哲罗鲑性别特异性标记筛选

研究通过比对哲罗鲑Hucho taimen (Pallas)基因组草图与虹鳟(Oncorhynchus mykiss)Y染色体序列, 获得哲罗鲑性别相关的候选序列, 并设计3对PCR扩增引物, 以此筛选哲罗鲑性别特异性标记。初步筛选结果显示, 在设计的3对引物中, 引物ST2在雌鱼中无扩增条带, 在雄鱼中有153 bp的扩增条带, 可作为哲罗鲑雄性特异性候选标记。为了消除样本降解及失误等因素导致的条带缺失, 研究以12S rRNA为参照, 采用双重PCR法, 在12S rRNA引物扩增出条带的前提下, 用ST2引物条带的有无来判断性别, 雌鱼为单带, 无ST2引物条带; 雄鱼为双带, 有ST2引物条带。同时为了验证本方法的可靠性, 对已知性别的哲罗鲑48尾雌、雄样本进行了检测, 结果显示该方法遗传性别鉴别准确率为100%。用此标记筛选哲罗鲑雌、雄鱼简单易行, 为哲罗鲑遗传学研究、单性养殖和性别控制育种等研究奠定了基础。

CHARACTERIZATION OF SEX-SPECIFIC MARKER IN HUCHO TAIMEN (PALLAS)

Hucho taimen belongs to Salmoniformes, Salmonidae, and Hucho. It is one of the rare and precious cold-water fishes with rapid growth, strong disease resistance, and high nutritional value. Due to dramatically declined resources from the 1950s, it has been listed in the China Red Data Book of Endangered Species-Pisces since 1998 and has also been recorded as vulnerable species in the IUCN Red List of Threatened Species. For the conservation and exploitation of this precious fish, researchers successfully carried out an artificial propagation program and established cultivation technology. Given its unique and superior characteristics, taimen has become an excellent aquaculture species and cultured around China since 2003. Despite the conversation or aquaculture of taimen, the central issue is how to produce high-quality eggs and larvae. However, it is difficult to distinguish the male and female individuals by morphology, especially in young fish, which hindered the selection of high-quality parents for propagation, it is essential to develop a simple method to efficiently characterize the male and female taimen. Sex-specific molecular markers could be satisfied with the requirement. Sex-specific markers are an essential tool for mono-sexual fish cultivation, optimization of sex ratio of broodstock, and sex-controlled breeding. To identify sex-specific markers in Hucho taimen, the sex-related candidate sequences were obtained by alignment of the draft genome of Hucho taimen against the Y chromosome of rainbow trout (Oncorhynchus mykiss). Three pairs of primers were designed based on this candidate sequence to characterize the sex-specific markers. The results indicated that one pair of primers (ST2) might be the male-specific marker of Hucho taimen, with a PCR product of 153 bp in male samples but not in female samples. To eliminate the effects of DNA degradation and artificial faults in PCR amplification failure, a duplex PCR assay was established by introducing the 12S rRNA as a reference. Samples were classified into males when ST2 and 12S rRNA were amplified successfully, whereas samples were classified into females when only 12S rRNA was amplified. All 48 samples were correctly classified with the accuracy of 100%. Furthermore, the PCR product amplified by ST2 was sequenced to obtain a 153 bp, and then aligned to taimen draft genome and Hucho hucho genome. This marker was close to sdY gene in Hucho taimen genome, while it matched lrp4 gene in Hucho hucho genome. The gene sdY is the sex determining locus of 15 salmon species, including Oncorhynchus mykiss, Salmo salar, and Hucho hucho, and it needs to determine whether lrp4 gene is related to sex determination. In conclusion, this study characterized a male-specific marker and established a genetic sex identification method to distinguish female from male taimen in a simple and efficient way, which provide a foundation for genetic research, monosexual culture and sex-controlled breeding of Hucho taimen.