Development and Application of gSSRs Markers in Macadamia from Genomic Sequences

Macadamia (Macadamia spp.) is an economically important nut crop worldwide, but genomic simple sequence repeat (SSR) markers remain limited, especially for germplasm from South China, hindering comprehensive genetic diversity assessment and population structure analysis. In this study, we developed and characterized gSSR markers based on leaf genomic sequence data. A total of 76 primer pairs were initially designed, of which 65 were successfully amplified, and 18 highly polymorphic markers were selected for further analysis. Using these 18 gSSR markers, we genotyped 104 Macadamia individuals from 15 populations in South China. The markers detected a total of 118 alleles, with the number of alleles per locus ranging from 2.056 to 7.167 (mean = 2.911). The observed heterozygosity (H_o) and expected heterozygosity (H_e) varied from 0.556 to 0.697 and from 0.389 to 0.669, respectively. Analysis of molecular variance (AMOVA) revealed significant genetic differentiation among populations (F_ST = 0.304, p < 0.01), with 16.39% of the variation occurring between populations and 83.61% within populations. Population structure analysis based on Structure, PCoA, and UPGMA clustering consistently divided the 15 populations into two distinct genetic groups. Relative migration analysis revealed substantial gene flow among the four populations examined in this study, with bidirectional relative migration rates all exceeding 0.7. One outlier locus (MacChr13-2) showing a positive selection signal was identified, suggesting potential adaptive divergence. These newly developed gSSR markers provide a valuable tool for genetic diversity assessment, germplasm management and cultivar improvement, and marker-assisted breeding of Macadamia.