Development and application of genomic SSR markers in Rosa persica

Rosa persica is the only single-leaf species of Rosa genus, which has been classified as the second class of national endangered plants. The development of efficient molecular markers can provide important data support for the analysis of the genetic diversity of R. persica populations and the study of the growth pattern of clones, as well as theoretical guidance for the conservation of genetic resources of its populations.

SSR loci with 1-6 nucleotide repeats in the whole genome sequence of R. persica were searched and analyzed by Krait v1.3 software, and then primers were designed by Primer Premier3.0 software. Sixteen samples of R. persica were selected to test the validity and polymorphism of the primers by agarose gel electrophoresis and capillary electrophoresis. Finally, the data of amplification products of high polymorphism primers were analyzed by POPGENE32 software and PowerMarker3.25 software for genetic parameters, and the 16 R. persica samples were analyzed by clustering using the software NTSYS-pc 2.10.

A total of 142 083 SSR loci were identified on the genome sequence of R. persica, with the largest number of dinucleotide repeat types, accounting for 46.95%. The units with the highest percentage from mononucleotide to hexanucleotide repeat types were A/T, AT/AT, AAG/CTT, AAAT/ATTT, AAAAT/ATTT and AAAAAG/CTTTTTT, which fully indicated that A/T were the dominant unit. The SSR sequence lengths of R. persica genome varied from 12 to 1 026 bp, and the different nucleotide repeat types all showed the pattern of the longer length and the lower number, and the number of SSR loci with interval lengths of 10-15 bp was the largest, accounting for 47.42%. Among the 140 pairs of primers synthesized, 112 pairs of primers could obtain clear bands, and the primer efficiency was 80%; finally, 14 pairs of primers with high polymorphism and good stability were screened out and 58 alleles were detected in 16 R. persica samples. The PIC value ranged from 0.314 3 to 0.675 9. The average allele number (Na), effective alleles (Ne), shannon diversity index (I) and polymorphic information content (PIC) were 4.142 9, 2.576 9, 1.080 8 and 0.529 2, respectively. Pearson correlation analysis showed that there was no significant correlation between the PIC values of the screened primers and the lengths of the SSRs. Cluster analysis revealed that the 14 pairs of primers screened were able to better differentiate R. persica based on different populations, with genetic similarity coefficients ranging from 0.45 to 0.86.

The large-scale development of SSR markers using the whole genome of R. persica is rich in number and diverse in type, and the highly polymorphic SSR loci screened will play an important role in the study of the genetic diversity of R. persica populations.