海带配子体性别连锁分子标记的细胞遗传学研究

Macroscopic monoecious sporophytes (2n) of Saccharina japonica can produce meiospores which are able to germinate microscopic female and male gametophytes (n). The 1:1 ratio of female to male gametophytes and all the female offsprings from the parthenogenetic sporophytes predict that the kelp has got a X/Y-like sex determination. Three molecular markers related to the sex of kelp gametophytes were developed by us in order to identify their gender since the chromosomes so small that a karyogram cannot be easily obtained. But are these markers linked to sex and where do they locate are to be investigated in detail prior to applications to sex identification. In this project, the speciality to the gametophytes is to be shown by Southern blot at the same time when new markers are developed by use of ISSR. The genetic linkage of these markers to the sex will be examined by special PCR in the parent gametophytes, their crossbred sporophytes and the offsprings released from the sporophytes as well. The location of them on the chromosomes is focused on by use of fluorescence in situ hybridization. This research will not only develop sex-linkage markers to the kelp gametophytes, but also lay a foundation for sex differentiation, sex chromosome and sex determination of this kelp.

雌雄同体二倍性的海带孢子体，经减数分裂产生孢子并分别发育成单倍性的雌、雄配子体，其性别比为1:1。该比值及"孤雌生殖"孢子体产生的游孢子所萌发配子体均为雌性的结果都预示着海带可能存在着类似XY性别决定系统。针对海带染色体小且难以进行核型分析以准确鉴定配子体性别，本课题组率先开发出3个性别相关的分子标记，但它们的性别连锁及细胞遗传学问题，值得深入探讨。为此，本项目在利用ISSR等技术开发新分子标记的同时，计划通过Southern杂交技术分析这些标记在雌、雄配子体中的特异性；借助海带的卵式生殖并辅以单性生殖方式，培养亲本配子体-F1代孢子体-F1代配子体，利用它们来探讨这些标记的性别连锁遗传规律；重点地利用荧光原位杂交（FISH）技术对这些性连锁标记进行染色体定位。本研究任务的完成不仅能开发出海带配子体性连锁分子标记，还能为海带配子体性别分化、性染色体是否存在等研究奠定扎实的技术与理论基础。

雌雄同体二倍性的海带孢子体，经减数分裂产生孢子并分别发育成单倍性的雌、雄配子体，其性别比为：1:1。该比值及“孤雌生殖”孢子体产生的游孢子所萌发配子体均为雌性的结果都预示着海带可能存在着类似XY性别决定系统。针对海带染色体小且难以进行核型分析以准确鉴定配子体性别，本课题组在已开发出的海带配子体性别相关分子标记的基础上，利用ISSR方法新开发出一个雌配子体特异标记，并将其转化为SCAR标记，命名为FRML-1488。利用海带雌、雄配子体lhcf6基因5’侧翼序列差异片段设计引物，开发出一个雄配子体特异分子标记RM-437。通过Southern杂交技术对对FRML-494、FRML-1488和RM-437分子标记进行进一步的雌、雄配子体特异性鉴定，与PCR特异扩增结果一致，再次证实了这些分子标记的性别特异性。遗传稳定性分析发现它们在雌、雄配子体杂合成孢子体的过程中没有发生位点突变，可以完整地遗传至子代。最后，利用FISH技术对这些分子标记进行染色体定位，杂交结果显示以FRML-494和FRML-1488做探针时，仅在雌配子体和孢子体染色体中出现杂交信号，雄配子中没有杂交信号；而以RM-437分子标记做探针则仅在雄配子体中出现杂交信号，雌配子体染色体中无杂交信号，进一步从细胞学角度证实了它们的性别特异性。另外，将两个雌配子体特异标记FRML-494和FRML-1488通过FISH技术进行海带孢子体染色体共定位发现它们分别位于不同的染色体上，说明这两个分子标记为不同的雌染色体特异标记。本项目在世界上首次将海带配子体特性的分子标记定位于染色体上，为后续海带染色体核型分析等工作的开展奠定基础。