花生高含油量重要候选基因的关联分析及其功能研究

Development of varieties with high oil content is an important objective in peanut (Arachis hypogaea L.) breeding program in China. However, to date there is still low breeding efficiency in high oil peanut breeding, partly because of a lack of functional markers associated with high oil content and key genes involved in triacylglycerols (TAGs) biosynthesis. One of the best approaches for developing functional markers is to conduct the association analysis between the polymorphisms of high oil candidate genes and oil content phenotypic traits. The multigene family encoding proteins related to lysophosphatidyl acyltransferases (LPATs) plays pivotal roles in TAGs biosynthesis. In our previous studies, the overexpression of AhLPAT2 in transgenic seeds is associated with increased TAGs. In view of the above, in this study, a mini core set including the peanut germplasm and some elite breeding resources with diverse genetic backgrounds and different levels of seed oil content will be considered as the natural population. In addition, LPAT family genes will be identified and their sequences will be obtained in cultivated peanut. Furthermore, the sequences diversity and linkage disequilibrium (LD) of LPAT genes considered as high oil content candidate genes will be evaluated. Subsequently, association test between LPATs polymorphisms with phenotype variations of oil content will be performed in order to identify functionally favorable alleles that are responsible to variations of seed oil content. According to the detected superior haplotypes associated with high oil content, the functional markers expected to be useful in marker-assisted breeding of high oil content in peanut and improvement novel elite germplasm with high oil content will be developed. Finally, to better understand the potential functions of superior haplotypes, their expression patterns will be analysis in seed at different developmental stages between low and high oil content genotypes of peanut. Meanwhile, to validate the effects of superior haplotypes identified in the association analysis, we will verify their function by transgenic analysis in peanut. Therefore, all the results obtained in our present study will facilitate to provide systemic theoretical basis and technical support for the improvement of seed oil content in peanut and other oil crops.

培育高油品种是我国花生品质改良的重要目标。但因缺少高油功能标记和关键基因资源，导致目前高油花生育种效率很低，而探明高油性状相关重要候选基因的多态性与含油量的关系是开发功能标记的最佳方法。LPAT是植物油脂合成的关键基因。我们前期发现花生LPAT家族成员AhLPAT2过表达能显著提高种子的含油量。据此，本研究将以前期构建的花生自然群体和关键育种材料为基础，筛选出遗传背景差异大且含油量具有多样性的种质作为核心研究材料，利用前期鉴定的花生LPAT家族为候选基因，通过测序检测候选基因在研究材料基因组序列中的多态性和连锁不平衡结构，在分析材料群体结构和鉴定含油量的基础上进行关联分析，挖掘与高油性状密切相关的优异单倍型及其SNPs，继而开发与高油显著关联的功能标记；最后，通过基因表达分析和遗传转化验证，进一步解析这些基因的功能。本研究成果可为高油花生品种的高效选育提供重要理论依据和技术支持。

花生是我国重要的油料作物和经济作物，在保障我国食用植物油安全供给中发挥不可替代的作用。长期以来，提高花生品种的含油量是我国花生品种改良的重要目标。通过育种选择使花生种子的含油量大幅提高，然而这种选择背后的潜在基因仍然未知。本项目旨在挖掘花生高油形成的关键候选基因，采用候选基因关联分析途径，解析对油脂积累具有显著增值效应的优异等位变异或功能单倍型，继而开发与高含油量紧密关联的功能标记，为提高高油花生品种育种效率和实现突破性种质创新奠定资源基础和提供技术支撑。本项目构建了花生含油量遗传研究核心群体，经群体结构分析和连锁不平衡分析，证实该群体具有高遗传多样性。克隆获得花生AhLPAT基因家族10个成员的基因全长序列，基于基因时空表达特性、基因表达丰度与种子油脂积累相关性、及在高低含油量极端材料中的差异表达，证实了AhLPAT2在花生种子油脂积累中起重要作用。对不同品种的AhLPAT2基因进行启动子区和编码区测序，发现该基因具有2种启动子单倍型和2种编码区单倍型，组合形成4种AhLPAT2基因单倍型。对AhLPAT2进行选择性检验发现，该基因的全长序列和编码区均受到显著正选择压力，而非编码区则未受到选择作用，推测该基因在进化中存在编码蛋白的功能分化。比较了具有不同AhLPAT2启动子和编码区单倍型花生种质的含油量，发现单倍型Hap4、启动子单倍型Pro2、编码区单倍型CDS2是控制花生种子含油量的优异/增效等位变异类型。利用重组自交系群体中不同单倍型纯合家系的含油量比较，充分证实Pro2和CDS2是AhLPAT2基因的优异等位变异类型。将AhLPAT2的优异变异类型在种子特异表达载体驱动下过量表达，转基因种子的含油量被显著提高，同时油脂中各脂肪酸成分也显著提高。联合应用PCR和酶切方法建立了优异单倍型特异的CAPS标记，成功应用于高油花生材料的检测和筛选中，具有稳定性强、检测效率高等突出特点，在高油花生育种中具有重要应用价值。