Identification of sex‐specific markers and heterogametic XX/XY sex determination system by 2b‐RAD sequencing in redtail catfish (Mystus wyckioides)

Sex‐specific markers provide significant molecular basis for sex control breeding biotechnology to produce all‐male or all‐female fish in commercial breeding. Redtail catfish (Mystus wyckioides), one of the commercial bagrid catfishes distributed in Southeast Asian, which have a long sexual maturation period that can last 3–5 years and males have apparent growth advantage over females, but its sex determination system remains unknown. In this study, we first applied 2b‐RAD‐seq approach to identify three male‐specific 2b‐RAD‐tags and one male heterogametic SNP locus and validated by blast to the genome survey sequences and PCR amplification in both wild and breeding populations. To get longer sex‐specific region, we performed genome walking and obtained a 4,630 bp of Y‐specific sequence and 4,581 bp of X‐specific sequence from the 2b‐RAD‐tag ref189950 with 92.19% nucleotide identity between them. And 9,923 bp/3,935 bp of Y‐specific sequences and 8,491 bp/5,172 bp of X‐specific sequences were also identified with 77.49% and 57.07% nucleotide identity in ref208528 and ref210837, respectively. Subsequently, three different kinds of sex‐specific primers with different length products were designed based on the detected highly sex differentiated regions and could be used to distinguish males and females both in wild and artificially bred populations. What is more, the X‐specific fragment was discovered to produce the dosage effect association in females and in males. The data suggest that male heterogametic XX/XY sex determination system should exist in the redtail catfish. More significantly, the sex‐specific markers are of great value to protect wild resources and improve the efficiency of all‐male breeding practices for aquaculture in the redtail catfish.     

Patterns of sex ratio response to water temperature during sex determination in honmoroko Gnathopogon caerulescens

ABSTRACT:   The sex ratios of the offspring of males from broods of honmoroko Gnathopogon caerulescens that displayed thermolabile sex determination (TSD), and those from eggs collected from the wild, strongly suggest that the combination of parents plays an important supplementary role in TSD in this species. The proportion of females in all broods from eight pairings of fish captured in the field decreased significantly at 30°C compared to 20°C, and one brood was entirely female at the lower temperature. These results suggest that phenotypic males (XX-males) exist in nature, probably as a result of sex change from genetic females caused by TSD. Sex ratios in relation to water temperature fall into five patterns, two of which seem to result from normal pairings of XX-females and XY-males, and from pairings of XX-males and XX-females with non-thermosensitivity. Two other patterns are thought to result from the same types of pairing, but with thermosensitivity. The last pattern, which shows male bias at both low and high temperature, is not explained only by the combination of genotypic sex determination (XX/XY) and TSD. Fluctuating temperatures close to natural conditions showed little potential to masculinize broods laid in the field between April and early June. However, the sex ratios of fish spawned in early June showed male bias at low and high temperatures. The sex determination mechanism in G. caerulescens may involve the interaction between temperature and sex-determining genetic factors, and the relative importance of each component differs with breeding season.     

雌雄同体鱼类性别分化及性转变研究进展

在脊椎动物中,鱼类具有多样的性别分化方式,大致可分为雌雄异体、雌雄同体以及单性生殖3类.一般情况下,鱼类性别决定后,性腺可分化为卵巢或精巢,并且在整个生命周期内保持不变.而在雌雄同体鱼类,其性别可以从雌性转变为雄性、雄性转变为雌性或者在雌雄两种性别间进行多次转变.雌雄同体鱼类具有多种性别转变形式,是研究脊椎动物性别决定...     

鱼类性别决定的遗传基础研究概况

动物从受精卵发育到具有不同性别特征的个体是一个奇妙而又严谨的过程,是人类长期以来试图揭示的自然现象。上世纪90年代初在人类Y染色体上发现了性别决定基因SRY[1],进而发现了一个新的Sox基因家族[2]。上述基因的发现,促进了以哺乳类为代表的动物性别决定和分化机制研究。由于鱼类在脊椎动物中的特殊进化地位、庞大的种类数量以及显著的社会经济价值,鱼类的性别决定研究一直受到遗传和发育学者的重视。尽管离最终阐明鱼类性别决定的机制还有距离,但近20多年来鱼类性别决定的遗传基础研究已取得不少重要进展。本文试图根据现有文献资料,…     

