山核桃寡核苷酸探针开发及其应用  

Development and Application of Oligonucleotide Probes for the Genus Carya

作　　者：苑轲 黄坚钦[1] 王克涛 夏国华[1] 张启香[1] 徐川梅 Yuan Ke;Huang Jianqin;Wang Ketao;Xia Guohua;Zhang Qixiang;Xu Chuanmei(State Key Laboratory of Subtropical Silviculture Jointly Built by the Province and Ministry Zhejiang A&F University,Hangzhou 311300)

机构地区：[1]浙江农林大学省部共建亚热带森林培育国家重点实验室,杭州311300

出　　处：《林业科学》2023年第5期88-99,共12页Scientia Silvae Sinicae

基　　金：浙江省农业新品种选育重大科技专项(2021C02066-12);安徽省科技厅长三角科技创新联合攻关专项(202004G01020005)。

摘　　要：【目的】利用山核桃全基因组测序数据,为山核桃开发一些寡核苷酸类型的染色体物理标记,并建立起山核桃寡核苷酸探针开发方法,以期为其他山核桃品种相关研究提供参考。【方法】以山核桃、湖南山核桃、云南山核桃、大别山山核桃、贵州山核桃、薄壳山核桃及喙核桃为材料,利用Tandem Repeat Finder软件分析山核桃基因组中的重复序列,按照Period size>4,Copy number>100,且Period size*Copy number>3000的筛选条件对重复序列进行筛选,根据筛选结果合成了约60条寡核苷酸探针,利用荧光原位杂交技术对这些探针进行筛选。【结果】1)sht-2、sht-3、sht-4、sht-5、sht-5S及sht-45S均可在山核桃染色体上产生相应的荧光信号,且sht-2、sht-3、sht-4及sht-5在山核桃染色体上的分布模式相同,有2对强弱不同的信号存在,sht-45S在山核桃染色体上有1对信号存在,sht-5S仅在山核桃单条染色体上有1个信号位点分布,这些探针均位于染色体的近着丝粒区域。2)sht-5的2对信号位点中,其中较强的1对位点与sht-45S的信号位点在山核桃染色体上分布位置完全重叠。3)45SrDNA与sht-45S在山核桃染色体上分布位置完全重叠,5SrDNA与sht-5S在山核桃染色体上的分布位置也完全重叠。4)sht-5在湖南山核桃、云南山核桃、大别山山核桃及贵州山核桃染色体上均有信号分布,且与山核桃分布模式相似,但sht-5在喙核桃及薄壳山核桃染色体上没有信号。5)sht-5S及sht-45S在湖南山核桃、云南山核桃、大别山山核桃、贵州山核桃及薄壳山核桃染色体上均有信号分布,但在喙核桃染色体上没有信号分布。6)sht-45S在湖南山核桃、大别山山核桃及贵州山核桃染色体上的分布模式相似,均只有1对位点存在,sht-45S在薄壳山核桃染色体上有2对位点存在,位于2对同源染色体的近着丝粒区域。7)sht-5S在云南山核桃、贵州山核桃、大别山山核桃及薄壳【Objective】The lack of chromosome physical markers seriously hinders the development of cytogenetics research in the genus Carya.In this study,we used the whole genome sequencing data to develop some oligonucleotide type chromosomal physical markers and established a development method of oligonucleotide probes for Carya cathayensis,in order to provide reference for the related research of other species in Carya.【Method】The repetitive sequences in the genome of C.cathayensis were analyzed by the Tandem Repeat Finder software and filtered out according to the principle of Period size greater than 4,Copy number more than 100,and Period size*Copy number more than 3000.About 60 oligonucleotide probes were designed and synthesized,and then these probes were further analyzed and screened by the technology of fluorescence in situ hybridization in C.cathayensis,C.hunanensis,C.tonkinensis,C.dabieshanensis,C.kweichowensis,C.illinoinensis and C.sinensis.【Result】1)The oligonucleotide probes of sht-2,sht-3,sht-4,sht-5,sht-5S and sht-45S were able to produce fluorescence signals in C.cathayensis.The probes of sht-2,sht-3,sht-4 and sht-5 had the same distribution patterns on the chromosome,and there were two pairs of signals with different fluorescence intensities.The sht-45S produced one pair of signals on the C.cathayensis chromosome,while sht-5S only had one signal site on the single chromosome of C.cathayensis.These probes were all located in the near centromere region of the chromosome.2)Among the two pairs of signal sites of sht-5,the stronger pair was completely overlapped with the distribution of the signal sites of sht-45S on the chromosome of C.cathayensis.3)The distribution positions of 45S rDNA and sht-45S on the chromosomes of C.cathayensis were completely overlapped,and the positions of 5S rDNA and sht-5S were also completely overlapped.4)The oligonucleotide probe of sht-5 was able to produce fluorescence signals in C.hunanensis,C.tonkinensis,C.dabieshanensis and C.kweichowensis,and the distribution pa

关 键 词：山核桃 重复序列 寡核苷探针 染色体 寡核苷酸荧光原位杂交 

分 类 号：S718.43[农业科学—林学]