栉孔扇贝和虾夷扇贝杂交过程中的细胞与分子遗传学分析
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摘要
本文在顺利进行栉孔扇贝和虾夷扇贝杂交的基础上，采用染色体核型分析、GISH、FISH等细胞遗传学手段和RAPD、ISSR、DNA测序、PCR-RFLP及同工酶电泳等分子生物学手段对两种扇贝在杂交过程中产生的一些重要的遗传学现象进行研究。得出如下结果：
1．对栉孔扇贝和虾夷扇贝正反杂交中的配子受精和子代早期胚胎发育进行观察。结果表明，两种扇贝间不存在明显的配子前生殖隔离，异种配子间的受精能力和杂种胚胎发育过程与亲本自交组相当；但后来的GISH分析表明：两种扇贝配子间可能存在亚生殖隔离方式，即同种配子优先受精现象，表现为当同源精子和异源精子同时存在的情况下，扇贝的卵子优先选择同源的精子受精，说明两种扇贝的配子识别机制还是存在差异的。
2．采用染色体核型分析技术对扇贝正反杂交子代早期胚胎的染色体构成进行研究，结果发现，在染色体数目上，绝大多数杂交子代的染色体数目为38条，仅有少数个体为单倍体、三倍体或非整倍体，核型分析表明杂交子代染色体组型为2n=6m+11sm+18st+3t，基本为双亲染色体核型的综合。引入基因组荧光原位杂交技术，采用优化的GISH条件对杂交子代早期胚胎的染色体构成进行分析，结果发现优化后的GISH技术能够更直观地对杂种子代进行鉴定，绝大部分杂交子代中有一半的染色体(n=19)被一方亲本的基因组探针标记上，而另一半染色体能被另一方亲本的基因组探针标记上，因而说明杂交子代为真正精卵结合水平的杂交种，然而有少数杂交后代为母本类型的个体或不对称继承类型的个体。构建了种属特异性ITS1基因探针，采用优化的FISH条件对杂交扇贝染色体构成进行进一步的分析，结果表明：优化后的FISH技术不仅能通过染色体上的杂交信号对杂交子代进行鉴定，而且还可以通过间期细胞上的杂交信号对杂种进行鉴定，无论用哪方亲本的ITS1基因作探针，杂交子代染色体和分裂相上均产生了亲本半数的杂交信号，进一步证明了栉孔扇贝和虾夷扇贝杂交子代早期胚胎为真正精卵水平的杂交种。
The cytogenetic and molecular analysis technologies, such as karyotype, GISH, FISH, RAPD, ISSR, DNA sequencing, PCR-RFLP and isozyme electrophoresis, were applied to investigate the important phenomenon during the hybridization between Chlamys farreri and Patinopecten yessoensis. The results were as follows. 1. The direct observation of fertilization and early embryo development of hybrids from the cross of C. farreri and P. yessoensis showed that there was no pre-zygote isolation between the two scallop species. The fertilization between gametes of C. farreri and P. yessoensis and early embryo development of hybrids were usually quite normal with no obvious difference confirmed between the cross group and their parental control. But later studies with GISH analysis showed that there might be a subtype of fertilization barrier functioning in the hybridization between the two
scallop species------assortative fertilization. This subtype barrier was represented like
this: when the scallop eggs were fertilized competitively with both conspecific and heterospecific sperm, preference may occur with the conspecific sperm were usually preferentially selected. This may suggest that there is some difference in the sperm-egg recognition system between the two scallop species. 2.Karyotype analysis showed that most of the progenies from the cross of C. farreri× P. yessoensis were diploid hybrids with each individual possessing 38 chromosomes, Only small portion of hybrids proved to be haploid, triploid or aneuploidy. The karyotype formula of the hybrids was 2n=6m+11sm+18st+3t, which was almost the combination of the karyotype of their parents. Genome in situ hybridization technology was developed and optimized to investigate the genomic composition of hybrid scallops. The result showed that GISH analysis was able to distinguish the hybrids easily from their parents. Most of the hybrid progenies possessed half set of chromosomes (n=19), which can be labeled with genome probe of one parent, and
