牙鲆Paralichthys olivaceus是我国重要的海水养殖鱼类，其育种技术的研究与应用一直是行业关注的重点。染色体操作技术包括雌核发育及三倍体人工诱导等，可用于单性养殖、性别控制或者快速纯化性状等，与常规育种技术等结合能够进行新种创制，是现代遗传育种的重要技术途径。近年来在很多鱼类中进行了广泛应用，但主要集中在淡水鱼类中，海水鱼类的相关研究和应用相对较少也不系统。本研究以牙鲆为研究对象，通过体尺性状的长期监测与比较，明确了同质/异质雌核发育和三倍体牙鲆的生长与外形特征；借助显微镜电镜观察、性激素水平检测与转录组分析等，对比了三倍体和二倍体牙鲆性腺发育的组织细胞学、内分泌和分子水平上的差异，明确了三倍体牙鲆的不育性。这些结果为牙鲆雌核发育与三倍体在育种中的应用提供了基础数据和参考。具体研究结果如下：

1、对2016年和2017年诱导获得的异质雌核发育（MEG）和同质雌核发育（MIG）牙鲆群体，分别在3、6、9、12、18和24 月龄（mph）进行了体尺性状的测量和分析。结果表明，MEG和MIG群体与二倍体对照群体具有相似的生长趋势，并且增长曲线的拐点在12 mph，三种群体从12到18 mph都表现出快速增长（P < 0.05）。总体而言，MEG群体显示出与对照二倍体相似的生长趋势，而MIG群体虽然个体间生长差异很大，但在一定程度上具有较好的生长性能。不同群体之间的体重特定生长率（SGRw）的差异表现出了它们的异速生长。对2017年的MEG、MIG和对照群体的形态学变化进行了对比。分析表明，与对照相比，MEG群体的体型更细、更长，而MIG群体的体型更圆、更短，且显示出较高的形状分散性，所以，MIG诱导和培育更适合筛选具有特定性状的个体。故MEG和MIG群体在其生长特性上都比较正常甚至具有一些优势，可用于牙鲆的选育。同时，分析了MEG和MIG牙鲆性腺发育状况，发现其卵巢虽有少部分发育不良，但是大多数发育正常，且可以产生正常的卵子；几乎所有MEG牙鲆精巢发育正常，都可以产生正常精子，而MIG雄性比例较低，但也有可产生成熟精子的个体。对于发育成熟的雌核发育群体进行人工繁殖，均可以正常受精得到可育的下一代。

2、对人工诱导的三倍体以及二倍体对照牙鲆群体，经倍性鉴定后，分别在同等条件下培育，并从6 mph后进行荧光标记后等比例予以混养。于3、6、9、12、18和24 mph进行了体尺性状的测量和分析。结果显示，二、三倍体牙鲆在24 mph前，无论是混养前还是混养期间，均表现出相同的生长规律，但在各项生长指标上并无明显差异。同时，对18、24和36 mph的二、三倍体牙鲆进行了性腺形态和组织切片观察。基本来说，三倍体性腺发育严重受阻。在外观上，其性腺明显萎缩不饱满，组织学上性腺发育迟缓，发育阶段较二倍体滞后。当二倍体卵巢为Ⅳ-Ⅴ期时，三倍体仍处于Ⅰ-Ⅱ期；二倍体精巢发育到Ⅴ期时，三倍体精巢处于Ⅳ期。进一步的透射电镜观察发现，三倍体牙鲆的雌雄性腺中均发生了细胞自噬和凋亡现象，出现大量凋亡小体，这可能是导致其生殖细胞发育受阻的原因。另外，36 mph的三倍体牙鲆虽然可以产生精子，但是通过镜检观察发现其精子极少有运动的，活力也很低。倍性检测发现三倍体精液呈现1、1-2和3倍，甚至4-5倍非整倍体的倍性。再经扫描电镜下观察，其精子与二倍体精子相比形态异常，呈现头部大小不一、线粒体缺失以及尾部短小等现象。对24 mph的二、三倍体牙鲆血液与性腺中的性激素—雌二醇和睾酮水平进行检测，发现在三倍体卵巢中两种性激素的含量均低于二倍体，但精巢中雌二醇的含量则显著高于二倍体（P < 0.05）。借助转录组分析，比较了18和24 mph的牙鲆二、三倍体性腺转录水平的差异，发现三倍体相对于二倍体在mRNA表达水平上存在明显不同。差异表达基因多为性腺发育相关基因，三倍体精巢中与精子发生相关的基因如mns1、dnah1和dnah5等出现下调；三倍体卵巢中与精巢发育相关的基因如amh、amhr2和wnt4等出现上调，这也为组织学上观察到的三倍体卵巢偏雄现象提供了分子生物学依据。不同月龄二、三倍体性腺的差异表达基因主要富集在代谢和细胞功能（如增殖，分化和迁移）等通路上，表明三倍体牙鲆性腺发育过程中基因表达和信号通路响应与二倍体不同。这些证据也进一步说明了三倍体牙鲆性腺为何发育迟缓、低育甚至不育的缘由。

