切花菊重要性状分子标记关联分析及相关等位基因挖掘

Chrysanthemum is one of the ten traditional important ornamental flowers in China and one of the four most popular cut flowers in the world , chrysanthemum is of high ornamental and economic value. However, the molecular genetic mechanisms of numerous important traits made slow progress because the majority of important traits of chrysanthemum belong to complicated quantity traits. Therefore,in this propasal, a natural population of cut chrysanthemum representative cultivars is employed to testify main morphological and physiological traits at different locations for several years. Genome-wide association strategy will be adopted by means of molecular markers (SRAP, SCoT and AFLP). Population structrue and the extent of linkage disequilibrium in the cut chrysanthemum germplasm will be detected using STUCTURE and TASSEL softwares. Markers significantly related to those important ornamental and abiotic tolerant traits will be discovered. On the other hand, candidate genes association analysis will be conducted as well. Candidate genes related to abiotic stress tolerance will be cloned and sequenced, from which SNPs or Indel will be detected. Diversity of the aligned sequences and the level of linkage disequilibrium will be analyzed, and the associations between nucleotide diversity and abiotic stress tolerance will be identified, from which excellent alleles will be detected using TASSEL software. Additionally, haplotype analysis will be performed using the Arlequin software followed by discovery of the haplotypes with high abiotic stress tolerance. The proposal is expected to develop a batch of molecular markers, gene alleles and haplotypes associated with main important traits. Meanwhile, the study will shade light on the molecular genetic mechanisms underlying the important traits of chrysanthemum,which will lay a theoretical foundation for further molecular breeding of chrysanthemum.

菊花是我国十大传统名花和世界四大切花之一，观赏和经济价值极高。其重要性状多为复杂的数量性状，重要性状相关分子遗传机制研究进展缓慢。为此，本申请项目拟采集切花菊自然群体代表性品种多年多点重要性状表型数据，借助SRAP、SCoT和AFLP等分子标记进行全基因组扫描，采用STRUCTURE 和TASSEL 等软件进行群体结构和连锁不平衡水平分析；发掘与重要性状（观赏性、抗逆性）相关联的分子标记。另一方面，采用候选基因关联分析策略，进行抗逆候选基因的克隆、测序、序列比对，发掘SNPs（或Indel），并用TASSEL软件分析候选基因序列多态性与LD水平及序列多态性与抗逆性的关联，挖掘优异等位基因；采用Arlequin软件进行单体型分析，发掘优异抗逆单体型。本项目的实施可发掘一批重要性状相关联的分子标记、等位基因及优异单体型，初步揭示菊花重要性状的分子遗传机理，为菊花分子设计育种提供理论依据。

菊花是我国十大传统名花和世界四大切花之一，观赏和经济价值极高。本研究选择了159个切花菊代表性品种的自然群体，对多个重要观赏性状及非生物抗性进行连续两年的观测和鉴定评价，获得了表型数据并鉴定出多个抗性优异的切花菊品种。应用SRAP、SCoT和SSR等分子标记进行了全基因组扫描，通过软件分析出该群体的群体结构主要分为两个亚群，平均亲缘关系系数为0.074，连锁不平衡水平分析结果显示1.3%标记位点组合的r2> 0.1（P< 0.01）。关联分析检测到54个标记位点同时与2011年和2012年的11个观赏性状显著关联，检测到的关联位点的表型变异解释率（PVE）范围为4.31%-21.80%，平均为5.71%。其中关联显著性最大的为与舌状花轮数（NRFR）相关联的SRAP标记位点E19M21-3（P=4.42E-08），且其两年的PVE也最大。检测到其中有17个标记同时与两个性状关联。通过对所试品种扦插生根苗的非生物抗性评价得到抗旱性很强的切花菊品种16个，耐盐性很强的切花菊品种7个。两年同时检测到的与抗旱性关联的标记位点8个（P < 0.01），关联位点的PVE范围为4.40%-7.57%，平均为5.63%，其中EST-SSR27-8显著性最大（P = 7.00E-04）且PVE也最大；EST-SSR34-3两年均有显著的正向表型效益，且表型效益值最大。两年各检测到3个标记位点与品种的耐盐性显著关联，没有两年同时检测到的关联位点，PVE范围为4.48%-8.35%，平均为5.95%，其中PVE最大的是EST-SSR187-2。采用候选基因关联分析策略，对抗逆候选基因TIP和SAP进行克隆、测序、序列比对，挖掘了TIP与耐盐性显著相关的SNP 3个，SAP与耐盐性、抗旱性显著相关的SNP分别有7个、2个，PVE均在11%以上。在本项目资助下，培养研究生5名，发表SCI论文8篇，申请国家发明专利2项，获得华耐园艺科技奖1项，多个品种获世界园艺博览会、全国菊花展览会金奖。