脐橙与葡萄柚体细胞杂种的基因组倍性效应及果实品质特征

To solve those reproductive barriers such as nucellar polyembryony in citrus conventional breeding and to promote citrus variety improvement in China, the applicant have created a large number of citrus somatic hybrids and other new germplasm in the past years. These new germplasm have successively flowered and set fruits, and were already used in breeding practice. The feedback information in the field indicated that many characters of these somatic hybrid fruits showed heterobeltiosis or parental bias and mid-parent characters were not presented in some cases. This phenomenon might be correlated with genomic ploidy effect and allocytoplasmic effect in the process of allopolyploidization. Aiming to reveal the genomic ploidy effect and fruit quality characteristics, comparisons will be made between two diploid parents, i.e. Bonanza navel orange (Citrus sinensis Osb.) which possessed high sugar and low organic acid with orange yellow pulp, Red Blush grapefruit (Citrus paradisi Macf.) with low sugar, high organic acid and red pulp, and their respective somatic hybrid and diploid cybrid obtained from protoplast fusion. We will analyse related genome sequences, retrotransposon activation, DNA methylation level and miRNAs expression profile and evaluate the content of sugar, organic acid and carotenoid related gene expression and metabolite profiling respectively. We also intend to investigate the possible relationship between fruit quality and genomic ploidy effect. Based on strong pre-study foundation and special experimental material, this project could provide a strong theoretical basis for guiding applying cell engineering technology to citrus improvement.

针对柑橘常规育种中存在的珠心胚干扰等生殖障碍，以及我国特色柑橘的遗传改良，申请人多年来创制了一大批柑橘体细胞杂种，这些新种质已陆续开花结果并用于育种实践。田间反馈的信息表明，柑橘体细胞杂种果实的许多性状未出现预期的亲中值，而表现超亲或亲本偏向性现象；该现象可能与异源多倍体化过程中的基因组倍性效应和异源胞质有关。本项目拟以"朋娜脐橙（高糖、低酸、果肉橙黄）+ 红玉葡萄柚（低糖、高酸、果肉红色）"异源四倍体体细胞杂种及其二倍体胞质杂种为试材，揭示其基因组倍性效应（通过基因组相关序列、反转录转座子活动、甲基化水平和miRNAs表达谱分析）和果实品质特征（通过糖、酸、类胡萝卜素含量测定及相关基因表达、果实代谢谱分析），并探讨基因组倍性效应和异源胞质（线粒体）与果实品质之间的联系。本项目前期研究基础扎实，试材独特，将为细胞工程技术进一步用于柑橘品种改良提供理论支撑。

人工合成的柑橘异源四倍体体细胞杂种为研究异源多倍体化过程中的遗传和表观遗传提供了珍贵试材。本项目以“Nova橘柚 + HB柚”（NH）等柑橘异源四倍体体细胞杂种为试材，其扩增片段多态性（AFLP）分析表明，叶肉亲本基因组变异相对愈伤组织亲本基因组变异较小；甲基化敏感扩增多态性（MSAP）分析表明体细胞杂种基因组中愈伤组织亲本基因组随机CCGG位点甲基化变化高于叶肉亲本，且体细胞杂种基因组中愈伤组织亲本基因组反转录转座子附近CCGG位点甲基化变化也高于叶肉亲本；SSR基因分型表明，体细胞杂种NH遗传模式可能为一种包含二体遗传、四体遗传和中间型遗传的混合遗传模式；NH等异源四倍体体细胞杂种果实代谢存在广泛变异，较多代谢物含量偏向HB柚亲本，少部分代谢物含量偏向Nova橘柚亲本，同时部分代谢物含量表现超亲，尤其是苦味黄酮，而这种代谢的广泛变异由基因表达变异决定。研究结果不仅丰富了高等植物异源多倍体化过程中基因组倍性效应理论，而且为细胞工程进一步用于柑橘遗传改良提供了借鉴，理论上和实践上均具有重要意义。本项目已在Tree Genetics & Genomes、Plant Cell Tissue & Organ Culture、园艺学报等刊物发表论文3篇，培养毕业博士生4人、硕士生2人。