Integrated agro-morphological and molecular characterization for progeny testing to enhance alfalfa breeding in arid regions of Tunisia

Alfalfa ( Medicago sativa L.) is a vital forage crop in arid and semi-arid regions, where improving its productivity and resilience is essential for sustainable agriculture. This study is part of a long-term breeding program aimed at improving the productivity and drought tolerance of the local “Gabssia” alfalfa germplasm from arid regions of southern Tunisia. Initiated in 2004, the program introduced exotic genotypes (Sardi10, Ameristand801S, ABT805, and Siciliano Ecotipo) to mitigate inbreeding depression and enhance genetic diversity. This research aimed to assess the genetic variability and agro-morphological performance of five alfalfa families (L39, E34, ABT32, A73, S71) and their progenies using a combined molecular and phenotypic approach. Phenotypic evaluation revealed significant variability in key agronomic traits, including leaf morphology, flower color, biomass yield, and seed characteristics. Fresh and dry biomass yields varied notably, with progenies A73-D2 and L39-D2 identified as the highest-yielding genotypes. Family A73 exhibited superior seed production, demonstrating high seed number per pod and 1000-seed weight. Molecular characterization using six Simple Sequence Repeat (SSR) markers (MTIC185, MTIC249, MTIC259, MTIC297, Endo20, and SucS1) showed moderate genetic diversity with Polymorphism Information Content (PIC) values of 0.215–0.373, supporting the potential for effective selection in breeding programs. Multivariate analysis classified genotypes into distinct performance groups tailored to breeding objectives, such as optimizing seed production or biomass yield. Notably, progenies from E34 and L39 exhibited superior forage productivity, while those from S71 and A73 demonstrated high variability, offering potential for improving drought tolerance and biomass quality. The integration of molecular and phenotypic data proved to be an efficient strategy for progeny testing and elite genotype selection. These findings highlight the value of marker-assisted selection in accelerating breeding efforts and developing stress-resilient alfalfa varieties, contributing to sustainable forage production in arid and semi-arid environments. • Integrated agro-morphological traits and SSR markers to improve progeny testing in alfalfa breeding. • Identified high-yielding genotypes with enhanced biomass and seed traits for arid regions. • Highlighted the importance of the support of SSR markers to enhance progeny testing in alfalfa breeding.