多倍体鲫鲂的形成及其生物学特征研究
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摘要
远缘杂交能有效的增加杂交后代的遗传变异,通过这种方法还能获得不同倍性的后代,包括杂交四倍体、杂交三倍体和雌核发育二倍体后代。本研究中,我们利用红鲫(♀)与团头鲂((?))进行亚科间杂交首次获得了大量不同倍性的后代,并对不同倍性鱼的形成机制和生物学特征进行了系统的研究。具体研究内容如下：
1.利用不同亚科且染色体数目也不相同的红鲫(2n=100)(♀)与团头鲂(2n=48)((?))杂交,成功的获得不育三倍体鲫鲂(3n=124)和两性可育四倍体鲫鲂(4n=148)。三倍体鲫鲂来源于受精卵第二极体的排除被抑制,而四倍体鲫鲂则来源于受精卵的第一次有丝分裂被抑制。四倍体鲫鲂两年性成熟,性成熟后的雌性四倍体鲫鲂能产生减数(2n)和不减数(4n)的卵子,并与雄性团头鲂回交获得了五倍体鲫鲂(5n=172)。四倍体鲫鲂产生不减数配子的现象与减数分裂前核内复制或者核内有丝分裂或者生殖细胞融合有关。本研究通过染色体数目、核型、平均DNA含量以及红细胞核体积大小确定了这些多倍体鲫鲂的倍性水平和染色体组成。另外,还通过扩增出相同和不同的DNA片段并且测序来揭示杂交多倍体鲫鲂与其亲本间的遗传关系和遗传标记。
2.F1代四倍体鲫鲂自交获得两种不同类型的鲫鲂F2代(A型、B型),分别自交后获得了F3代。A型鲫鲂F2、F3代个体都是染色体数目为148的四倍体,包含2套红鲫染色体和2套团头鲂染色体；B型鲫鲂F2、F3代个体都是染色体数目为200的四倍体,包含4套红鲫染色体；A、B型鲫鲂F2、F3代均两性可育且一年性成熟,并且能稳定的产生二倍体配子。利用B型四倍体鲫鲂((?))与红鲫(♀)杂交制备出生长速度快的不育三倍体鱼(3n=150)。
3.用遗传灭活的团头鲂精子诱导F1代四倍体鲫鲂产生的卵子,无需染色体加倍处理,获得了存活的雌核发育后代,其中包括染色体数目为148的雌核发育四倍体和染色体数目为100的雌核发育二倍体后代。染色体数目为148的雌核发育后代只需要一年性成熟,而且能产生四倍体卵子。
4.红鲫(2n=100)(♀)与团头鲂(2n=48)((?))杂交第一代中获得了雌、雄两性的天然雌核发育红鲫(GRCC),而且性别比例接近1：1。GRCC两性可育,自交后形成了两性可育的青色和红色两种类型的鲫鱼后代(GGCC和GRCC1)。通过染色体数目检测以及核型分析发现,GRCC、GGCC和GRCC1都为二倍体(2n=100)并且含有1—3个微小染色体。包含有雄性决定基因的微小染色体形成时父本遗传渗漏造成了雄性雌核发育鱼的形成。团头鲂精子诱导红鲫卵子之后失活并最终降解,但是会有微小染色体留在卵子中。激活的红鲫卵子拥有50条染色体,通过自然加倍后变成100条染色体,并最终发育成为雄性的二倍体雌核发育红鲫。
5.通过FISH杂交、sox-HMG DNA标记和微卫星DNA标记来揭示天然雌核发育红鲫(GRCC)与其亲本(红鲫和团头鲂)间的遗传关系和遗传变异,并进一步从分子水平证明GRCC、GGCC和GRCC1染色体分裂相中的微小染色体来源于父本。相关结果也证明了远缘杂交能够获得由父本引起遗传变异的两性可育二倍体雌核发育鱼。
6.对红鲫(2n=100)、团头鲂(2n=48)及其远缘杂交所获得四倍体(4n=148)、三倍体(3n=124)后代的5S rDNA的编码区序列(5S)和非转录间隔区序列(NTS)进行比较研究。母本红鲫基因组内有三种不同大小的5S rDNA结构单元,其NTS序列分别为83、220和357bp。父本团头鲂基因组内只有一种5S rDNA结构单元,NTS序列为68bp。在红鲫与团头鲂的远缘杂交多倍体后代中,四倍体鲫鲂继承了母本部分的5S rDNA结构单位,自身还形成了一种明显不同于亲本的独特5S rDNA序列,而且父本特有的5S rDNA结构单元在四倍体鲫鲂基因组中完全丢失,表现出明显的进化特征。三倍体鲫鲂不仅完全继承了亲本所特有的5S rDNA结构单元,而且核酸序列没有明显的重组和变异,稳定的保留了5S rDNA的亲本结构特征。
The distant crossing is an effective means to increase genetic variation in the hybrid progeny. With this method, it is possible to form the different ploidy offsprings including the tetraploid hybrids, triploid hybrids, and gynogenetic diploid. In this study, we first obtained many different ploidy offsprings of red crucian carp (RCC)(♀)×blunt snout bream (BSB)(♂), and their biological characteristics and the formation mechanism were studied. The major results were presented as follows:
1. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (2n=100)(♀)×blunt snout bream (2n=48)(♂), which belonged to a different subfamily of fish and had different chromosome numbers in the catalog. The triploid hybrids resulted from the retention of the second polar body of the fertilized eggs and the tetraploid hybrids resulted from the inhibition of the first cleavage of the fertilized eggs.The 2-year-old female tetraploid hybrids reached maturity, and produced unreduced (4n) and reduced (2n) eggs. Pentaploid hybrids were found in the progeny of 4nRB(♀)×BSB (♂). The formation of the unreduced tetraploid eggs was also due to premeiotic endoreduplication, endomitosis, or fusion of germ cells of oogonia. The ploidy levels and chromosome composition of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers.
