Diversity analysis and cross-species amplification of custard apple (Annona squamosa) using simple sequence repeats developed via next-generation sequencing technology

In this study, we isolated microsatellite markers for Annona squamosa through partial genome sequencing using next-generation sequencing (NGS) technology. From the NGS data, a total of 179,080 microsatellites were identified. Seventy randomly selected microsatellite markers were standardised for PCR amplification of A. squamosa DNA, and diversity was assessed in 40 genotypes. The number of alleles per locus ranged from 15 (ASIIHR38) to 46 (ASIIHR19, ASIIHR31 and ASIIHR35), with a mean value of 26.82. The polymorphic information content (PIC) values ranged from 0.748 (ASIIHR21) to 0.965 (ASIIHR40), with an average of 0.90. Dendrogram analysis classified the accessions into two major groups, which were further subdivided into multiple subgroups based on genetic relatedness. Most of the markers exhibited high levels of cross-species transferability, successfully amplifying in related species such as Annona cherimola (94.2%), Annona reticulata (95.7%), Annona glabra (97.1%), Annona muricata (90%) and Annona atemoya (80%). The results of this study demonstrate that species-specific genomic simple sequence repeats (SSRs) are more accurate for assessing genetic diversity. The SSR markers developed in this study will be valuable for diversity analysis, species identification, linkage map construction, DNA fingerprinting and crop improvement.