兜兰高通量SSR分子遗传图谱的构建及开花时间性状QTLs定位与分析

Paphiopedilum orchids are world-famous ornamental plants. The flowers are the most important characteristic in Paphiopedilum plants, where the ornamental value is in the flower. Flowering time directly determines longevities of flowers traits for view and admire, and potential possibilities of market development. Studies on cross-breeding activities have focused on flower traits, such as flower organ, and resulted in many reports about new hybrid cultivars. Yet, the inheritance determinism of flowering time traits is poorly understood in Paphiopedilum orchids. In preliminary studies, through transcriptomes analyses using high-troughput sequencing and simple sequence repeat (SSR) loci detection in Paphiopedilum orchids, we have identified that the sequences of Paphiopedilum orchids transcriptomes are abundant in SSR loci, and that developed SSR markers from the Paphiopedilum orchids transcriptomes have high levels of polymorphism, stability, and their transferability from one species to another within the Paphiopedilum genus. Based on previous studies, we employed tessellated leaves with abaxial entirely purple species P. concolor owning the year-round flowering pattern grown under greenhouse, and green leaves with abaxial green species P. hirsutissimum with summer-flowering habits in greenhouse as the parents to develop an interspecific F1 segregating population in this study. We constructed interspecific molecular genetic maps in Paphiopedilum orchids by using the F1 population and SSR markers developed from the sequences in Paphiopedilum orchids transcriptomes. On the results above, flowering time traits were investigated in the parents and F1 population based on intensive measurements of phenological and morphological traits in greenhouse cultivation. In order to search QTLs of flowering time traits and their ranges of contribution ratios for phenotypic variance, QTLs for flowering time traits were localized and analyzed, respectively. Our objective was to parse the genetic determinism of flowering time in Paphiopedilum, and to provide some theoretical basis for future molecular breeding of flower time traits in Paphiopedilum.

兜兰是世界花卉名品，其观赏价值最重要部分是花。开花时间直接决定着兜兰花部性状的观赏时间和市场开发潜力。兜兰杂交育种研究长期以来集中在花器官等性状上，相关新品种的报道较多，而关于其花期性状的遗传机制却知之甚少。前期研究，通过兜兰转录组测序分析和SSR位点检测，证实兜兰转录组序列中含丰富SSR位点，且已开发的SSR标记存在高的多态性、稳定性和种属内可转移性。本项目将在前期基础上，以斑叶、叶背有深紫色斑、温室种植四季开花的同色兜兰和绿叶、温室种植夏季开花的带叶兜兰为亲本，以构建的F1分离群体为作图群体，兜兰转录组序列中检测的SSR标记为作图标记，构建兜兰种间SSR分子遗传图谱。在此基础上，调查亲本和F1群体的开花时间性状，并分别对开花时间性状进行QTL定位和分析，旨在寻找与兜兰开花时间性状的QTLs及其对表型变异的贡献率，解析兜兰开花时间的遗传基础，以期为今后兜兰花期性状的分子育种提供理论依据。

本项目以斑叶、叶背有深紫色斑、温室种植具四季开花习性的同色兜兰和绿叶、温室种植具夏季开花习性的带叶兜兰为亲本，构建F1分离作图群体，兜兰叶片转录组unigenes中检测的SNP标记为作图标记，构建兜兰种间的高密度SNP遗传图谱，以期为今后兜兰重要观赏性状的分子育种提供理论依据和重要观赏性状相关基因的精细定位奠定基础。主要结论如下：.（1）以带叶兜兰为母本（♀）、同色兜兰为父本（♂），通过人工授粉杂交育成F1新品种“红霞兜兰”，属斑叶类，抗性强，花期3～5月，开花期长达50～60天；花梗长5.5～7.8 cm，紫褐色，披白色短柔毛；花1～2朵，直径10～12 cm，紫红色具紫褐色斑点；中萼片宽卵形，长3.5～4.3 cm，宽3.2～4.0 cm，紫红色，边缘具淡黄色晕和紫褐色斑点；合萼片宽卵形，稍小于中萼片，长3.0～3.5 cm，宽2.5～3.0 cm，淡黄白色，具深浅不一的紫红色斑点或斑块；花瓣椭圆形，边缘波状具白色缘毛，长5.0～5.5 cm，宽3.5～4.0 cm；唇瓣兜状，长3.5～4.5 cm，宽1.9～2.1 cm，黄褐色，具紫红色斑点；退化雄蕊黄色，中心具紫褐色晕。花梗长度中等，避免了母本花梗过长易倒伏和父本花梗过短贴着盆生长的缺点，具有较好的观赏价值，对兜兰属植物资源的种质创新和保护具有重要意义。.（2）通过杂交试验获得了在花色、花型和叶色有明显分离的F1群体同色兜兰（♀）×带叶兜兰（♂）。在此基础上，利用RNA-Sequencing技术对兜兰双亲的叶片进行高通量测序，双亲都获得了18G以上的clean data，并对双亲的clean data进行unigene的组装、注释和SNP标记的开发，共获得504,936个SNP标记编码基因型，其中有165,196个SNP标记成功编码，进一步筛选用于遗传图谱作图的SNP标记6,874个。将筛选出的6,874个SNP标记，通过两两标记之间计算MLOD值，过滤掉一些低质量的SNP标记，最后构建了兜兰密度最大的一张遗传连锁图谱，共有13条连锁群，包含5,656个SNP标记，平均遗传距离为0.28cm，总图距为1606.27 cm。其中13号连锁群上最多为1,225个，平均遗传距离为0.09 cm；其次是11号连锁群上为1,085个，平均遗传距离为0.11 cm。最终SNP标记在F1群体中的平均完整度为99.76%。