核质不相容在新疆杨树杂交带物种维持机制中的作用研究

How species maintains their integrity in the face of frequent hybridization is one of the central questions in speciation. Reduced fitness of hybrids due to the breakdown of co-adapted nuclear gene complexes was often used to explain it. However, the role of cytonuclear incompatibility in hybrid breakdown and speciation has been largely overlooked, especially in plant. In current research, we intend to clarify the role of cytonuclear incompatibility in isolating interspecific gene exchange in Populus, by exploring the reproductive isolation mechanisms in a hybrid zone of Populus alba and P. tremula, two ecologically divergent species that hybridize frequently, along the Irtysh River system in the Altai region, northwest China, where significant cytonuclear disequilibrium was detected previously. First, we will analysis the genetic structure of this hybrid zone in more detail, using both nuclear microsatellite markers and chloroplast DNA sequences, to determine the possibility of nuclear genome incompatibility between P. alba and P. tremula, and to further ascertain the occurrence of cytonuclear disequilibrium. Then, we will estimate the contribution of assortative mating on such cytonuclear disequilibrium as well as on isolating interspecifc gene flow in this hybrid zone, by analyzing the natural mating patterns of hybrids and parental taxa using microsatellite markers, while natural selection against hybrids will be estimated by comparing the frequencies of each hybrid class between natural pollinated seeds and adult trees. And then, we will ascertain the occurrence of cytonuclear incompatibility by comparing the reproductive success between inter and intra cytoplastic and nuclear combinations via hand pollination in lab, and examine whether and how it works on reduced fitness of hybrids through comparing the performance of offspring among these combinations in different parental environment. At last, genes involved in cytonuclear incompatibility will be identified by analyzing compatible and incompatible seedlings using genome and transcriptome sequencing. The results from all above analyses would help us to fully understand the reproductive isolation mechanisms between hybridizing Populus and the contribution of cytonuclear incompatibility on isolating interspecific gene exchange. Our research will provide insights into whether cytonuclear incompatibilities provide sufficiently strong barriers to gene flow to maintain divergence in nature and how these intrinsic mechanisms can facilitate further adaptive divergence to extrinsic ecological environments. This would be very important for our understanding the role of cytonuclear incompatibilities in plant speciation. In addition, our research would be also very important for us to consider how to avoid cytonuclear incompatibility in future hybridization breeding of Populus.

频繁杂交的两个物种如何维持其分化是物种形成的核心问题。核基因组不相容导致杂交后代适合度下降被认为在杂交物种地位维持甚至物种形成中起重要作用，但是核基因组与胞质基因组不相容在植物物种形成中的影响却常被忽视。本项目拟通过对新疆额尔齐斯河流域欧洲山杨和银白杨杂交带繁殖隔离机制的全面解析，明确核质不相容在杨树物种形成中的作用。我们拟利用微卫星标记进行父本分析，确定选型交配对杂交带物种分化的贡献；比较种子和成树中各杂合类型的比例，确定是否存在对杂合基因型的选择淘汰；结合人工授粉及同质生物园实验明确是否存在核质不相容，及其如何起作用；并利用二代测序技术进行全基因组重测序和转录组测序，找到核质不相容涉及的基因。该研究不仅有利于我们全面了解自然杂交带的物种维持机制，还将有助于我们认识，在物种形成过程中，内在隔离机制如何通过外部生态环境来完成物种的适应性进化；同时也为我们后期进行杨树杂交选育提供科学依。

频繁杂交的两个物种如何维持其分化是物种形成的核心问题。在植物中，质体和核基因组之间的相互作用被发现可作用于维持杂交物种的完整性。在新疆额尔齐斯河流域，欧洲山杨和银白杨频繁杂交产生银灰杨，然而杂交亲本物种仍然维持明显分化。本项目对该地区欧洲山杨和银白杨杂交带繁殖隔离机制的解析，确定核基因组与胞质基因组不相容在维持杨树物种完整性中的作用。.对额尔齐斯河流域银灰杨的全面调查和遗传组成的进一步分析表明，该地区的银灰杨个体主要为欧洲山杨和银白杨F1代杂交后代，回交个体很少。人工授粉实验和自然授粉种子的亲本分析，明确了银灰杨与欧洲山杨、银白杨都具有亲和性；银灰杨雌株能接受双亲的花粉产生大量回交的种子，且与所携带的叶绿体类型关系不大。在自然授粉情况下，并未发现选型交配在银灰杨与欧洲山杨和银白杨回交过程中起作用，与亲本雄株的距离可能是影响银灰杨雌株回交的重要因素。人工授粉回交后代的初步分析，并未发现核质不相容对子代的生长和叶绿体含量有重要影响。.完成了欧洲山杨和银白杨不同叶绿体单倍型个体的叶绿体基因组测序和种间比较，发现一些欧洲山杨和银白杨种间差异明显的叶绿体基因。利用7个杨属和1个柳属植物转录组和基因组数据，分析了植物编码PEP全酶亚基的叶绿体RANP基因与核sigma基因家族在杨柳科植物中协同进化情况；发现所研究的8种杨柳科植物中，这两个家族在进化速率相关性并不明显高于其他对照基因，表明在这些植物中叶绿体RANP基因与核sigma基因家族并不存在协同进化。.本研究的结果并未发现核质不相容在新疆额尔齐斯河流域的欧洲山杨和银白杨杂交带物种维持机制中起作用，杂交后代与亲本的距离可能是影响回交的重要因素，推测欧洲山杨和银白杨的生态位不同可能是其分化的重要原因。然而，更完整的答案，还需更多人工授粉和自然授粉实验和子代分析的验证。此外，杨柳科核基因和叶绿体基因之间的协同进化还有待该科更多物种的进一步研究。