I,J) Spermatogonia and spermatocytes in the testis-like gonad of GFYT hybrids. (K,L) Spermatids with heteromorphous nuclei in the testis-like gonad of GFYT hybrids. F, H, J, and L, respectively present the amplification of the dashed box in E, G, I, and K.

FIGURE 5

Apoptosis assay and spermatogenesis-related gene expression in the testis of GF and GFYT hybrids. (A) TUNEL analysis of the testis of GF and testis-like gonad of GFYT hybrids, Bar = 20 μM. (B) Real-time PCR quantification of p53, bcl-2, bax, and caspase9. (C) Real-time PCR quantification of dmc1, spo11, rad51, mns1, and meig1. Columns labeled with different symbols (# or *) mean significant difference (p < 0.05).

FIGURE 6

Schematic presenting the four types of formation mechanisms of the heterogeneous hybridization offspring. (A) Allodiploid resulting from the fusion of haploid gametes. (B) Allotriploid deriving from double fertilization. (C) Allotriploid resulting from the expulsion inhibition of the second pole. (D) Allotetraploid deriving from the impeding of the first cleavage.