大黄鱼性别特异SNP标记的开发与验证

大黄鱼是我国养殖量最大的海水经济鱼类,其雌鱼生长显著快于雄鱼,但两性的外部形态差异不明显,也没有异形性染色体,依靠传统方法无法对其活体准确进行生理性别和遗传性别的判别与鉴定,需要开发性别特异的分子标记。本研究从2尾雌鱼和2尾雄鱼、以及分别由50雌鱼与50尾雄鱼组成的2个混合样品的基因组重测序数据比较中筛选与性别显著关联的SNP位点,对其中11个位点分别设计引物在15尾雌鱼和15尾雄鱼中扩增出PCR产物进行Sanger测序验证,鉴定出1个与性别完全连锁的位点(SNP6,15尾雌鱼均为纯合、15尾雄鱼均为杂合)。然后,设计等位基因特异性PCR引物,其中包括2条雌性与雄性通用引物和1条雄性特异引物,在闽—粤东族与岱衢族大黄鱼合计近2 200个个体中进行扩增,结果在全部雌鱼中都只扩增出1个348 bp的条带,而在全部雄鱼中还扩增出1个194 bp的Y染色体特异条带,检出率达到100%。研究表明,大黄鱼属于XX♀-XY♂类型的性别决定。本研究鉴定出一个雄性特异SNP标记,并建立了一种新的大黄鱼遗传性别鉴定技术,为大黄鱼单性育种、基因组选择育种和性别决定分子机制研究提供了重要的技术手段。     

Sex differentiation and sex determination in eels: consequences for management

The aim of this review is to present an overview of the sex differentiation and sex determination processes in eels in relation to the urgent need to provide scientific knowledge to better protect and manage the Anguilla genus. Indeed, the global decline of the three main temperate eel stocks, Anguilla anguilla, Anguillidae (Fisheries Management and Ecology, 2003, 10, 365); Anguilla japonica, Anguillidae (Casselman, Eel Biology, Springer Japan, 2003, 293) and Anguilla rostrata, Anguillidae (Tatsukawa, Eel Biology, Springer, Japan, 2003, 255), raises concerns about the necessity to better understand all stages of the life cycle of eels (Righton and Walker, Journal of Fish Biology, 2013, 83, 754). Little is known about the mechanisms involved in the production of males and females in this species with environmental sex determination. Previous reviews identifying the density of individuals as the major factor influencing sex determination were undertaken (Krueger and Oliveira, Environmental Biology of Fishes, 1999, 55, 381; Reviews in Fish Biology and Fisheries, 2005, 15, 37). Here, we review the current advances on the subject, focusing on the roles of early growth rate and interindividual relationships, which are mechanisms underpinned by density, as well as the sex differentiation process, and we question how this knowledge might influence global conservation measures.     

贝类性别决定与分化相关基因研究进展

性别决定与分化是生命发育的基本事件,性别决定与分化机制一直是生命科学研究的热点问题。贝类具有雌雄同体、雌雄异体、雄性先熟和性转换等复杂的性别类型,是研究无脊椎动物性别决定与分化机制及其演化进程的理想动物类群。挖掘贝类性别决定与分化调控基因,阐明相关基因的调控作用,对于揭示贝类性别决定与分化的分子机制具有重要意义。本文就贝类性别决定与分化相关基因的研究进展进行了综述,并对该研究领域进行展望,以期为贝类的性别决定和分化机制、生殖操作和遗传改良等研究提供参考。     

鱼类性别决定的研究进展

有关鱼类性别决定的研究主要集中在温度、性激素、芳香化酶以及随机重复序列、核受体基因等对性别分化的调控方面。由于鱼类所处分类地位较低 ,生活环境千差万别 ,鱼类性别决定没有一个普遍的模式 ,目前研究的鱼类又各不相同 ,因此象哺乳动物那样的性别决定级联模式还没能阐述。本综述旨在阐述近几年有关鱼类性别决定机制方面的研究动态和进展 ,为系统研究鱼类性别决定机制提供参考     

基于转录组分析不同温度下池蝶蚌性别相关基因的表达

性别决定与分化是生命发育的基本事件,性别决定与分化机制一直是生命科学研究的热点问题。贝类具有雌雄同体、雌雄异体、雄性先熟和性转换等复杂的性别类型,是研究无脊椎动物性别决定与分化机制及其演化进程的理想动物类群。挖掘贝类性别决定与分化调控基因,阐明相关基因的调控作用,对于揭示贝类性别决定与分化的分子机制具有重要意义。本文就贝类性别决定与分化相关基因的研究进展进行了综述,并对该研究领域进行展望,以期为贝类的性别决定和分化机制、生殖操作和遗传改良等研究提供参考。     

Effects of estradiol-17β and 17α-methyltestosterone on gonadal sex differentiation in the F2 hybrid sturgeon, the bester

