Production of Tiger Puffer Takifugu rubripes Offspring from Triploid Grass Puffer Takifugu niphobles Parents.

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2-5 kg) much larger than average market size (0.6-1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.

The genetic architecture of growth rate in juvenile Takifugu species.

Closely related species have often evolved dramatic differences in body size. Takifugu rubripes (fugu) is a large marine pufferfish whose genome has been sequenced, whereas T. niphobles is the smallest species among Takifugu. We show that, unsurprisingly, the juvenile growth rate of T. rubripes is higher than that of T. niphobles in a laboratory setting. We produced F(2) progenies of their F(1) hybrids and found one quantitative trait locus (QTL) significantly associated with variation in juvenile body size. This QTL region (3.5 Mb) contains no known genes directly related to growth phenotype (such as IGFs) except Fgf21, which inhibits growth hormone signaling in mouse. The QTL in Takifugu spp. is distinct from the region previously known to control body size variations in stickleback or tilapia. Our results suggest that in the fish tested herein, genomic regions underlying body size evolution might have different genetic origins. They also suggest that many diverse traits in Takifugu spp. are amenable to genetic mapping.

Gonadal development and fertility of triploid grass puffer Takifugu niphobles induced by cold shock treatment.

Tiger puffer Takifugu rubripes is one of the most valuable fish species in Japan; however, there has not been much progress in their selective breeding until recently despite their potential in aquaculture. Their long generation time and the large body size of their broodstock make breeding difficult. Recently, we made a surrogate broodstock, which produced gametes of different species in salmonids. Therefore, by using closely related recipients, which have small body sizes and short generation times, it is possible to accelerate breeding of the tiger puffer. Thus, we considered the grass puffer Takifugu niphobles, which has a short generation time and a small maturation size, as a potential recipient for gamete production of the tiger puffer. Furthermore, if sterile triploid individuals are used as recipients, the resulting surrogate broodstock would produce only donor-derived gametes. Therefore, we examined conditions for inducing triploidy by suppressing meiosis II to retain the second polar body in grass puffer. We found that cold shock treatment, which is 5°C for 30 min starting from 5 min after fertilization, is optimal to obtain high triploidization and hatching rates. Although the resulting triploid grass puffers produced small amounts of gametes in both sexes, the offspring derived from the gametes could not live for over 3 days. Furthermore, we found that triploid grass puffer showed normal plasma sex steroid levels compared with diploids. These are important characteristics of triploid grass puffer as surrogate recipients used for germ cell transplantation.

A genetic linkage map for the tiger pufferfish, Takifugu rubripes.

The compact genome of the tiger pufferfish, Takifugu rubripes (fugu), has been sequenced to the "draft" level and annotated to identify all the genes. However, the assembly of the draft genome sequence is highly fragmented due to the lack of a genetic or a physical map. To determine the long-range linkage relationship of the sequences, we have constructed the first genetic linkage map for fugu. The maps for the male and female spanning 697.1 and 1213.5 cM, respectively, were arranged into 22 linkage groups by markers heterozygous in both parents. The resulting map consists of 200 microsatellite loci physically linked to genome sequences spanning approximately 39 Mb in total. Comparisons of the genome maps of fugu, other teleosts, and mammals suggest that syntenic relationship is more conserved in the teleost lineage than in the mammalian lineage. Map comparisons also show a pufferfish lineage-specific rearrangement of the genome resulting in colocalization of two Hox gene clusters in one linkage group. This map provides a foundation for development of a complete physical map, a basis for comparison of long-range linkage of genes with other vertebrates, and a resource for mapping loci responsible for phenotypic differences among Takifugu species.