Gene characterization of Willd. and L. accessions: unmasking genetic diversity.

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The genus L. includes the domesticated seed crop Willd. and the semi-domesticated seed/fodder  L., both valued for their high protein content and high-quality grains. This study investigates the morphological and molecular characteristics of starch granules in 50 accessions including Willd. and L, to elucidate variations in amylose content and genetic markers. Starch granules were analyzed using scanning electron microscopy, revealing primarily angular to polygonal shapes with an average size of ~ 1.5 µm. Dynamic light scattering showed size variation: L. granules ranged from 115.1 ± 8.09 to 192.5 ± 5.11 nm, while Willd. granules from 204.5 ± 21.45 to 263.9 ± 12.48 nm. Apparent amylose content (AAC) was categorized via iodine staining into high (> 25%), intermediate (19-25%), low (11-19%), and very low (5-12%) classes. The results demonstrated a wide AAC range, with L. displaying a broader spectrum compared to Willd. The molecular characterization of the locus, crucial for amylose synthesis, was performed using PCR and sequencing. The locus, consisting of 13 exons and 12 introns, showed significant sequence similarity with species. The key single nucleotide polymorphisms (SNPs) associated with AAC levels were identified, including variations in exons 1, 4, 6, 9, and 13. A 100 bp deletion in intron 9 was specific to L, facilitating the development of an allele-specific PCR marker to distinguish between Willd. and L. The phylogenetic analysis of sequences divided accessions into two primary clusters, reflecting their A-genome and B-genome origins. The study enhances understanding of genetic diversity and offers insights for breeding applications in species.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s13205-024-04173-6.