Development of InDel markers associated with leaf protein content in mulberry based on whole-genome resequencing

Mulberry (Morus alba L.), as a crucial dual-purpose crop for medicinal and forage applications, requires genetic improvement of leaf protein content to substantially enhance its feed value. This study utilized 46 mulberry germplasms as test materials, with 29 accessions subjected to whole-genome resequencing on the Illumina HiSeq platform. Based on the resequencing data, Indel primers were designed and screened to analyze genetic diversity and develop Indel markers tightly linked to crude protein content in mulberry leaves. The results showed that a total of 1 155 585 InDel loci were detected in the 29 accessions, with 11 401 InDels located in coding regions (0.99%). Analysis of functional annotations in extreme-phenotype germplasm revealed that 2 563 InDel-containing genes within coding sequence (CDS) regions of high-protein accessions were significantly enriched in catalytic activity and metabolic processes. Further utilizing InDel variations within CDS regions, 98 pairs of InDel primers were designed, yielding a novel linked marker (Chr1-3-204) with distinct amplification patterns between high- and low-protein accessions. Validation in 46 accessions showed 89.1% accuracy (r = 0.726, P < 0.01). Sanger sequencing confirmed its high accuracy (97.7% similarity to reference), with 84% identity to Morus motabilis. This study is the first to develop a linked marker for crude protein content in mulberry leaves, providing a technical reference for the breeding of high-protein mulberry varieties.N.