The effects of giving oral estradiol-17β (E₂) and 17α-methyltestosterone (MT) on gonadal sex differentiation in the F₂ hybrid sturgeon, the bester ( Huso huso female ×  Acipenser ruthenus male), are investigated. Giving E₂ at 10 μg/g diet to fish from 14 months until 31 months of age induced incomplete feminization and resulted in approximately 40% abnormal ovary development in which oocytes were observed without ovarian lamellar structures and gonadal shape was similar to normal testis. Giving MT at 25 μg/g diet for the same duration failed to induce masculinization, and resulted in approximately 30% undeveloped gonads even at 30–37 months of age. In contrast, E₂ and MT at only 1 μg/g diet given from 3 to 18 months of age was sufficient to induce feminization and masculinization, respectively. In these fish, feminization and masculinization were observed at 9 months, when most putative ovaries and testes were histologically distinguishable by the shape of the gonadal surface. These results indicate that sex reversal can be induced in these fish by hormone treatment that is started at 3 months age, before morphological differentiation occurs on the stroma of the gonads.     

Allelic segregation of sex‐linked microsatellite markers in Nile tilapia (Oreochromis niloticus) and validation of inheritance in YY population

Stocking of all‐male fingerling produced by direct administration of male hormone 17‐α‐methyltestosterone is the most preferred method for present‐day aquaculture of the Nile tilapia Oreochromis niloticus. However, due to the growing concern of negative impact of steroid hormone in food fish, production of ‘genetically male’ tilapia, which depends on the concrete and thorough understanding of sex determination, has long been a scientific curiosity. The objective of the present study was to identify reliable sex‐linked markers and to evaluate the applicability of those markers in terms of monosex production approach. ‘XY’ neofemales were produced by using synthetic oestrogen and identified through selective breeding and progeny testing. Three females with progeny not deviating from 3:1 sex ratio (male:female) were designated as ‘XY’ neofemales and were used subsequently to produce putative YY progeny. Among the fifteen microsatellite markers tested, marker ARO172 was most informative in differentiating male and female genotypes. Twenty‐seven F₂ fish from three families were identified as putative YY males based on marker genotyping, and four of them were crossed to produce F₃ to validate marker association by progeny testing. The YY males produced 86%–100% male progeny indicating ARO172 a unique sex‐linked marker applicable in marker‐assisted selection.     

低龄中华鲟外科手术性别鉴定技术

取 5 1尾人工养殖 1.5～ 5 .6年龄、体重 2 .6～ 35 .5kg的中华鲟 (Acipensersinensis) ,通过腹腔外科手术检查性腺并取得性腺组织材料。 5～ 5 .6年龄 (体重 18～ 35 .5kg)的中华鲟性腺都能通过肉眼分辩雌雄 ,是进行性别鉴定的较佳时期。手术时 ,水温 14 .3～ 30℃、切口位于腹中线或偏离腹中线 1～ 4cm、切口长 4～ 5 .5cm(体重 2 .6～ 9.7kg)或 3.8～ 7.5cm(13.5～ 35 .5kg)、切口采用医用丝线行交叉缝合或一针一结缝合、出池后的全程操作时间 2 0～ 30min ,可较圆满完成性腺检查和性腺组织取材 ,94 .1% (4 8/5 1)的受试鱼存活 ,伤口在 15～ 4 0d天愈合 ,缝合线自行消失。水温高、切口短、采用一针一结缝合均能加快伤口的愈合     

湖鲟微卫星引物在三种鲟鱼及杂交子代的通用性研究

利用12对湖鲟(Acipenser fulvescens Rafinesque)微卫星引物对小体鲟(Acipenser ruthenus Linnaeus)、施氏鲟(Acipenser schrenckii Brandt)、西伯利亚鲟(Acipenserbaeri Brandt)及三种杂交鲟进行种间的通用性研究.结果表明:12对湖鲟微卫星引物在六种鲟鱼中有较高的通用性,有6对引物(50%)在6种鲟鱼中扩增出PCR产物,但仅有4对在6种鲟鱼中得到清晰可辨且有多态性的条带,可以直接用来分析几种鲟鱼的遗传多样性和进行杂交鲟的辅助育种工作.其中,位点LS-68在三种纯种鲟鱼中的等位基因大小范围上有明显差异,可作为三种纯种鲟鱼的分子标记位点;位点AfuG112在除施氏鲟外的其他鲟鱼中都得到特异扩增产物,可以用于检测施氏鲟群体的纯度(施氏鲟无带,怎么能用来检测纯度),人工辅助育种的品种选择;位点AfuG122仅在三种杂交鲟中得到清晰可辨的条带,可以作为区分杂交种与纯种的分子标记位点.     

