实验海洋生物学重点实验室知识库

        黄海绿潮自2007年起至今已连续15年暴发，规模居世界首位，浒苔（Ulva prolifera）是黄海绿潮主要的优势种，结合其形态与生理特征以及遗传分析结果发现，黄海绿潮系由浒苔的单一群体—漂浮生态型（floating ecotype）引发，提示其生物学特性是绿潮暴发的重要内因。尽管在实验室条件下，浒苔漂浮生态型群体与定生群体间可以实现杂交，但其历年间始终保持遗传均一性与特殊性，提示可能具有独特的生活史策略。
        浒苔具有复杂的生活史和多样化的繁殖方式，其有性生活史属于同型世代交替类型，很难从形态上区分孢子体世代与配子体世代，给浒苔生活史研究造成很大困难。因此，需要建立适用大样本量、能够快速准确鉴定藻体世代和性别的方法，以取代费时费力的传统方法，获得具有生态学意义的调查数据。本研究首次开发了针对浒苔性别的分子标记并进行了验证，进一步研究了新标记在浒苔多种繁殖方式中的遗传规律，以此为基础，对连续五年的绿潮漂浮浒苔成体、现场采集微繁体的萌发藻体、以及室内诱导放散微繁体的萌发藻体开展了标记检测，探讨了浒苔的生活史策略，取得以下结果：
        1，成功开发了浒苔不同交配型的特异分子标记
选取来自中国与日本多个浒苔不同群体的大量样本，结合四个石莼属非浒苔样本为实验材料，根据U. partita中预测的性别相关区域，在浒苔中通过同源克隆PRA1基因，开发了两对针对浒苔不同交配型的特异分子标记。标记通过在实验室构建的浒苔有性生活史中检测，证实是等位基因并与交配型相关。标记具有以下优点：1）可同时区分样本的倍性与性别；2）仅通过一步PCR即可完成检测，操作简单；3）适用于样本的高通量同步检测。
         2，明确了新标记在浒苔多种繁殖方式中的遗传规律，在亚细胞水平首次描述了原位体细胞再生这一特殊繁殖方式
         除了有性生殖，还构建了无减数分裂生殖（apomeiosis）、单性生殖（parthenogenesis）、以及原位体细胞再生（in situ somatic embryogenesis）共三种浒苔非典型的繁殖方式，阐明了新标记在总计四种繁殖方式中的遗传规律，并结合流式细胞技术进行了验证。证实仅在有性生殖方式中，存在两性标记的分离与聚合，而在三种非典型繁殖方式中，子代藻体的倍性或性别均与母本一致。另外，首次从亚细胞水平观察描述了原位体细胞再生这一特殊繁殖方式的具体特征，发现其本质上是一种营养繁殖，澄清了之前报导的错误认识，证实这种繁殖方式可在浒苔孢子体、两性配子体、以及其它石莼属物种中广泛发生。
         3，揭示了连续五年漂浮浒苔成体的世代与性别分布及其年际变化特征
利用新标记，对2016-2020年大量漂浮生态型藻体进行了检测，发现：1）2016年为孢子体配子体混杂，2017、2018年为孢子体绝对优势，2019、2020年为配子体绝对优势；2）在一个暴发周期内的不同阶段，藻体的世代及性别分布特征基本保持一致，提示绿潮生物量的增加主要依靠生长与营养繁殖；3）在绿潮暴发年际间，藻体世代及性别分布特征常表现出明显的年际变化，结合对微繁体萌发藻体的检测结果，证实存在活跃的有性生殖过程；4）无论成体的世代组成特征如何，其微繁体均来自多种繁殖方式，其萌发藻体均接近等世代分布（孢子体∶配子体 = 1∶1）；5）世代分布在连续四年（2017-2020）中呈现高度同步化，这不符合同型世代交替生物的典型特征，提示海域环境对优势世代具有强选择性。
         4，提出了浒苔漂浮生态型的生活史策略
在每个暴发年度，浒苔漂浮生态型均利用多种繁殖方式产生等世代分布的两类微繁体（2N/N），推测二者的萌发受到环境因子的强烈选择，从而影响成体的世代分布，同时确保浒苔能够有效应对变化的海域环境，以实现连续多年的稳定暴发。另外，浒苔漂浮生态型既能够发生活跃的有性生殖过程，又能保持其遗传特殊性，提示具有特殊机制，以实现与定生群体间事实上的生殖隔离。
        上述研究结果可为深入揭示黄海浒苔绿潮的生物学成因提供重要理论支撑。

        The Yellow Sea green tide has occurred in successive years since 2007. The floating U. prolifera consists of a genetically unique ecotype, different from other attached populations located in the intertidal zones around the Yellow Sea. Although these two ecotypes could hybridize under laboratory conditions, the floating ecotype of U. prolifera has remained its genetically distinct for years, strongly suggesting a special reproductive strategy that might be related to its blooming.
