Sunflower (Helianthus annuus L.), known for its adaptability and high yield potential, is vital in global edible oil production. Estimating genetic diversity is a key pre-breeding activity in crop breeding. The current study comprised of 48 genotypes which were assessed for their biometrical traits at department of Oilseeds, Tamil Nadu Agricultural University, during the rainy season of 2022. The lines were subsequently characterised using 103 simple sequence repeat (SSR) markers for molecular diversity analysis. The results indicated that the net nucleotide distances indicated varying genetic divergence, with subpopulations II and V showing the highest (0.056) and I and IV the lowest (0.014). Subpopulation IV exhibited the highest heterozygosity (0.352), while subpopulation III had the lowest heterozygosity and a low Fst (0.173). Principal components analysis (PCA) and hierarchical cluster analysis were employed for assessing the morphological diversity, facilitating genotype grouping and parent selection for breeding programs. The first four components cumulatively accounted for 86.72% of the total variation. Cluster Analysis grouped 48 sunflower genotypes into three clusters based on genetic diversity. COSF 13B stands out for its high head diameter, oil content, seed yield, and oil yield based on mean performance of morphological data. Principal coordinate analysis (PCoA) mirrored the groupings from the Neighbor Joining method, with the first three components explaining 27.24% of the total variation. Molecular data analysis identified five distinct clusters among the germplasm. By integrating morphological and molecular marker data with genetic distance analysis, substantial diversity was revealed with the genotypes RHA 273 and GMU 325 consistently demonstrated high oil yield per plant. The genotypes GMU 477, GMU 450, COSF 13B, RHA 102, CMS 1103B, and RHA GPR 58 have been identified as suitable parents for enhancing oil content in sunflower breeding programs. These findings also aid in selecting SSR markers for genotype characterization and in choosing diverse parents for breeding programs.
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