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题名: 鱼类性别连锁DNA标记的分离及其用于性别控制育种研究
作者: 王达
答辩日期: 2009-05-30
导师: 桂建芳
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 黄颡鱼 ; 银鲫 ; X和Y染色体连锁DNA标记 ; 全雄鱼 ; 雄性特异DNA标记 ; 雄鱼起源
其他题名: Studies on isolation of sex-linked DNA markers and application in artificial sex control in fish
摘要: 黄颡鱼和银鲫均为我国重要的淡水养殖鱼类。黄颡鱼雌、雄生长速度差异显著,其雄鱼生长速度比雌鱼快近两倍。因此,如果能够在育种中人工控制黄颡鱼的性别,培育出全雄黄颡鱼,将大大提高养殖效益。本研究利用AFLP技术筛选得到黄颡鱼X和Y染色体连锁的DNA标记,并基于此提出了一个分子标记辅助的全雄黄颡鱼培育路线,可用于全雄黄颡鱼的持续生产。而银鲫不仅仅是我国重要的经济鱼类,更是研究进化生物学和发育生物学的一种兼具有雌核发育和有性生殖的独特三倍体鱼类。在银鲫的天然群体中含有5%~20%的雄性个体,然而目前对于银鲫的性别决定机制和雄鱼起源尚不清楚,本研究通过对一个雌核发育银鲫偏雄突变系的分析,从中分离得到一个银鲫雄性特异的等位基因,为银鲫的性别决定分子机制和雄鱼起源的研究打下基础。 在黄颡鱼的研究中,基于两性繁殖得到的黄颡鱼家系和人工雌核发育获得的XX雌鱼、XY雄鱼和YY超雄鱼,结合AFLP分子标记技术和BSA分析方法,我们成功地筛选得到6个黄颡鱼X和Y染色体连锁的AFLP片段,并最终将其中的4个X和Y染色体连锁的AFLP片段转换为SCAR标记,从而建立了黄颡鱼遗传性别PCR鉴定方法。首先,基于AFLP技术、BSA分析方法和个体筛选,对黄颡鱼两性繁殖雌、雄鱼,以及雌核发育XX雌鱼、XY雄鱼和YY超雄鱼基因组进行扫描和对比分析,我们筛选得到6个黄颡鱼X和Y染色体连锁的AFLP片段,并克隆了这6个黄颡鱼性染色体连锁的AFLP片段;序列比对显示其中的4个片段(Pf62-Y和Pf62-X;Pf33-Y和Pf63-X)分别为两对等位基因,并分别命名为Pf62和Pf33;其次,通过基因组步移技术,我们克隆得到等位基因Pf62的核苷酸序列共3578bp和等位基因Pf33的核苷酸序列共1987bp;为分析这两个等位基因分别在黄颡鱼X和Y染色体上拷贝间的序列差异,我们分别于黄颡鱼XX、XY和YY基因组中扩增这两个等位基因的部分序列,结果克隆得到Pf33的两个等位序列Pf33-Y 和Pf33-X(片段大小分别为1503bp和1502bp),以及Pf62的三个等位序列Pf62-Y,Pf62-X和Pf62-Xs(片段大小分别为1382bp,1415bp和1102bp),Pf62-X和Pf62-Xs序列差异为一个313bp的插入/缺失序列;其中,Pf33-Y 和Pf62-Y为Y染色体连锁的序列,Pf33-X、Pf62-X和Pf62-Xs为X染色体连锁的序列;通过序列比对分析,我们在这些等位序列上发现了一些X或Y染色体特异的多态位点;基于这些多态位点,设计了4对特异引物,成功地将它们转化为黄颡鱼X和Y染色体连锁的SCAR标记,建立了黄颡鱼遗传性别PCR鉴定方法,并以此为基础提出了一套分子标记辅助的全雄黄颡鱼培育路线。 在银鲫的研究工作中,我们在人工繁殖实验中发现了一个具有97.2%雄性子代的雌核发育银鲫偏雄突变系;通过对这个偏雄突变系的分析,最终从中分离得到一个具有转录活性的银鲫雄性特异等位基因。首先,通过免疫组化技术揭示了银鲫偏雄突变系中雄鱼的精巢结构及其精子发生都是正常的;并通过测交实验证实这些雄鱼的雄性决定遗传物质是可以遗传至下一代的,且突变系中2.8%的雌鱼为遗传上的雌鱼;其次,使用AFLP技术对突变系的亲本及其子代进行遗传背景分析,证实突变系中的雌、雄鱼确实为同一母本的雌核发育子代,雌雄子代间具有非常接近的遗传背景,特别适于筛选性别特异DNA标记;因此,结合BSA分析方法,使用256个AFLP引物组合对雌雄子代基因组进行扩增比较,并分离得到一个大小为86bp的银鲫雄性特异AFLP片段,命名为CagM86;通过基因组步移技术从雄性银鲫基因组中克隆得到该雄性特异片段及其侧翼序列共4016bp,并将其转化为雄性特异的SCAR标记;blastx分析显示该雄性特异序列为一个含有SET Domain的新基因,因此将该基因命名为CagSET-M;随后,我们在银鲫D系、A系和二倍体彩鲫的雌雄基因组中分别克隆得到该基因的多个同源序列,基于聚类分析结果,我们发现从彩鲫基因组中克隆得到的同源序列Ca显示出进化上与银鲫雄性特异等位基因CagSET-M的关系最近;接着,通过RT-PCR检测,发现银鲫雄性特异等位基因CagSET-M是具有转录活性的,并且特异于银鲫精巢中转录;最后,通过RACE技术于银鲫精巢Smart cDNA文库中克隆得到CagSET-M共2134nt的cDNA全长,结合基因组序列,确立了该基因的结构。根据以上数据,分析了形成雌核发育银鲫偏雄突变系的可能原因,讨论了银鲫雄鱼起源的可能途径。
英文摘要: The yellow catfish (Pelteobagrus fulvidraco Richardson) and the gibel carp (Carassius auratus gibelio) are both commercially important fish species in China. In yellow catfish (Pelteobagrus fulvidraco), adult males grow about 3 times larger than female adults. Therefore, studies on the sex-linked marker, sex-determining mechanism and genetic manipulation for producing all-male population would be important and interesting subject for this species, and would be of significant benefit for yellow catfish aquaculture. Speaking to the gibel carp, in addition to its popularity in the fishery and aquarium industries, the genetic background of this species is noteworthy because it is a uniquely gynogenetic species with minor ratio of males in natural habitats, and is able to reproduce both by gynogenetic and gonochoristic modes. Hence, isolation of sex-linked markers in gibel carp would help us to explore the male origin and sex determination mechanisms in this species. This dissertation consists of two sections. In studies on yellow catfish, a Y-linked and X-linked allele marker-assisted sex control technique for mass production of