马铃薯薯形Ro基因的图位克隆与功能分析

Tuber shape is an important appearance quality of potato and also important target trait in potato breeding. The major tuber shape gene Ro was located on the chromosome 10 with still un-cloned so far. In our previous study, a preliminary physical map of Ro gene was constructed based on a diploid potato population. Moreover, the electronic physical map of Ro gene was constructed through the PacBio sequencing of BAC-end library. In the present study, we will intend to use the BAC-end sequences to develop new molecular markers for further screening BAC library and finish construction of the complete physical map of Ro gene region. Then we could perform the sequencing and gene annotation of all BAC clones existed in Ro gene target region. Based on these genome sequences information of BAC clones, we could design new primers to develop the linkage or co-segregated marker, which can be used to finally identify the candidate BAC clones of Ro gene. Next, through constructing and screening the sub-clone library, the positive sub-clone could be identified and verified the function by genetic transformation to finally clone the Ro gene. The cloning of Ro gene would lay a foundation for the genetic mechanism of potato tuber formation, and also is significant to improve potato tuber shape and improve the efficiency of potato directed breeding.

薯形是马铃薯块茎重要的外观品质，也是马铃薯育种的重要目标性状。控制薯形性状的主效基因Ro位于第10号染色体上，该基因目前尚未被克隆。前期研究中，本项目组以二倍体马铃薯薯形分离群体为材料，初步构建了Ro基因位点的物理图谱，并基于BAC-end文库PacBio测序获得了Ro区域的电子物理图谱。本研究拟在此基础上，进一步利用Ro区域的BAC末端序列开发标记，筛选BAC文库，完成目标区域Ro基因位点的完整物理图谱构建，并进行该位点区域内BAC克隆的测序分析和基因注释。进一步根据目标区域内BAC克隆的基因组序列，设计引物，开发标记，找出与Ro基因紧密连锁或共分离的分子标记，结合目标区域内的基因注释，确定候选BAC克隆并构建亚克隆文库，筛选获得阳性亚克隆进行转基因功能验证，获得Ro基因。为进一步解析马铃薯薯形遗传机制奠定基础，对于马铃薯薯形改良，提高马铃薯定向育种效率也具有重要意义。

薯形是马铃薯外观最重要的品质性状之一。由于不良或不规则的马铃薯形状会增加加工成本，并影响消费者的消费意愿，因此培育形状规则均匀的马铃薯品种非常重要。以往的研究表明，圆薯形主效控制位点Ro位于10号染色体上。然而，薯形基因的精细定位和克隆尚未见报道。.在本研究中，通过组织切片和转录组测序分析表明，圆形和长薯形之间的发育差异早在匍匐茎弯钩期就已经开始了。为了精确定位并克隆薯形调控基因，我们构建了一个二倍体薯形分离群体，并通过开发薯形连锁标记构建了遗传连锁图，该遗传连锁图谱总长度为25.8cM，平均标记间隔为1.98cm。结合2014-2017年收集的表型数据，确定了薯形的一个主要数量性状位点（QTL），该位点解释了61.7%-72.9%的薯形变异。通过对重组个体的基因分型和表型研究，将薯形调控基因Ro定位在一个193.43kb的区间，该区间包含18个基因。根据组织切片和转录组测序的结果，初步预测了5个候选基因。通过对候选区间内基因的序列及功能分析，确定候选基因，并构建过表达载体及敲除载体，进行了转基因功能验证。StOFP基因过表达载体转入长薯形DM植株中获得薯形表型的变化。本研究对马铃薯薯形遗传机制的解析奠定了基础，对于马铃薯薯形改良，提高马铃薯定向育种效率也具有重要意义。