2. The females and males tetraploid hybrids of red crucian carp (RCC)(♀)×blunt snout bream (BSB)(♂) mated each other to generate the two types of F2(F2-A, F2-B). F2-A and F2-B fertilized with each other to form F3-A and F3-B, respectively. F2-A and F3-A were both tetraploid hybrids with 148 chromosomes with two sets from RCC and two sets from BSB; F2-B and F3-B both were tetraploid hybrids with 200 chromosomes with four sets from RCC. F2-B, F3-B, F2-A, and F3-A matured at the age of one year, and steadily produced diploid gametes. F3-B(♂) were mated with RCC(♀) to produce sterile triple crucian carp (3n=150) with fast-growth.
3. Following activation by UV-irradiated sperm of blunt snout bream, without the cold-shocking, tetraploid eggs of the allotetraploid hybrids (red crucian carp (♀)×blunt snout bream (♂)) developed into normal live gynogenetic offsprings. All these gynogenetic progenies were female, including diploids with 100 chromosomes and tetraploid with 148 chromosomes. The gynogenetic progenies with 148 chromosomes matured at the age of one year, which produced tetraploid eggs.
4. The females and unexpected males of natural gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (♀,2n=100)×blunt snout bream (♂,2n=48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC1) and another variational gray crucian carps (GGCC). The GRCC and their offsprings were proved to be diploids (2n=100) with one to three microchromosomes by examining the chromosomal metaphases and karyotypes. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish.
5. Means of fluorescence in situ hybridization (FISH), Sox-HMG DNA markers, and microsatellite DNA markers were performed in bisexual GRCC and their parents, revealing their molecular genetic relationship and genetic variation, and further indicating the microchromosomes derived from the paternal fish. Our results proved that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish
6. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) were conducted in red crucian carp, blunt snout bream, and their polyploid offspring. The three monomeric 5S rDNA classes of RCC were characterized by distinct NTS types with 83, 220 and 357bp, respectively. In BSB, only one monomeric 5S rDNA was observed, which was characterized by one NTS type with 68bp. In the polyploid offspring, the tetraploid hybrids partially inherited 5S rDNA classes from their female parent; however, they also possessed a unique 5S rDNA sequence with a novel NTS sequence. The characteristic paternal 5S rDNA sequences were not observed. The 5S rDNA of triploid hybrids was completely inherited from the parental species, and generally preserved the parental 5S rDNA structural organization.