The olive flounder Paralichthys olivaceus is an important mariculture fish in China, and the research and application of its breeding technology have always been concerned. Chromosome manipulation techniques including artificial induction and breeding of gynogenesis and triploids can be used for mono-sex culture, sex control, or rapid purification of traits. Combined with conventional breeding methods, the techniques could create a new strain and have been important ways of modern genetic breeding. In recent years, Chromosome manipulation techniques have been widely used in many fish species, but mainly in freshwater fish. The related research and application in marine fish are lack and not systematic. In this study, growth and body shape characteristics of the artificially induced meio-/mito-gynogenetic and triploid flounder stocks were clarified through long-term observation and comparison of their morphological traits. By observation under microscope and electron microscope, sex hormone level detection, and transcriptome analysis, the differences of the gonadal development of the triploid and diploid flounder were compared at histocytological, endocrinological, and molecular levels, which showed sterility of the triploid flounder. The researches would provide basic data and references for the application of gynogenesis and triploid of the flounder and other fish in genetic breeding. The main results are as follows:

1. The growth and morphological characteristics of the meio-gynogenesis (MEG) and meio-gynogenesis (MIG) flounder stocks induced in 2016 and 2017 at 3, 6, 9, 12, 18, and 24 months post hatching (mph) were measured and analyzed, respectively. The results showed that the growth trends of both the MEG and MIG stocks were similar to the control stocks, and the inflection point of growth curve was at 12 mph. They all presented fast growth from 12 to 18 mph (P < 0.05). Overall, the MEG fish showed a similar growth with the control diploids, while the MIG fish had a better growth performance to some extent although their growth was quite different. The difference of the specific growth rate in body weight (SGR_w) among different stocks indicated their allometric growth. In terms of the morphological characteristics, different stocks showed different shapes. Compared with the control stock, the body shape of the MEG stock is thinner and longer, while that of the MIG stock is mellower. The frame data indicated that the cumulative contributive proportions of two main principal components extracted with principal component analysis were 79.75% and 78.88% in the MIG and MEG stocks at 12 mph, respectively. And the shape dispersion of the MIG stock was higher, which indicated that MIG induction and cultivation are more suitable for screening individuals with specific traits. Therefore, these results revealed that both the MEG and MIG ones have normal or even some advantages on their growth characteristics, which could be used for the flounder breeding. At the same time, gonadal development of the MEG and MIG flounder was studied, and the results showed that although a small part of their ovaries were underdeveloped, most of the ovaries and almost all of the testes of MEG developed normally, thought the proportion of MIG males is low, but there are also individuals who can produce mature sperm. All of them can produce normal eggs or normal sperm. The mature gynogenetic flounder could be used for artificial propagation to obtain the fertile offspring.

2. The artificially induced triploid and diploid control flounder stocks, after ploidy identification, were respectively cultured in different culture tanks under similar culture condition. At 6 mph, the triploid and diploid fish were respectively labeled with different fluorescence markers and then mixed for culturing in equal proportion in the same tank. The growth and morphological characteristics of them at 3, 6, 9, 12, 18, and 24 mph were studied. The results showed that the diploid and triploid flounder of 3 – 24 mph presented the similar growth patterns both before or during mixed-culture, and there was no significant difference in various growth indexes. In the meantime, observation of the external morphology and histological sections of the gonads at 18, 24, and 36 mph of the diploid and triploid flounder were performed. Generally, development of the triploid gonads was severely blocked. In appearance, the gonads were obvious atrophy and not full. Histologically, the gonadal development was retarded and lagged behind the diploid. When the ovaries of the diploid reached stages Ⅳ-Ⅴ, the triploid ones were still at stages Ⅰ-Ⅱ. And when the testes of the diploid arrived at stage Ⅴ, the triploid ones were at stage Ⅳ. According to observation under transmission electron microscope, autophagy and apoptosis occurred, and a large number of apoptotic bodies appeared in both the male and female gonads of the triploid, which may lead to the inhibition of the triploid germ cell development. Although the 36 mph triploid flounder could produce sperm, very few sperm moved under microscope, and the motility was almost not detected. Ploidy test presented that the triploid sperm appeared haploid, haploid – diplid, and triploid, even 4-5 aneuploidy. Further observation by using scanning electron microscope was performed and the results showed that, compared with the diploid sperm, morphology of the sperm in the triploid group was almost abnormal, mainly showing different sizes of the head, deletion of the mitochondrial, and short tail. The levels of the sex hormones estradiol and testosterone in the blood and gonads of the diploid and triploid flounder at 24 mph were detected. The results presented that the levels in the gonads of the triploid females were lower than those of the diploid ones, while the levels of E2 in the triploid testes were higher than those in the diploid ones (P < 0.05). The differential expression of genes of the flounder diploid and triploid gonads at 18 and 24 mph was also compared with transcriptome analysis. Transcriptome data showed that the mRNA expression levels of the triploids and diploids are quiet different. Many genes related to spermatogenesis in the triploid testis, such as mns1, dnah1 and dnah5, were down-regulated; and in the triploid ovary, genes related to testicular development, such as amh, amhr2 and wnt4, were up-regulated, which also provided molecular evidence with the testicular-like gonads observed in the histology of the triploid ovary. As for the gonads of the diploid and triploid at 18 and 24 mph, the enriched pathways were concerned with metabolism and cell function, which are related to cell proliferation, differentiation, and migration. These results give the further evidences to explain why the gonads of the flounder triploid are developmental retardation, low fertility, and even infertility.

