17 Omer Idris Musa Olom Zhenwu Wei Jiaqing Li Nana Liu Ahmed Mohammed Mustafa Lazim Journal of Animal and Plant Sciences JAPS 20262026 36 5 1018-7081 https://doi.org/10.36899/JAPS.2026.5.0103 <p style="text-align: justify; line-height: 1.1;"><span style="font-size: 12pt;"><span style="line-height: 115%; font-family: Calibri, sans-serif; color: black;">The multifoliate leaf trait in alfalfa is a key agronomic trait associated with enhanced forage yield and quality. <span style="background: white;">In this study, a backcross (BC</span></span><span style="line-height: 115%; font-family: 'Cambria Math', serif; color: black; background: white;">₃</span><span style="line-height: 115%; font-family: Calibri, sans-serif; color: black; background: white;">) progeny was developed from crosses between alfalfa (<em>Medicago sativa</em> L. cv. Huaiyin) and PL34HQ multifoliate leaves to analyze the leaf morphology and population structure using SSR markers. Leaf morphological traits were observed and recorded at different growth stages to assess structural and seasonal variations. Genomic DNA was extracted from each progeny, and Simple Sequence Repeat (SSR) markers were employed to evaluate the genetic diversity and population structure of the 147 BC</span><span style="line-height: 115%; font-family: 'Cambria Math', serif; color: black; background: white;">₃</span><span style="line-height: 115%; font-family: Calibri, sans-serif; color: black; background: white;"> progeny and their two parents. The observations of leaf morphology revealed diverse compound leaf types (with 4, 5, 6, or 7 leaflets) on the main stem of multifoliate plants. The frequency of multifoliate leaves was notably higher in spring and autumn than in winter and summer, with an overall multifoliate rate of 90.48% in the BC</span><span style="line-height: 115%; font-family: 'Cambria Math', serif; color: black; background: white;">₃</span><span style="line-height: 115%; font-family: Calibri, sans-serif; color: black; background: white;"> progeny. The highest and lowest rates of multifoliate plants were 88.75% and 5.35%, respectively. Using 15 SSR primers, a total of 68 bands were amplified across the 147 hybrids, with an average of 4.53 alleles per locus. Primer MES 3 generated the highest number of alleles (7), while MES 37 and MES 64 produced the fewest (2). The polymorphism rate ranged from 66.67% (W6007) to 100% (MES 37 and MES 64). The average PIC and gene diversity values were 0.672 and 0.224, respectively. Population structure analysis using STRUCTURE, </span><span style="line-height: 115%; font-family: Calibri, sans-serif; color: black;">principal coordinate analysis<span style="background: white;"> PCoA, and neighbor-joining methods identified three distinct genetic groups. Group III exhibited the greatest genetic diversity (0.2543) and highest Shannon index (0.3831), whereas Group I had the lowest values (0.2413 and 0.3560, respectively). These findings provide valuable insights for developing mapping populations, selecting breeding parents, and evaluating SSR marker polymorphism in alfalfa improvement programs.</span></span></span></p> Alfalfa; Microsatellite; Population structure; Genetic diversity; BC3 progeny Pakistan Agricultural Scientists Forum https://thejaps.org.pk/AbstractView.aspx?mid=2025-JAPS-411