Genetic Diversity Assessment of Lotus corniculatus L. in Khuzestan Using Molecular Markers and DNA Barcoding

Document Type : Research Paper

Authors

Department of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

10.22103/jab.2025.24998.1681

Abstract

Abstract
Objective
Lotus corniculatus L. is a widely distributed, tetraploid perennial legume valued for its adaptability to diverse environments and its applications in forage production, soil improvement, and medicine. It is essential to assess its genetic diversity using molecular markers for conservation and breeding strategies. This study presents the first molecular characterization of L. corniculatus accessions from Khuzestan province in southwestern Iran.
Materials and methods
Twenty-one L. corniculatus accessions were collected from eight geographically distinct sites. Genetic variation was assessed using 12 SCoT and 6 ISSR primers. Genetic structure and relationships among accessions were analyzed using cluster analysis, principal coordinate analysis (PCoA), and the STRUCTURE software. To complement these analyses, three genetically divergent accessions were selected for sequencing of two DNA barcode regions: the internal transcribed spacer (ITS) of nuclear ribosomal DNA and the trnH-psbA intergenic spacer of the chloroplast genome. Barcoded sequences were analyzed using BLAST and Maximum Likelihood phylogenetic reconstruction.
Results
SCoT and ISSR markers revealed high polymorphism (96.87% and 85.8%, respectively), indicating substantial genetic diversity among the accessions. Cluster and PCoA consistently grouped the accessions into two main clusters, with the Minoo Island accession forming a distinct and divergent group. STRUCTURE analysis supported the existence of three genetic clusters, reflecting both admixture and clear genetic differentiation. DNA barcoding revealed that all three sequenced accessions, including the genetically distinct Minoo Island accession, shared identical sequences in both the ITS and trnH-psbA regions, forming a single haplotype, here designated as the Iranian haplotype. The ITS sequence exhibited 99.71% identity with L. tenuis, while the trnH-psbA region showed 100% identity with L. japonicus. However, both barcode regions lacked sufficient resolution to distinguish intraspecific variation.
Conclusions
Both marker systems were effective in detecting genetic diversity, with SCoT markers showing higher average polymorphism and mean PIC values, and ISSR markers exhibiting stronger marker index and resolving power. The distinct clustering of the Minoo Island accession highlights the potential for local adaptation and underlines the importance of conserving regional germplasm. In contrast, the lack of variation in the barcode sequences emphasizes the limited ability of conserved regions such as ITS and trnH-psbA to resolve fine-scale genetic structure. Complementary tools such as chromosome counting and genome-wide markers (e.g., SNP genotyping, RAD-seq, WGS) are recommended to improve taxonomic resolution.

Keywords

References

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Agricultural Biotechnology Journal
Volume 17, Issue 3
August 2025
Pages 113-134

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