Date Published: May 24, 2018
Publisher: Public Library of Science
Author(s): He Zhou, Qi-Zheng Xu, Rui Zhang, Zi-Xin Zhuang, Yin-Qiang Ma, Wei Wang, Tian-Yu Ma, Yi Sui, Yang Liu, Xiaojuan Cao, Hanping Wang.
Hybrid triploid loaches (Misgurnus anguillicaudatus) were generated from natural tetraploid and diploid loaches. We studied the gonads of parents and offspring from direct and reciprocal crosses through histological and transcriptome analyses. The triploid offspring had inferior ability to form sperm and egg cells compared with diploid forms. After sequencing the transcriptomes, 655,109,158 clean reads were obtained, and 62,821 unigenes and 178,962 transcripts were assembled. Of these unigenes, 23,189 were annotated in the GO database, 18,525 in the KEGG database and 24,661 in the KOG database. 36 fertility-related genes were found to be differentially expressed between the direct cross (2n × 4n) progenies and their parents, while 53 fertility-related genes between the reciprocal cross (4n × 2n) progenies and their parents. Following protein-protein interaction network analyses, 54 differentially expressed genes, including PLCB4, cyp17a1 and Pla2g4d, were mined, yielding candidate genes involved in the poor fertility of hybrid triploid loaches. This is the first report of transcriptomes of gonads of hybrid triploid loaches and their parents, offering a substantial contribution to sequence resources for this species and providing a deep insight into the molecular mechanism controlling the fertility of hybrid triploid fish.
Loach (Misgurnus anguillicaudatus; Cypriniformes; Cobitidae) is one of the endemic fishes of Asia and a common small freshwater fish in China. The loach is distributed in all parts of China. For delicious taste and traditional Chinese medicine value, it has become one of the most important cultured fish species in China. The loach exhibits a range of polyploidy: in addition to diploid, there are natural triploid , tetraploid [2–5] and hexaploid  forms in China. This ploidy has received close attention by scholars internationally. Thus, Arai et al. [4,6–8] reported the distribution of natural triploid loach in Japan, but found no natural tetraploid forms. The tetraploid variety has the characteristics of fast growth, low oxygen consumption and high nutritional value. It has important germplasm value in genetics and breeding, and as such is a unique and valuable resource in China. The genome formation of natural tetraploid loach in China was studied systematically by Li [5,9–14] on cellular and molecular levels. These showed that the natural tetraploid loach is a genetic form (4n = 100) that produces normal 2n eggs and 2n spermatozoa. A new hybrid intraspecific triploid was prepared by crossing diploid and tetraploid loaches , in combination with cross-breeding and triploid breeding. In theory, triploid fish are expected to be sterile, but a few individuals can reach sexual maturity and produce small amounts of eggs and spermatozoa. Therefore, we have previously studied the chromosome numbers, DNA content, meiosis and reproductive characteristics of triploid loaches [15,16] with fertility as the endpoint. From the viewpoint of epigenetics, differences in DNA methylation levels and the control of key genes in hybrid triploid loaches and their parents have been analyzed . However, the molecular mechanisms controlling the fertility of these fish remain unclear.
Triploid fish have the characteristics of fast growth, good meat quality, strong disease resistance, but poor fertility, so its breeding is of great significance . These breeding characteristics have been receiving increasing attention in fishery resource management. It is generally accepted that triploid fish—being infertile—can transform the energy used in gonadal development into muscle growth, thus giving them a potential growth advantage. In addition, the sterility of triploid fish is of great significance for controlling the over-growth of fish and protecting natural germplasm resources. Moreover, triploid fish can also be used as ideal vectors for generating transgenic lines to address ecological safety problems and ethical concerns . Most artificially induced triploid fish proved to be sterile [28–39]. However, there have been differences between reports. Thus, Yang  found that male triploid rainbow trout can produce spermatozoa, but a large number of malformed embryos appeared after hybridization, and all the progeny died during maturation. Yin et al.  found that the gonadal functions of triploid catfish differ between genders, as there was no difference between the normal and diploid testis, but the female ovary showed arrest of egg development at the oogonium stage. Arai  studied direct and reciprocal hybridization by using tetraploid loaches and diploid loaches of unknown origin. The male hybrids were sterile but female hybrids were fertile, and produced large triploid eggs (3n), and small haploid eggs (1n). Both types of egg could be fertilized and produce viable offspring. In the present study, hybrid triploid offspring were obtained by hybridization between tetraploid and diploid loaches, which are unique to China. Histology of the testes showed that spermatogonia, spermatocytes and large numbers of mature spermatozoa could be observed in diploid and triploid testes, but that the mature sperm content was lower than in normal diploid fish. The diploid and triploid female ovaries also contained second, third, and fourth phase oocytes, yolk granules and follicular membranes. However, the molecular mechanisms controlling fertility in such triploid fish have not been reported so far.