1.利用不同亚科且染色体数目也不相同的红鲫(2n=100)(♀)与团头鲂(2n=48)((?))杂交,成功的获得不育三倍体鲫鲂(3n=124)和两性可育四倍体鲫鲂(4n=148)。三倍体鲫鲂来源于受精卵第二极体的排除被抑制,而四倍体鲫鲂则来源于受精卵的第一次有丝分裂被抑制。四倍体鲫鲂两年性成熟,性成熟后的雌性四倍体鲫鲂能产生减数(2n)和不减数(4n)的卵子,并与雄性团头鲂回交获得了五倍体鲫鲂(5n=172)。四倍体鲫鲂产生不减数配子的现象与减数分裂前核内复制或者核内有丝分裂或者生殖细胞融合有关。本研究通过染色体数目、核型、平均DNA含量以及红细胞核体积大小确定了这些多倍体鲫鲂的倍性水平和染色体组成。另外,还通过扩增出相同和不同的DNA片段并且测序来揭示杂交多倍体鲫鲂与其亲本间的遗传关系和遗传标记。
2.F1代四倍体鲫鲂自交获得两种不同类型的鲫鲂F2代(A型、B型),分别自交后获得了F3代。A型鲫鲂F2、F3代个体都是染色体数目为148的四倍体,包含2套红鲫染色体和2套团头鲂染色体；B型鲫鲂F2、F3代个体都是染色体数目为200的四倍体,包含4套红鲫染色体；A、B型鲫鲂F2、F3代均两性可育且一年性成熟,并且能稳定的产生二倍体配子。利用B型四倍体鲫鲂((?))与红鲫(♀)杂交制备出生长速度快的不育三倍体鱼(3n=150)。
3.用遗传灭活的团头鲂精子诱导F1代四倍体鲫鲂产生的卵子,无需染色体加倍处理,获得了存活的雌核发育后代,其中包括染色体数目为148的雌核发育四倍体和染色体数目为100的雌核发育二倍体后代。染色体数目为148的雌核发育后代只需要一年性成熟,而且能产生四倍体卵子。
4.红鲫(2n=100)(♀)与团头鲂(2n=48)((?))杂交第一代中获得了雌、雄两性的天然雌核发育红鲫(GRCC),而且性别比例接近1：1。GRCC两性可育,自交后形成了两性可育的青色和红色两种类型的鲫鱼后代(GGCC和GRCC1)。通过染色体数目检测以及核型分析发现,GRCC、GGCC和GRCC1都为二倍体(2n=100)并且含有1—3个微小染色体。包含有雄性决定基因的微小染色体形成时父本遗传渗漏造成了雄性雌核发育鱼的形成。团头鲂精子诱导红鲫卵子之后失活并最终降解,但是会有微小染色体留在卵子中。激活的红鲫卵子拥有50条染色体,通过自然加倍后变成100条染色体,并最终发育成为雄性的二倍体雌核发育红鲫。
5.通过FISH杂交、sox-HMG DNA标记和微卫星DNA标记来揭示天然雌核发育红鲫(GRCC)与其亲本(红鲫和团头鲂)间的遗传关系和遗传变异,并进一步从分子水平证明GRCC、GGCC和GRCC1染色体分裂相中的微小染色体来源于父本。相关结果也证明了远缘杂交能够获得由父本引起遗传变异的两性可育二倍体雌核发育鱼。
6.对红鲫(2n=100)、团头鲂(2n=48)及其远缘杂交所获得四倍体(4n=148)、三倍体(3n=124)后代的5S rDNA的编码区序列(5S)和非转录间隔区序列(NTS)进行比较研究。母本红鲫基因组内有三种不同大小的5S rDNA结构单元,其NTS序列分别为83、220和357bp。父本团头鲂基因组内只有一种5S rDNA结构单元,NTS序列为68bp。在红鲫与团头鲂的远缘杂交多倍体后代中,四倍体鲫鲂继承了母本部分的5S rDNA结构单位,自身还形成了一种明显不同于亲本的独特5S rDNA序列,而且父本特有的5S rDNA结构单元在四倍体鲫鲂基因组中完全丢失,表现出明显的进化特征。三倍体鲫鲂不仅完全继承了亲本所特有的5S rDNA结构单元,而且核酸序列没有明显的重组和变异,稳定的保留了5S rDNA的亲本结构特征。
The distant crossing is an effective means to increase genetic variation in the hybrid progeny. With this method, it is possible to form the different ploidy offsprings including the tetraploid hybrids, triploid hybrids, and gynogenetic diploid. In this study, we first obtained many different ploidy offsprings of red crucian carp (RCC)(♀)×blunt snout bream (BSB)(♂), and their biological characteristics and the formation mechanism were studied. The major results were presented as follows:
1. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (2n=100)(♀)×blunt snout bream (2n=48)(♂), which belonged to a different subfamily of fish and had different chromosome numbers in the catalog. The triploid hybrids resulted from the retention of the second polar body of the fertilized eggs and the tetraploid hybrids resulted from the inhibition of the first cleavage of the fertilized eggs.The 2-year-old female tetraploid hybrids reached maturity, and produced unreduced (4n) and reduced (2n) eggs. Pentaploid hybrids were found in the progeny of 4nRB(♀)×BSB (♂). The formation of the unreduced tetraploid eggs was also due to premeiotic endoreduplication, endomitosis, or fusion of germ cells of oogonia. The ploidy levels and chromosome composition of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers.