1．对栉孔扇贝和虾夷扇贝正反杂交中的配子受精和子代早期胚胎发育进行观察。结果表明，两种扇贝间不存在明显的配子前生殖隔离，异种配子间的受精能力和杂种胚胎发育过程与亲本自交组相当；但后来的GISH分析表明：两种扇贝配子间可能存在亚生殖隔离方式，即同种配子优先受精现象，表现为当同源精子和异源精子同时存在的情况下，扇贝的卵子优先选择同源的精子受精，说明两种扇贝的配子识别机制还是存在差异的。
2．采用染色体核型分析技术对扇贝正反杂交子代早期胚胎的染色体构成进行研究，结果发现，在染色体数目上，绝大多数杂交子代的染色体数目为38条，仅有少数个体为单倍体、三倍体或非整倍体，核型分析表明杂交子代染色体组型为2n=6m+11sm+18st+3t，基本为双亲染色体核型的综合。引入基因组荧光原位杂交技术，采用优化的GISH条件对杂交子代早期胚胎的染色体构成进行分析，结果发现优化后的GISH技术能够更直观地对杂种子代进行鉴定，绝大部分杂交子代中有一半的染色体(n=19)被一方亲本的基因组探针标记上，而另一半染色体能被另一方亲本的基因组探针标记上，因而说明杂交子代为真正精卵结合水平的杂交种，然而有少数杂交后代为母本类型的个体或不对称继承类型的个体。构建了种属特异性ITS1基因探针，采用优化的FISH条件对杂交扇贝染色体构成进行进一步的分析，结果表明：优化后的FISH技术不仅能通过染色体上的杂交信号对杂交子代进行鉴定，而且还可以通过间期细胞上的杂交信号对杂种进行鉴定，无论用哪方亲本的ITS1基因作探针，杂交子代染色体和分裂相上均产生了亲本半数的杂交信号，进一步证明了栉孔扇贝和虾夷扇贝杂交子代早期胚胎为真正精卵水平的杂交种。
The cytogenetic and molecular analysis technologies, such as karyotype, GISH, FISH, RAPD, ISSR, DNA sequencing, PCR-RFLP and isozyme electrophoresis, were applied to investigate the important phenomenon during the hybridization between Chlamys farreri and Patinopecten yessoensis. The results were as follows. 1. The direct observation of fertilization and early embryo development of hybrids from the cross of C. farreri and P. yessoensis showed that there was no pre-zygote isolation between the two scallop species. The fertilization between gametes of C. farreri and P. yessoensis and early embryo development of hybrids were usually quite normal with no obvious difference confirmed between the cross group and their parental control. But later studies with GISH analysis showed that there might be a subtype of fertilization barrier functioning in the hybridization between the two
scallop species------assortative fertilization. This subtype barrier was represented like
this: when the scallop eggs were fertilized competitively with both conspecific and heterospecific sperm, preference may occur with the conspecific sperm were usually preferentially selected. This may suggest that there is some difference in the sperm-egg recognition system between the two scallop species. 2.Karyotype analysis showed that most of the progenies from the cross of C. farreri× P. yessoensis were diploid hybrids with each individual possessing 38 chromosomes, Only small portion of hybrids proved to be haploid, triploid or aneuploidy. The karyotype formula of the hybrids was 2n=6m+11sm+18st+3t, which was almost the combination of the karyotype of their parents. Genome in situ hybridization technology was developed and optimized to investigate the genomic composition of hybrid scallops. The result showed that GISH analysis was able to distinguish the hybrids easily from their parents. Most of the hybrid progenies possessed half set of chromosomes (n=19), which can be labeled with genome probe of one parent, and
引文
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