Chili (Capsicum annuum L.) is a self-pollinated crop, with natural cross-pollination occurring below 4%–5%. It intends to have low heterosis. Developing cross-pollination in chili currently receives much attention to achieve diversity in trait improvement. Double-crossing becomes one of the alternatives to achieving this goal. In this study, three different parental chili genotypes (K, B, and T) gained crossing, with four populations (S2 K, F3 KB, F2 BTKB, and F2 KBBT) developed. Using 11 selected sequence-related amplified polymorphism (SRAP) combination markers that target Open Reading Frame (ORF) regions assessed molecular diversity in these chili populations. Results revealed the possibility of identifying diversity using SRAP markers based on primer profile information. The iMEC analysis showed high values of PIC (0.3381), discriminant power (0.882), and mean polymorphic value (97.88%). The highest similarity emerged between the populations BTKB and KBBT as the reciprocal. Then, the smallest similarity appeared between K and the double cross. Compared with the self-pollinated genotype, SRAP primers discovered that double crosses provided more variation based on Shannon’s index (I) and percentage of polymorphic loci (PPL). The genetic distance denotes maternal inheritance or extraneous involvement in progeny. However, multiple-parent hybridization authenticated the boost in genetic diversity.
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