Multi-locus GWAS mapping and candidate gene analysis of anticancer peptide lunasin in soybean (Glycine max L. Merr.)

Authors: LOCKLEAR, RIKKI - Fayetteville State University; KUSUMAH, JENNIFER - University Of Illinois; RASHAD, LAYLA - Fayetteville State University; LUGARO, FELECIA - Fayetteville State University; VIERA, SONIA - Fayetteville State University; KIPEGO, NATHAN - Fayetteville State University; KIPKOSGEI, FAITH - Fayetteville State University; JEROP, DAISY - Fayetteville State University; JACQUET, SHIRLEY - Fayetteville State University; KASSEM, ALDELMAJID - Fayetteville State University; DE MEJIA, ELVIRA - University Of Illinois; Mian, Rouf; JUAN, JIAZHENG - Fayetteville State University

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/26/2025
Publication Date: 7/14/2025
Citation: Locklear, R., Kusumah, J., Rashad, L., Lugaro, F., Viera, S., Kipego, N., Kipkosgei, F., Jerop, D., Jacquet, S., Kassem, A., De Mejia, E., Mian, R.M., Juan, J. 2025. Multi-locus GWAS mapping and candidate gene analysis of anticancer peptide lunasin in soybean (Glycine max L. Merr.). Plants. https://doi.org/10.3390/plants14142169.
DOI: https://doi.org/10.3390/plants14142169

Interpretive Summary: Lunasin is a phytochemical found in soybean seeds that demonstrated significant cancer-preventive, antioxidant, and other health benefits in various research. The objectives of the study were to identify genetic loci associated with lunasin content and linked candidate genes. A mapping panel of 144 diverse soybean lines was obtained from the USDA Soybean Germplasm Collection of diverse geographical origins and genetic backgrounds genotyped using SoySNP50K iSelect Beadchips. The lunasin content in soybean seeds was measured using the enzyme-linked immunosorbent assay (ELISA) method. The broad-sense heritability (h²) of seed lunasin content (mg/g of dry weight) across the two years was 27%. QTNs significantly related to lunasin content in soybean seeds were detected on 15 chromosomes, with logarithm of the odds (LOD) scores greater than 3.0, explaining phenotypic variations identified using the R package mrMLM (v4.0). For significant QTNs, the SNPs were relatively evenly distributed across soybean chromosomes, with the highest concentration on chromosomes 6 and 16 (4 SNPs each) and the lowest on chromosomes 4, 5, and 15 (1 SNP each). Sixteen significant QTNs were identified in 2003, and seventeen QTNs were identified in 2021. Significant QTNs on chromosomes 3, 13, 16, 18, and 20 were consistently identified across multiple models as being significantly associated with lunasin content, based on assessment data from two years. The candidate genes underlying significant QTNs for lunasin content in soybean seeds were annotated using the SoyBase JBrowser (soybase.org) with the Williams 82 (Wm82) genome assembly 5 (doi.org/10.1002/tpg2.20382). A total of 28 genes were found based on the soybean genome browser, utilizing data from two years of field assessments, with 12 candidate genes identified in seeds from 2003 and 15 from 2021. These candidate genes encode pentatricopeptide repeat (PPR) superfamily protein (9), transmembrane amino acid transporter proteins (5), ribosomal proteins (3), ATP binding (2), nudix hydrolase 1 (NUDT1) cluster, and others. These annotated candidate genes are located near or adjacent to the regions of significant SNPs. Our study is an important effort to understand the genetic basis and functional genes for lunasin production in soybean seeds. The genetic mapping of the loci conditioning lunasin production in soybean will be helpful in marker assisted breeding to improve lunasin contents of future soybean cultivars.

Technical Abstract: Soybean peptide lunasin exhibits significant cancer-preventive, antioxidant, and hypocholesterolemic effects. This study aimed to identify quantitative trait nucleotides (QTNs) associated with lunasin content and to annotate the candidate genes in the soybean genome. The mapping panel of 144 accessions was obtained from the USDA Soybean Germplasm Collection, encompassing diverse geographical origins and genetic backgrounds, and was genotyped using SoySNP50K iSelect Beadchips. The lunasin content in soybean seeds was measured using the enzyme-linked immunosorbent assay (ELISA) method, with lipid-adjusted soybean flour prepared from seeds obtained from the Germplasm Resource Information Network (GRIN) of USDA-ARS in 2003 and from North Carolina in 2021, respectively. The broad-sense heritability (h²) of seed lunasin content (mg/g of dry weight) across the two years was 27%. QTNs significantly related to lunasin content in soybean seeds were detected on 15 chromosomes, with LOD scores greater than 3.0, explaining phenotypic variations identified using the R package mrMLM (v4.0). For significant QTNs, the SNPs were relatively evenly distributed across soybean chromosomes, with the highest concentration on chromosomes 6 and 16 (4 SNPs each) and the lowest on chromosomes 4, 5, and 15 (1 SNP each). Sixteen significant QTNs were identified in 2003, and seventeen QTNs were identified in 2021. Significant QTNs on chromosomes 3, 13, 16, 18, and 20 were consistently identified across multiple models as being significantly associated with lunasin content, based on assessment data from two years. A total of five chromosomes contained QTNs identified by more than four different models, all with LOD scores greater than 3.0, explaining varying amounts of phenotypic variation over the two years of data. The candidate genes underlying significant QTNs for lunasin content in soybean seeds were annotated using the SoyBase JBrowser (soybase.org) with the Williams 82 (Wm82) genome assembly 5 (doi.org/10.1002/tpg2.20382). A total of 28 genes were found based on the soybean genome browser, utilizing data from two years of field assessments, with 12 candidate genes identified in seeds from 2003 and 15 from 2021. These candidate genes encode pentatricopeptide repeat (PPR) superfamily protein (9), transmembrane amino acid transporter proteins (5), ribosomal proteins (3), ATP binding (2), nudix hydrolase 1 (NUDT1) cluster, and others. These annotated candidate genes are located near or adjacent to the regions of significant SNPs. In summary, our study is an important effort to understand the genetic basis and functional genes for lunasin production in soybean seeds.