青钱柳全基因组SSR位点分析及多态性引物开发  

Analysis of SSR locus based on the whole genome sequences of Cyclocarya paliurus and the development of polymorphic primers

作　　者：刘莉[1,2] 瞿印权 余延浩 王倩 洑香香[1] LIU Li;QU Yinquan;YU Yanhao;WANG Qian;FU Xiangxiang(College of Forestry and Grassland,Nanjing Forestry University,Nanjing 210037,China;Shanghai Pudong Forestry Station,Shanghai 200120,China)

机构地区：[1]南京林业大学林草学院,江苏南京210037 [2]上海市浦东新区林业站,上海200120

出　　处：《南京林业大学学报（自然科学版）》2024年第4期67-75,共9页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：国家自然科学基金项目(31971642);中央财政林业科技推广示范项目(皖[2023]TG13号)。

摘　　要：【目的】通过对青钱柳全基因组序列分析,开发基因组SSR分子标记;尝试构建19个青钱柳优良药用无性系的DNA分子身份证,为后续种质资源评价、遗传多样性和种质鉴定提供技术支撑。【方法】利用MISA(microsatellite identification tool)软件对青钱柳全基因组进行SSR位点搜寻、筛选、识别及富集分析,采用Primer 3.0进行SSR引物设计;用重复性和稳定性高的SSR标记构建青钱柳无性系的识别系统。【结果】(1)从全基因组中共检测出89741个SSR位点,SSR位点的发生频率为62.07%。(2)基因组SSR位点中单核苷酸重复单元比例最高,占总SSR位点的62.67%;六核苷酸重复单元比例最低,占0.15%;SSR位点的重复基序大多以(A/T)n为主。(3)单核苷酸和二核苷酸重复类型的SSR位点基序重复次数集中在6~16次;随重复次数增加,各SSR位点重复类型出现频率均呈下降趋势。(4)基因组SSR序列长度介于10~476 bp,不同类型重复单元的SSR序列长度存在变异性;随着重复次数的增加,SSR序列出现的频率整体呈下降趋势。(5)利用Primer 3.0成功设计出78285对SSR引物;合成的377对中有75对引物可扩增出多态性条带;用5对单碱基重复的多态性SSR引物分析19个药用无性系,构建出无性系的二维码DNA分子身份证。【结论】青钱柳基因组SSR位点出现频率高,位点种类丰富,可为种质资源评价及指纹图谱的构建提供丰富的候选分子标记。【Objective】Genomic simple repeat sequence(SSR)loci were analyzed by screening the whole genome of Cyclocarya paliurus.DNA molecular ID cards of 19 excellent medicinal clones of C.paliurus were constructed based on the newly-developed SSR primers.These genomic SSR markers could support further research,such as the evaluation of the germplasm resource,analysis of genetic diversity,and identification of cultivars/clones.【Method】The SSR loci were screened along with the whole genome of C.paliurus and were enriched and analyzed using MISA software.Subsequently,SSR primers were designed using Primer 3.0.Furthermore,a system for identifying clones of C.6Epaliurus was constructed based on selected SSR markers with high reproducibility and stability.【Result】(1)We detected 89741 SSR loci from the whole genome,with an occurrence frequency of 62.07%.(2)Among all SSR loci,the proportion of SSRs with a mononucleotide motif was the highest(62.67%)and a hexa-nucleotide repeat was the lowest(0.15%).Most of the repeated motifs in the SSR loci were dominated by(A/T)n.(3)The repeat number of mono-nucleotide and di-nucleotide motifs ranged from 6 to 16.With the increase in the repeat number,the frequencies of various SSR repetition types displayed a downward trend.(4)The length of the SSR sequences varied from 10 to 476 bp,and this length variation existed in different repetitive motifs.Additionally,the frequency of SSR occurrence tended to decrease as the repeat number increased.(5)We successfully designed 78285 pairs of SSR primers using Primer 3.0.A total of 377 primer pairs were randomly synthesized for amplifying polymorphic SSR fragments,among which 75 pairs primers were successful.Moreover,quick response code DNA molecular ID cards for 19 medical-use clones of C.paliurus were constructed by five pairs of polymorphic SSR primers with a mono-nucleotide motif.【Conclusion】The frequency of genomic SSR loci was high,and there was variability in the type of SSR loci.Simple repeat sequences developed from the whole gen

关 键 词：青钱柳 简单重复序列(SSR) 全基因组 DNA分子身份证 

分 类 号：S722[农业科学—林木遗传育种] Q756[农业科学—林学]