苹果砧木耐涝主效QTL精细定位及候选基因鉴定与功能分析

Apple (Malus × domestica Borkh) is one of the most cultivated fruit crops in China. While apple tree frequently encounters waterlogging stress mainly due to excess rainfall, soil compaction, or poor soil drainage, resulting in yellowing leaf, declined fruit quality and yield in some region. In our previous study, the mapping population was constructed based the cross of high waterlogging tolerance of apple rootstock G41 and M. sieverii (Ledeb) Roem and was also finished the quantitative trait loci (QTL) mapping of the waterlogging tolerance of apple rootstock. The project is based on previous research, fine mapping of QTL locus will be conducted based on the results of specific length amplified fragment sequencing (SLAF-seq) and sequencing information of the reference genome of apple for developing new single nucleotide polymorphisms (SNP) and simple sequence repeats (SSR) markers. All genes located on the fine physical mapping corresponding to the major QTL will be predicted, annotated and classified by bioinformatics analysis. Combining SNP analysis and digital gene expression profiling between two parents, putative candidate genes involved in waterlogging tolerance of apple rootstock will be identified, then the response characteristics will be verified by reverse transcription-polymerase chain reaction (RT-PCR) method to find out the corresponding genes. Finally, cloning of full-length cDNA of important candidate genes for waterlogging tolerance using rapid amplification of cDNA ends (RACE) technique were cloned and transferred to Arabidopsis and apple for functional verification. The key genes and linkage markers involved in waterlogging tolerance of apple rootstock can be found out in this study, which can provide a theoretical basis for molecular-assisted selection breeding and better cultivating high quality and yield apple.

苹果（Malus × domestica Borkh）是我国主要栽培果树树种之一，但部分苹果产区由于夏、秋季的大量集中降雨和排水不良等造成果园积水，导致苹果树叶片黄化、脱落，果实品质和产量下降，已成为部分苹果产区亟待解决的问题。项目组前期已用耐涝性强的苹果砧木G41与不耐涝新疆野苹果构建了群体，并完成了苹果砧木耐涝QTLs初步定位。本项目拟在此基础之上，利用SLAF-seq和苹果全基因组测序信息，开发紧密连锁的SNP和SSR标记，加密局部遗传图谱，对QTL对应的精细物理图谱进行基因预测、注释分类及亲本间SNP分析，结合亲本差异表达基因分析，确定候选基因，通过RT- PCR验证其淹水胁迫响应特征；利用RACE技术克隆耐涝重要候选基因全长cDNA，并在拟南芥和苹果中做基因功能验证。通过本研究获得与苹果砧木耐涝紧密的连锁标记及关键基因，为苹果分子标记辅助育种和优质高产栽培奠定基础。

苹果（Malus×domestica Borkh）是我国主要栽培果树树种之一，但部分苹果产区由于夏、秋季的大量集中降雨和排水不良等造成果园涝害频繁发生，导致苹果树叶片黄化、脱落，果实品质和产量下降。鉴定苹果耐涝相关基因对苹果耐涝分子标记辅助育种和优质高产栽培具有重要意义。本研究以耐涝苹果砧木G41和不耐涝苹果砧木新疆野苹果（M. sieverii (Ledeb) Roem.）及其构建的包含495个F1杂交后代为材料，从F1杂交群体中挑选出耐涝和不耐涝株系各50株，构建两个极端性状DNA混池，采用简化基因组测序（SLAF-seq）技术，开发SLAF标签和SNP标记，结合苹果基因组信息和遗传关联性分析，对苹果耐涝基因进行定位及候选基因预测，并对候选基因在耐涝差异的株系中进行了淹水胁迫下的表达分析。以‘金冠’苹果为参考基因组，共开发119 072个SLAF标签，其中多态性SLAF有11 133个。通过序列分析和检测SNP 位点，共获得6 237 071个SNP，其中高质量SNP 有170 617个。通过ED和SNP -index方法关联分析，获得一个与耐涝性状紧密关联的候选区域，位于苹果第10号染色体1.94—3.25 Mb，关联区域大小为1.31 Mb，关联区域内包含120个基因。通过转录组分析发现MdADH1和MdSHN1可能与耐性有关，进一步通过转基因功能验证，表明MdSHN1正向调控耐涝性。研究结果为全面揭示苹果砧木耐涝的遗传机制和分子标记辅助选择耐涝苹果砧木提供理论依据，对我国苹果分子标记辅助育种和优质高产栽培具有重要理论和实际意义。