2. The females and males tetraploid hybrids of red crucian carp (RCC)(♀)×blunt snout bream (BSB)(♂) mated each other to generate the two types of F2(F2-A, F2-B). F2-A and F2-B fertilized with each other to form F3-A and F3-B, respectively. F2-A and F3-A were both tetraploid hybrids with 148 chromosomes with two sets from RCC and two sets from BSB; F2-B and F3-B both were tetraploid hybrids with 200 chromosomes with four sets from RCC. F2-B, F3-B, F2-A, and F3-A matured at the age of one year, and steadily produced diploid gametes. F3-B(♂) were mated with RCC(♀) to produce sterile triple crucian carp (3n=150) with fast-growth.
3. Following activation by UV-irradiated sperm of blunt snout bream, without the cold-shocking, tetraploid eggs of the allotetraploid hybrids (red crucian carp (♀)×blunt snout bream (♂)) developed into normal live gynogenetic offsprings. All these gynogenetic progenies were female, including diploids with 100 chromosomes and tetraploid with 148 chromosomes. The gynogenetic progenies with 148 chromosomes matured at the age of one year, which produced tetraploid eggs.
4. The females and unexpected males of natural gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (♀,2n=100)×blunt snout bream (♂,2n=48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC1) and another variational gray crucian carps (GGCC). The GRCC and their offsprings were proved to be diploids (2n=100) with one to three microchromosomes by examining the chromosomal metaphases and karyotypes. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish.
5. Means of fluorescence in situ hybridization (FISH), Sox-HMG DNA markers, and microsatellite DNA markers were performed in bisexual GRCC and their parents, revealing their molecular genetic relationship and genetic variation, and further indicating the microchromosomes derived from the paternal fish. Our results proved that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish
6. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) were conducted in red crucian carp, blunt snout bream, and their polyploid offspring. The three monomeric 5S rDNA classes of RCC were characterized by distinct NTS types with 83, 220 and 357bp, respectively. In BSB, only one monomeric 5S rDNA was observed, which was characterized by one NTS type with 68bp. In the polyploid offspring, the tetraploid hybrids partially inherited 5S rDNA classes from their female parent; however, they also possessed a unique 5S rDNA sequence with a novel NTS sequence. The characteristic paternal 5S rDNA sequences were not observed. The 5S rDNA of triploid hybrids was completely inherited from the parental species, and generally preserved the parental 5S rDNA structural organization.
引文
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