RAD-Seq-derived SSR markers: a new paradigm for genetic analysis and construction of genetically improved production populations in Pinus koraiensis.

Abstract

Background: The principal objective of this research is to develop highly specific polymorphic molecular markers, with the aim of addressing the paucity of simple sequence repeat (SSR) markers in Pinus koraiensis. The objective of this initiative is to facilitate the efficient management of genetic resources within Pinus koraiensis, enable precise pedigree identification and establish a foundational framework for subsequent whole-genome sequencing and assembly strategies. To achieve this objective, a simplified genome sequencing approach was employed, utiliZing RAD-Seq technology on a sample of 100 clones of Pinus koraiensis sourced from 6 seed orchards. The SSR sequences present within the contig were identified via TBtools-II software, which was also employed to generate a comprehensive summary and analysis of the sequence characteristics. On the Basis of these insights, primers were designed and subjected to meticulous screening using bioinformatics methodologies, and their efficacy was subsequently verified. The SSR markers were subsequently employed to examine the genetic diversity and structure of the plus tree population. The genetic data were then integrated with multiyear cone production records from the population of plus trees, thus facilitating the construction of a production population.

Results: A total of 80,539 SSR sites were identified among the 5,840,917 reads that were subjected to analysis. Notably, that the majority of these SSRs were dinucleotide to trinucleotide repeats, constituting 93.838% of the total. As the number of repeats increased, a gradual decline in the number of SSRs was observed. The most prevalent repetitive motif within the SSR loci was (AT/TA)_n, representing 30% of all loci. A total of 1,933 SSR sites were selected for primer design, resulting in the successful formulation of 1,162 primer pairs, representing a success rate of 60.114%. Among these, 205 SSR primer pairs exhibited incomplete coverage of the SSR positions, whereas 79 primer pairs demonstrated specificity, as confirmed by e-PCR. Further analysis revealed 27 primer pairs suitable for amplification of the target fragments, nine of which exhibited polymorphisms. The mean number of alleles per primer (N_A) was calculated to be 9.333, whereas the mean number of effective alleles (N_E) was 4.032. The mean polymorphic information content (PIC) was 0.530. The Shannon index (I) for the plus tree population was 1.159, with observed heterozygosity (H_O), expected heterozygosity (H_E), and unbiased expected heterozygosity (µH_E) values of 0.583, 0.530, and 0.533, respectively, and a fixation index (F) of -0.079. No statistically significant genetic differentiation was observed among the subgroups within the population, and the level of genetic diversity was comparable among subgroups delineated by different criteria. Significant differences in cone production were observed between the plus trees (P<0.01). A production population of 20 individuals was constructed via the simulated annealing algorithm, which exhibited a more reasonable mating system (F=-0.028) and demonstrated superior cone production compared with that of the plus tree population, with an increase of 79.6%.

Conclusions: The utilization of RAD-seq technology enables the development of SSR markers and the screening of polymorphic primers, thereby providing a robust tool for research pertaining to genetic diversity analysis and the identification of germplasm in Pinus koraiensis. The plus tree population in Xiaobeihu exhibits a considerable degree of genetic diversity, with no notable genetic differentiation. The construction of production populations via the developed SSR markers in combination with cone production from plus trees results in a reasonable population size and a mating system, thereby providing a scientific basis and technical support for the evaluation of Pinus koraiensis germplasm resources and advanced-generation improvement efforts. Furthermore, this study provides a reference and guidance for the development and application of SSR markers in other tree species.