Comparative analysis of reproductive performance between spring and autumn forms of Persian sturgeon,Acipenser persicus,Bordin, 1897: with an emphasis on sex steroids and milt quality

To compare the reproductive performance of spring and autumn forms of Persian sturgeon males, Acipenser persicus, the relative abundance of spermiating and non‐spermiating males, milt quality, sex steroids and cortisol were investigated after pituitary preparation (PP) treatment and at the time of spermiation. Our results showed that higher numbers of spring males are spermiated after PP treatment than autumn individuals. The autumn spermiating males had higher and lower values of sperm density and duration of motility than spring males respectively. The milt volume was higher in spring males than in autumn individuals. The percentage of motility was statistically equal in both the groups examined. In both forms of brooders, the serum levels of testosterone (T), 11‐ketotestosterone (11‐KT), progesterone (P), 17α, 20β, 21‐trihydroxy‐4‐pregnen‐3‐one (20βS) and cortisol (C) increased after PP treatment, although such increases did not occur in non‐spermiating males. Our results showed the probable involvement of 20βs, P, T, 11‐KT and C in the final maturation of Persian sturgeon. In addition, it was demonstrated that the spring form of Persian sturgeon shows better reproductive performance than the autumn form in terms of a response to artificial induction and milt quality.     

Male‐biased sex ratio of nonanadromous Oncorhynchus mykiss in a partially migratory population in California

Abstract – Differential rates of anadromy between males and females are common in partially migratory salmonid populations, but this pattern is not fully clear for Oncorhynchus mykiss (rainbow trout/steelhead) from the limited but mixed data available. In particular, there are very few data on sex ratios of juvenile and nonanadromous (resident) fish to help assess sex composition of various life stages and life‐history types. We used a recently developed Y‐chromosome genetic marker to assess sex ratio of stream‐dwelling (i.e., juvenile and nonanadromous) O. mykiss in a small coastal basin in central California, USA. We analysed 384 samples collected from three contiguous study reaches over 3 years. Sex ratio was 1:1 among juvenile‐sized O. mykiss (<150 mm) but highly male‐skewed (83%) among nonanadromous‐sized individuals (≥150 mm), and this sex ratio × size pattern did not differ among years or study reaches. Our results suggest that the rate of anadromy differs between males and females in this basin. Our study also demonstrates the application of new genetic markers to determine sex composition of immature and nonanadromous salmonids, which will help assess sex‐specific life‐history behaviour in partially migratory populations of O. mykiss and other species.     

半滑舌鳎的性腺分化和温度对性别决定的影响

半滑舌鳎(Cynoglossus semilaevisGünther)是近年来新开发的一种理想的增养殖对象,雌性个体生长速度比雄性快2～3倍。由于温度可影响多种鱼类的性腺分化,因而探索温度对该鱼的性别决定和性腺分化的影响,获得性逆转雄性亲本,用于培育全雌后代,具有重要的应用前景和经济效益。通过石蜡组织切片,对半滑舌鳎早期性腺分化进行组织学观察。发现在24℃饲养条件下,孵化后30 d半滑舌鳎性腺开始分化,其中具有裂隙的性腺原基未来发育为卵巢,而不具裂隙的原基未来发育为精巢。在孵化后25～100 d对半滑舌鳎进行不同温度(16℃、20℃、24℃、28℃、32℃)处理,在9月龄时利用石蜡组织切片鉴定表型性别,观察温度对性腺分化的影响,结果表明,28℃和32℃高温能显著提高群体中的雄性比例,使其分别达到69.2%和66.7%;而低温16℃和20℃对性腺分化影响不大,群体中雄性比例分别为56.5%和57.1%;24℃处理群体中雌雄个体比例接近1∶1。半滑舌鳎雌性特异的遗传性别鉴定技术也检测到高温和低温处理组出现了由基因型雌性向表型雄性逆转的雄性个体。这些结果表明温度影响了半滑舌鳎性腺分化方向。     

鲟鱼GtH放射免疫测定方法的建立及催产前后史氏鲟血清GtH含量的变化

用蛋白质快速液相层析(FPLC)分离俄罗斯鲟鱼脑垂体促性腺激素（GtH）,共获得GtH纯化蛋白4.6mg。用SDS-PAGE变性胶电泳图谱初步分析纯化蛋白,用反相高效液相色谱（rpHPLC）分离出两个亚基,质谱测定α亚基分子量：15603KD,两个β亚基分子量分别为14338KD和14694KD。制备了鲟鱼GtH的兔抗血清多克隆抗体,采用氯胺T法用125I标记抗原,用羊抗兔γ球蛋白做二抗,建立鲟鱼GtH的放射免疫测定方法。测定了催产前后不同时间史氏鲟血清中GtH含量的变化,统计分析表明成功排卵的雌鱼比未排卵和阴性对照组在催产后12小时和16小时GTH分泌量升高显著,最高达到234ng/ml;催产效应时间与催产后血清中GtH的含量紧密相关。