胡柚新品种01-7的AS-PCR和d CAPS标记开发与应用  

Development and application of AS-PCR and dCAPS markers for discriminating new cultivar 01-7 Huyou(Citrus changshanensis)

作　　者：吴伊静 张慧艺 苗长久 汪丽霞 杨兴良 陈文博 徐昌杰[1,2] 陈昆松 WU Yijing;ZHANG Huiyi;MIAO Changjiu;WANG Lixia;YANG Xingliang;CHEN Wenbo;XU Changjie;CHEN Kunsong(Department of Horticulture,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,Zhejiang,China;Zhejiang Key Laboratory of Horticultural Crop Quality Improvement,Hangzhou 310058,Zhejiang,China;Changshan Agricultural Characteristic Industry Development Center,Changshan 324200,Zhejiang,China)

机构地区：[1]浙江大学农业与生物技术学院园艺系,杭州310058 [2]浙江省全省园艺作物品质调控重点实验室,杭州310058 [3]常山县农业特色产业发展中心,浙江常山324200

出　　处：《果树学报》2024年第7期1310-1321,共12页Journal of Fruit Science

基　　金：浙江省“三农九方”科技协作计划项目(2022SNJF083);浙江大学全国农业科技现代化先行县(常山)建设项目(588970-Y12202);衢州市科技计划项目(2022K25)。

摘　　要：【目的】挖掘胡柚新品种01-7和普通胡柚间的差异单核苷酸多态性(SNP),据此开发可区分01-7与其他胡柚的分子标记。【方法】对01-7a和普通胡柚ZZ(祖宗树)进行全基因组重测序,利用生物信息学分析方法挖掘两份材料间的纯合SNP。采用PCR、克隆结合Sanger测序对SNP的正确性进行验证,进而开发等位基因特异PCR(AS-PCR)和衍生酶切扩增多态性(dCAPS)分子标记体系,最后应用12份胡柚材料就AS-PCR和dCAPS分子标记的普适性进行评估。【结果】对01-7a和普通胡柚ZZ重测序原始数据进行过滤,共获得高质量碱基数36.06 Gb。利用生物信息学手段挖掘出一个纯合SNP:Chr1_7111834_G/A。01-7a和普通胡柚ZZ在该SNP位点的基因型分别是A/A和G/G,这一结果得到了经典的基因克隆-Sanger测序确认。基于此SNP开发了AS-PCR和dCAPS分子标记,经对12份胡柚材料测试,表现符合预期:AS-PCR-F1在所有4份01-7中扩增出特异条带,而在其他胡柚材料中无扩增;AS-PCR-F2在01-7中无扩增,在其他胡柚材料(脆红除外)中扩增出特异条带;所有4份01-7材料在dCAPS分析中出现酶切条带,而在其他胡柚材料(脆红除外)中不被酶切。脆红因突变产生天然的酶切识别位点而被酶切。经典的基因克隆-Sanger测序结果确认两种标记正确区分了基因型,并发现脆红有着不同于其他胡柚的分子标记条带是由于它在该SNP位点侧翼还有额外的序列变异。【结论】基于全基因组重测序数据挖掘出01-7a胡柚和普通胡柚ZZ间的一个纯合SNP,据此开发了ASPCR和dCAPS分子标记,该两标记可区分01-7、脆红、夏红和其他胡柚。【Objective】Huyou(Citrus changshanensis K.S.Chen et C.X.Fu)is a local characteristic citrus species in China with a history of over a hundred years,and its main production area is in Changshan county,Quzhou City,Zhejiang province.After a long process of propagation by seeds and selection by seedlings,new cultivars/lines such as Cuihong,Hongrou huyou,Xiahong,Huyou elite plant a,Huyou elite plant b and 01-7 were obtained from the ordinary huyou population.Among them,01-7 has become the main huyou cultivar to be extended because of its strong cold resistance,as well as long storage longevity and slight-but-special bitter flavor in the fruit.However,there is a high degree of similarity between different accessions of huyou,and it is difficult to distinguish among them with naked eyes in terms of sapling morphology.With the progresses in techniques on genome sequencing and associated single nucleotide polymorphism(SNP)analysis,SNP mining and marker development are becoming an efficient and accurate way to discriminate close cultivars.However,to date,there has been no study on SNP based molecular markers for discriminating huyou accessions.The aim of this study was to mine homozygous SNP(s)present between 01-7 and ordinary huyou,and to develop efficient and accurate molecular markers for identifying 01-7.【Methods】Whole genome resequencing was performed on 01-7a and ordinary huyou ZZ(ancestral tree),and after obtaining high quality sequencing data,bioinformatics analysis tools were utilized to identify homozygous SNPs between these two accessions.The authenticity of SNPs predicted in silico was verified by PCR,cloning combined with Sanger sequencing.Then allele-specific polymerase chain reaction(AS-PCR)and derived cleaved amplified polymorphic sequences(dCAPS)molecular markers were developed based on the obtained SNP information,and finally,the applicability of molecular markers was evaluated by the performance of ASPCR and dCAPS molecular markers in 12 huyou accessions covering main production bases and main cul

关 键 词：胡柚 全基因组重测序 SNP AS-PCR dCAPS 

分 类 号：S666.3[农业科学—果树学]