基于纤毛鹅观草特异的卫星重复序列开发寡核苷酸探针  

Development of oligonucleotide probes specific to Roegneria ciliaris chromosomes based on satellite repeats

作　　者：程梦豪 Miroslava Karafiatova 孙昊杰 Kate Arina Holušva Jaroslav Dolezel 宋新颖 王海燕[1] 王秀娥[1] CHENG Menghao;Miroslava Karafiatova;SUN Haojie;Kate Arina Holušova;Jaroslav Dolezel;SONG Xinying;WANG Haiyan;WANG Xiu’e(State Key Laboratory of Crop Genetics and Germplasm Enhancement/Cytogenetics Institute/Collaborative Innovation Center for Modern Crop Production Co-sponsored by Province and Ministry(CIC-MCP),Nanjing Agricultural University,Nanjing 210095,China;Institute of Experimental Botany,Czech Academy of Sciences/Centre of the Region Hanafor Biotechnological and Agricultural Research,77900 Olomouc,Czech Republic)

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室/细胞遗传研究所/现代作物生产省部共建协同创新中心,江苏南京210095 [2]捷克科学院实验植物学研究所/哈纳地区生物技术和农业研究中心,捷克共和国奥洛穆茨77900

出　　处：《南京农业大学学报》2022年第3期442-452,共11页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金国际(地区)合作与交流项目(31661143005);国家重点研发计划项目(2016YFD0102001-004);江苏省现代农业产业技术体系(JATS〔2020〕411)。

摘　　要：[目的]本文旨在开发纤毛鹅观草特异的寡核苷酸(oligonucleotide,oligo)探针,进一步完善纤毛鹅观草染色体鉴定技术。[方法]利用前期选育的普通小麦-纤毛鹅观草异附加系DA2S^(c)L和DA6S^(c),通过流式分拣和基因组二代测序获得纤毛鹅观草染色体6S^(c)和染色体臂2S^(c)长臂的基因组序列,利用Repeatexplorer2/TAREAN软件从中鉴定出卫星重复序列并设计成oligo探针,通过荧光原位杂交(fluorescence in situ hybridization,FISH)分析所开发oligo探针的应用价值。[结果]从6S^(c)和2S^(c)L基因组序列中共鉴定出11个卫星重复序列并开发成11个oligo探针,oligo-FISH结果表明,其中6个探针可以在纤毛鹅观草染色体上产生明显杂交信号,而在小麦染色体上未产生明显杂交信号,可用于特异鉴定小麦背景中的纤毛鹅观草染色体或片段。对1套普通小麦-纤毛鹅观草异附加系oligo-FISH分析发现,oligo-2S^(c)L^(-1)63和oligo-6S^(c)-111仅在S^(c)基因组染色体上产生明显信号,可作为纤毛鹅观草S^(c)组特异探针;将oligo-2S^(c)L-161和oligo-2S^(c)L-163组合,对1整套二体异附加系进行双色oligo-FISH,构建了纤毛鹅观草染色体的oligo-FISH核型。[结论]本研究提供了纤毛鹅观草重复序列组成的初步信息,开发的探针能特异鉴定纤毛鹅观草染色体,阐明纤毛鹅观草2个基因组的来源和分化,对推动纤毛鹅观草优异基因的转移和利用有重要意义。[Objectives]The purpose of this paper was to develop oligonucleotide(oligo)probes specific to Roegneria ciliaris chromosomes,and further improve the efficiency for distinguishing R.ciliaris chromosomes.[Methods]Using wheat-R.ciliaris disomic addition lines(DALs)DA2S^(c)L and DA6S^(c),the genome sequences of chromosome 6S^(c) and chromosome arm 2S^(c)L were obtained by flow cytometry and next-generation sequencing.Satellite repeats were identified using Repeatexplorer2/TAREAN and their corresponding oligo probes were designed and synthesized.The potential application value of the newly developed probes was investigated by oligo-FISH.[Results]Eleven satellite repeats were identified from the genome sequences of chromosome 6S^(c) and chromosome arm 2S^(c)L.Eleven oligo probes were developed based on 11 satellite repeats and were used for oligo-FISH to test their genome specificity.We confirmed that 6 of the 11 probes could produce obvious FISH signals only on the R.ciliaris chromosomes,and no obvious signal was observed on any of the wheat chromosomes.This indicated that they were genome specific and could be used to identify the chromosomes or chromosome segments of R.ciliaris in wheat background.Single oligo probe or probe combinations were further used for oligo-FISH analysis using a complete set of wheat-R.ciliaris DALs.The results showed that two probes,oligo-2S^(c)L-163 and oligo-6S^(c)-111,produced obvious signals only on all S^(c) genome chromosomes,and the two probes were S^(c) genome specific.By dual-color oligo-FISH of the wheat-R.ciliaris DALs using probe combination oligo-2S^(c)L-161 and oligo-2S^(c)L-)63,we constructed an oligo-FISH karyotype of R.ciliaris chromosomes.[Conclusions]This is the first preliminary analysis on the information on the constitution of repetitive sequences in R.ciliaris genome.The developed oligo probes in this study have potential application in identifying R.ciliaris chromosomes,clarifying the origin and differentiation of the two genomes of R.ciliaris.These will accelerate t

关 键 词：纤毛鹅观草 普通小麦-纤毛鹅观草异附加系 染色体分拣 卫星重复序列 寡聚核苷酸探针 荧光原位杂交 

分 类 号：S512[农业科学—作物学]