        U. prolifera has a complex life cycle and a variety of reproductive methods. The sexual life cycle of U. prolifera is diplohaplontic and isomorphic, consisting of alternating generations of dioecious, and haploid gametophytes and diploid sporophytes. Because these two generations have identically shaped thalli, it is hard to discriminate them based on morphological characteristics, especially during their vegetative growth stages, which makes it very difficult to study life history of U. prolifera. It is necessary to set up a method which can identify the generation and sex of algae quickly and accurately, and replace the time-consuming and laborious traditional method to obtain the survey data with real ecological significance. In this study, we first developed and validated the sex-specific molecular markers of U. prolifera, and further studied the genetic rule of the new markers in various reproductive modes of U. prolifera. Based on the above results, the marker was detected the green tide floating U. prolifera adults from 2016 to 2020, the germination of micro-propagules collected during the green tide, and the offspring algae induced by laboratory reproduction. The results are as follows:
        1. Successfully developed specific molecular markers for different mating types of U. prolifera. 
       A large number of samples were collected from different populations of U. prolifera from China and Japan, together with four individuals from four other Ulva species were selected for the development of MT-related markers. Based on the sex-related region reported in U. partita, two pairs of specific molecular markers for different mating types were developed by homologous cloning PRA1 gene from U. prolifera. The marker was tested in the sexual life history of U. partita which was constructed in the laboratory, and the results confirmed that the amplified product was an allele and related to mating type. Compared with existing methods for identifying generation or sex, these MT-related molecular markers have obvious advantages: 1) it can distinguish the generation and sex of the sample at the same time; 2) it can be detected by one-step PCR, and the operation is simple; 3) it is suitable for the high-throughput synchronous detection of the sample. 
        2. The genetic rule of the new marker in various reproduction modes of U. prolifera was clarified, and the special reproduction mode of in situ somatic cell regeneration was described for the first time at subcellular level.
       In addition to sexual reproduction, three atypical reproductive methods, apomeiosis, parthenogenesis, and in situ somatic embryogenesis were constructed, the genetic rule of the new marker in the total four propagation modes was elucidated and verified by flow cytometry. The results showed that only in sexual reproduction, there were segregation and aggregation of markers, and in the three atypical reproduction modes, the Ploidy or sex of the progenies was the same as that of the female parent. In addition, it is the first time to describe the specific characteristics of in situ somatic cell regeneration at the subcellular level, which is essentially a vegetative propagation, it was confirmed that this type of reproduction can occur in sporophytes, gametophytes and other species of Ulva.
        3. The distribution of generation and sex of floating U. prolifera for 5 years and its inter-annual variation were revealed.
        A large number of floating ecotype of U. prolifera were detected from 2016 to 2020 by using the new markers. The results showed that: 1) The sporophytes and gametophytes were mixed in 2016, the sporophytes were absolutely dominant in 2017 and 2018, and the gametophytes were absolutely dominant in 2019 and 2020; 2) At different stages of the green tide outbreak, the generation and sex distribution of the algae were basically consistent, suggesting that the increase of green tide biomass was mainly dependent on growth and vegetative propagation; 3) The characteristics of generation and sex distribution of algal often show obvious inter-annual changes. Combined with the detection results of germination of micro-propagules, it is confirmed that there is an active sexual reproduction process; 4) No matter what the generation composition of green tide floating algae is, the micro-propagules came from various reproductive modes, and the generation distribution of germinating algal were sporophytes : gametophytes = 1 : 1; 5) The generation distribution was highly synchronized in four consecutive years (2017-2020) , which does not accord with the typical characteristics of alternating generations, suggesting that the marine environment has strong selectivity to the dominant generation.
        4. A life-history strategy for the floating ecotype of U. prolifera is proposed
      At the outbreak of green tide each year, the floating ecotypes of U. prolifera produced two types of micro-propagules (sporophytes : gametophytes = 1 : 1) by various reproduction methods. It is inferred that the germination of the two types of micro-propagules was strongly selected by environmental factors, thus affecting the generation distribution of the green tide floating algae, at the same time, the same biomass of the two generations in the micro-propagules ensures that the floating ecotypes of U. prolifera can effectively cope with the changing marine environment to achieve a stable outbreak for many years. In addition, the floating ecotypes of U. prolifera can not only have sexual reproduction, but also maintain their genetic specificity, suggesting that it have special mechanisms to achieve de facto reproductive isolation from attached populations. 
       The above research can provide important theoretical support for revealing the biological causes of the green tide of U. prolifera in the Yellow Sea.