The simple sequence repeat (SSR) survey of ‘Tunisia’ genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with ‘Tunisia’ genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the ‘Tunisia’ genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.

Pomegranate (Punica granatum, L.) is a fruit tree that is increasingly popular worldwide due to the health-related properties of the fruit juice. While several studies highlighted the rich phytochemical diversity, few efforts have been devoted to an integrative understanding of the level of diversity of this species. This study investigated the diversity of 40 pomegranate accessions in an Indian ex situ collection by using twenty-nine morphological traits, six biochemical parameters, and twenty-nine Simple Sequence Repeats (SSR) markers. Among the evaluated traits, fruit volume (23.34% CV), fruit weight (21.12% CV), and fruit color (*a) (22.69 % CV) largely contributed to the morphological classification. Based on Mahalanobis D² distance and Tocher’s clustering, the 40 pomegranate accessions were grouped into eight clusters, partly consistent with their origin. Specifically, cultivars introduced from foreign countries were present in distinct clusters. The SSR marker analysis generated 66 alleles. The observed heterozygosity values ranged from 0.05 to 0.63, with a mean value of 0.30. Maximum molecular genetic dissimilarity was observed between ‘IC-318720′ and ‘Gul-e-Shah Red’ (0.30). The neighbor-joining dendrogram separated wild accessions from cultivated varieties. The combination of morphological, biochemical, and molecular characterization allowed for comprehensively characterizing the pomegranate diversity and provided information on the relationships between the different aspects of the diversity. This work also suggests that the origin of the accessions is an important factor of discrimination and that the level of admixture between local and foreign material is currently limited.