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

  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 46 999 SSR loci were detected among the 45 850 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. 38 507 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.