基于RAD-seq技术的金银花SSR标记开发及鉴定

Development and identification of SSR markers based on RAD-seq of Lonicera japonica

【目的】通过开发特异性高的多态性分子标记位点,以弥补金银花在简单重复序列(SSR)标记方面的匮乏,推动金银花在遗传资源管理及良种鉴定等方向的研究,为后续全基因组测序及组装策略奠定基础。【方法】运用RAD-seq技术对2份金银花样品进行简化基因组测序,利用MISA软件识别contig上的SSR序列并对各类型序列特征进行归纳分析,进而设计引物并利用生物信息学的方法进行引物筛选和有效性检测。【结果】对‘九丰一号’和‘亚特’金银花进行简化基因组测序,分别获得27.805 Mbp和42.560 Mbp干净读长。在组装的45850个基因序列中共检测到46999个SSR位点,其中单核苷酸重复单元比例最高(49.15%),六核苷酸重复单元最少(0.20%)。SSR位点的重复基序以(A/T)n为主,具有偏向性。除单碱基和二碱基重复类型外,SSR位点基序的重复次数集中在5~6次,且随重复次数增加,SSR位点重复类型出现的频率呈下降趋势。金银花基因组SSR序列长度介于10~310 bp之间,随重复次数增加,各SSR序列出现的频率逐渐下降。利用Primer3.0成功设计出38507对SSR引物,设计成功率为81.93%。对挑选的35对SSR引物进行多态性分析,等位基因数、观测杂合度和多态性信息含量(PIC)的平均值分别为5.057、0.363和0.568,其中高多态信息位点26个,中度多态信息位点9个,均未偏离哈迪–温伯格平衡。【结论】利用RAD-seq技术可实现SSR标记的大量开发和多态性SSR引物的筛选,并为金银花在遗传多样性分析和种质鉴定等相关方面的研究提供数据支持。

[Objective]Through the development of polymorphic molecular markers to make up for the lack of Lonicera japonica in SSR markers,this paper aims to promote the research in genetic resource management and variety identification,and lay the foundation for future de novo assembling of Lonicera japonica genome.[Method]RAD-seq(restriction-site associated DNA sequencing)was used to conduct simplified genome sequencing for two Lonicera japonica samples.The SSR sequences were identified by MISA and the characterizations of SSR sequences and their polymorphisms were analyzed by bioinformatics methods.[Result]27.805 Mbp and 42.560 Mbp clean reads were obtained in‘Jiufengyihao’and‘Yate’using restriction-site associated DNA(RAD).A total of 46999 SSR loci were detected among the 45850 gene sequences assembled,with the highest proportion of mono-nucleotide repeat SSRs(49.15%)and the lowest proportion of hexa-nucleotide repeat SSRs(0.20%).The repeated motifs at SSR loci were dominated by(A/T)n and showed a bias.Apart from mono-nucleotide and di-nucleotide repetition types,the repeat counts of SSR motifs mainly ranged from 5 to 6.The frequency of SSRs repetition types showed a downward trend with the increase of repeat number.The range of SSR length ranged from 10 to 310 bp,and the frequency of SSR sequence tended to decrease as the number of repetitions increasing.38507 pairs of SSR primers were successfully designed,with a design success rate of 81.93%.35 pairs of SSR primers were analyzed for polymorphism,in which the average number of alleles,observed heterozygosity and PIC were 5.057,0.363 and 0.568,among which there were 26 highly polymorphic loci and 9 moderately polymorphic loci,all of which did not deviate from the Hardy-Weinberg equilibrium.[Conclusion]The large-scale development of SSR markers and the screening of polymorphic SSR primers can be realized based on RAD-seq technology,and provide data support for the research of Lonicera japonica in genetic diversity analysis and germplasm identification.