目 录

第一章 绪论. 1

1.1鱼类育种技术. 1

1.2 雌核发育. 1

1.2.1 生长与形态学特征. 9

1.2.2 性腺发育特征. 9

1.3 三倍体. 10

1.3.1 生长特征. 17

1.3.2 性腺发育的变化. 18

1.4 鱼类性腺发育规律. 19

1.4.1 HPG轴相关基因研究现状. 19

1.4.1.1 促性激素释放激素. 21

1.4.1.2 促性腺激素. 22

1.4.1.3 促性激素受体. 23

1.4.1.4性腺中的相关因子. 24

1.4.2 三倍体鱼类生长激素及其分子生物学研究. 25

1.4.3 三倍体鱼类性腺发育及其分子生物学研究. 25

1.4.4 三倍体鱼类性腺发育的组学研究. 26

1.5 牙鲆相关研究现状. 28

1.5.1 雌核发育牙鲆. 28

1.5.2 三倍体牙鲆. 29

1.6 研究目的与意义. 29

第二章 雌核发育牙鲆生长和性腺发育特征. 31

2.1前言. 31

2.2 材料与方法. 31

2.2.1 实验材料. 31

2.2.1.1 实验鱼的诱导与养殖管理. 31

2.2.1.2主要试剂与仪器. 32

2.2.2 实验方法. 33

2.2.2.1 生长指标测量和性别检测. 33

2.2.2.2 形态学指标测量方法. 34

2.2.2.3 组织切片. 34

2.2.2.4 数据处理. 35

2.3 结果. 35

2.3.1 雌核发育牙鲆生长特征. 35

2.3.1.1 MEG和MIG群体的生长特征. 35

2.3.1.2 MEG和MIG群体的形态学特征. 39

2.3.2雌核发育牙鲆性腺发育特征. 44

2.3.2.1 性腺发育状况. 44

2.3.2.2 育性评估. 50

2.4 讨论. 51

2.4.1 雌核发育牙鲆生长规律. 51

2.4.2两种雌核发育的形态差异. 52

2.4.3 雌核发育在鱼类育种中的应用. 54

2.5 小结. 55

第三章 三倍体牙鲆生长和性腺发育特征. 57

3.1 前言. 57

3.2 材料与方法. 58

3.2.1 实验材料. 58

3.2.1.1 实验鱼的诱导与养殖管理. 58

3.2.1.2 主要试剂与仪器. 58

3.2.2 实验方法. 59

3.2.2.1 样品采集. 59

3.2.2.2 倍性测定. 59

3.2.2.3 生长指标的测定. 59

3.2.2.4 组织切片与电镜超薄切片. 59

3.2.2.5性类固醇激素测定. 60

3.2.2.6精子质量评估及形态观察. 61

3.2.2.7牙鲆性腺总RNA提取及cDNA合成. 61

3.2.2.8转录组高通量测序. 62

3.2.2.9荧光实时定量PCR（qPCR）. 65

3.2.2.10统计分析. 67

3.3 结果. 68

3.3.1三倍体牙鲆生长规律. 68

3.3.2 二、三倍体牙鲆性腺发育的差异. 70

3.3.3 三倍体牙鲆性腺转录组分析. 78

3.3.3.1 二、三倍体转录组差异. 82

3.3.3.2 不同月龄二、三倍体性腺转录组的差异分析. 88

3.3.3.3 二、三倍体性腺转录组雌雄间的差异分析. 91

3.3.3.4 转录组数据验证. 96

3.4 讨论. 98

3.4.1 三倍体鱼类的诱导与倍性检测方法的差异. 98

3.4.2 三倍体牙鲆的生长. 98

3.4.3 三倍体鱼类性腺的不育特征. 99

3.4.4 二、三倍体牙鲆性腺转录水平的变化. 101

3.5 小结. 105

第四章 结论与展望. 107

4.1 结论. 107

4.2 展望. 108

参考文献. 109

致谢. 141

作者简介及学位攻读期间发表学术论文情况. 143