脐橙早熟芽变及其早熟性状回复型材料的AFLP和MSAP分析  

AFLP and MSAP analysis of early ripening bud mutant and its revertant materials of navel orange

作　　者：赖春旺 周小娟 米兰芳[1] 陈健美[1] 欧阳欢 钟八莲[1] LAI Chunwang;ZHOU Xiaojuan;MI Lanfang;CHEN Jianmei;OUYANG Huan;ZHONG Balian(Gannan Normal University/National Navel Orange Engineering Research Center,Ganzhou 341000,Jiangxi,China;Agricultural and Rural Affairs Bureau of Ganzhou Jiangxi,Ganzhou 341000,Jiangxi,China)

机构地区：[1]赣南师范大学·国家脐橙工程技术研究中心,江西赣州341000 [2]江西省赣州市农业农村局,江西赣州341000

出　　处：《果树学报》2022年第8期1346-1357,共12页Journal of Fruit Science

基　　金：国家自然科学基金项目(32060667)。

摘　　要：【目的】探究不同成熟期的脐橙在基因组、甲基化修饰水平和变异模式方面的差异,为挖掘早熟性状分子标记,探讨脐橙早熟芽变性状与DNA甲基化的关系及早熟性状相关候选基因的筛选奠定基础。【方法】以纽荷尔脐橙、纽荷尔早熟芽变品种赣南早脐橙及赣南早的早熟性状回复型突变体(性状回复型)为试材,采用扩增片段长度多态性技术(amplified fragment length polymorphism,AFLP)和甲基化敏感扩增多态性分析(methylation sensitive amplification polymorphism,MSAP)对其在基因组、甲基化修饰水平和变异模式方面的差异与脐橙早熟性状之间的关系进行探究。【结果】采用AFLP与MSAP技术均发现较多多态性条带,其中AFLP的多态性占比(5.5046%~5.5556%)高于MSAP(1.3699%~3.2110%),发现7对引物共8条多态性条带与脐橙熟期性状共分离。试材DNA甲基化发生频率为13.7614%~16.8182%,平均14.9116%,全甲基化为主要的甲基化方式。试材超甲基化发生频率显著高于去甲基化发生频率,性状回复型果实熟期回复推测与CHG去甲基化相关。【结论】在基因层面证明性状回复型为赣南早的回复型突变体。脐橙果树在形成不同熟期芽变材料过程中,基因组DNA的超甲基化与去甲基化同时发生,但以超甲基化为主。本研究为进一步阐明脐橙果实熟期性状的发生发展机制提供指导。【Objective】Gannanzao is an early ripening bud sport material of Newhall,and its fruit maturity was over 30 days earlier than Newhall l.Early ripening revertant was the bud sport material of Gannanzao,and its fruit maturity was consistent with that of Newhall.The purpose of this study was to detect the differences of genome,methylation modification level and variation mode among the Newhall,Gannanzao and revertant.The relationship between these differences and early ripening character of navel orange was analyzed to further clarify the occurrence and development mechanism of fruit ripening character of the navel orange.【Methods】In this study,the Newhall,Gannanzao and revertant mutant were used as experimental materials.The amplified fragment length polymorphism(AFLP)and methylation sensitive amplified polymorphism analysis(MSAP)were employed to study the genome,methylation modification level and variation mode of the three different materials.The materials were collected from the orchard in Yudu county,Ganzhou city and the“citrus germplasm resource nursery”of Gannan Normal University.We used the improved CTAB method for the extraction of the genomic DNA of the samples.The quality and concentration of genomic DNA were detected by agarose gel electrophoresis combined with ultramicro nucleic acid protein analyzer(Thermo Scientific,USA).The genomic DNA meeting the requirements was diluted to 100 ng·μL^(-1) and stored in the refrigerator at-20℃for standby.Both AFLP and MSAP markers needed double enzyme digestion,ligation,pre-amplification and selective amplification.However,the endonuclease used in AFLP labeling was different from that used in MSAP labeling.EcoRⅠ-MseⅠwere used in AFLP,EcoRⅠ-HpaⅡand EcoRⅠ-MspⅠwere used in MSAP.AFLP marker and MSAP marker selective amplification products were added with 20μL mineral oil to prevent samples volatilization and put into Qsep 100TM type automatic nucleic acid protein analyzer(Bioptic,Inc.Taiwan).High Resolution Cartridge(S1)was used for electropho

关 键 词：脐橙 果实熟期 芽变 AFLP MSAP 差异分析 

分 类 号：S666.4[农业科学—果树学]