all-male yellow catfish has been developed through AFLP approach. Based on the artificial propagation families, and gynogenetic XX, XY and YY individuals obtained previously, six Y-linked and X-linked AFLP fragments were screened by sex-genotype pool bulked segregant analysis and individual screening. Interestingly, sequence analysis revealed two pairs of allelic genes from them. Furthermore, the flanking sequences were cloned and sequenced from a XY male, and a pair of locus-specific primers for Pf33 and Pf62 was respectively designed to amplify their copies from 8 XX, 8 XY and 8 YY genomes. Significantly, two alleles of Pf33 were identified and named Pf33-Y and Pf33-X with 1503bp and 1502bp, and three alleles of Pf62 were identified, and named Pf62-Y, Pf62-X, Pf62-Xs and with 1382bp, 1415bp and 1102bp respectively. According to the variations in the sequences, four Y-linked or X-linked SCAR primer pairs were designed and converted into Y-linked and X-linked SCAR markers (YSM and XSM). Consequently, the YSM and XSM were successfully used to identify genetic sex and YY super-male, and applied to all-male population production. According to the results, a novel and simple technique system for commercial production of YY super-males and all-male populations was suggested in the yellow catfish. The other part is about the gibel carp. In this study, a male-biased mutant family was discovered from the gynogenetic gibel carp, and a male-specific allele was identified from the mutant family. Firstly, normal spermatogenesis was observed in the testes of males in the mutant family by immunofluorescence histochemistry. Testing cross demonstrate that the 2.8% females were genetic females and the male-determined genetic materials in the mutant males can transmit to the next generation. Subsequently, nearly identical AFLP profiles were observed between males and females in the male-biased mutant family, but a male-specific 86bp AFLP fragment was screened by sex-pool bulked segregant analysis and individual screening. Based on the 86bp male-specific AFLP fragment, a total of 4016bp flanking sequences of this male-specific AFLP fragment were obtained by genome walking, and a male-specific SCAR marker was designed, and the male-specific DNA fragment was confirmed to be steadily transmitted to the next generation and to be consistently detected only in males. Furthermore, six homologous sequences of the male-specific sequence were identified from genomes of male and female Gibel carp clone D and clone A and diploid Carassius auratus, and phylogenetic analysis of these homologous sequences indicated that the male-specific allele of Gibel carp was genetically more related to the homologous sequence identified from diploid Carassius auratus. Moreover, the 4016bp male-specific sequence revealed a novel SET domain gene CagSET-M, which was subsequently identified to be specially transcribed in testis of adult male Gibel carp. Then, a Smart cDNA library of testis of Gibel carp was constructed, and the full length of CagSET-M was cloned by RACE approach. Finally, the male determination and male origin of gibel carp were discussed.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12494
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
鱼类性别连锁DNA标记的分离及其用于性别控制育种研究.王达[d].中国科学院水生生物研究所,2009.20-25
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