Molecular and genetic analyses of a major gene for seed protein content in soybean

Abstract

Seeds store nutrients for conservation and propagation of genetic materials between two generations and propagation. Seed crops are cultivated to supply those nutrients. Soybean is important seed crop since it has high protein content to maintain body and generate energy. Continuous efforts of soybean breeders are directed to improve the protein quantity by identifying linked markers and quantitative trait loci (QTL). Danbaekkong has 48% of seed protein content that its major protein QTL was founded on Chr 20, surrounding region of Satt239 and Satt496 marker interval. We selected three residual heterozygous lines heterozygous in the Satt239-Satt496 region from two recombinant inbred line populations of Sinpaldalkong 2 x Danbaekkong and Daewonkong x Danbaekkong soybean crosses to develop two sets of near isogenic lines (NILs), Prot_highSD/Prot_lowSD and Prot_highDD/Prot_lowDD with different seed protein content. The maternal parent cultivar Danbaekkong, carrying a high protein content allele, showed high seed protein content. In the genomic region harboring the introgressed Danbaekkong segment on Chr 20, we identified nucleotide differences between low- and high-protein NILs through whole genome sequencing. From the major protein QTL region, we found 66 non-synonymous single nucleotide polymorphisms and one frameshifts are overlapped between both low- and high-protein lines. After elimination of genes by expression during seed stages, we identified two genes, calcium-dependent protein kinase and exocyst subunit exo70 family protein, are more likely involved in see protein accumulation in soybean. The gene expression changes of developing seed between Prot_lowDD and Prot_highDD represent the pattern of up-regulation of many genes in Prot_highDD at 2 week after flowering (WAF), stage of early maturation. Up-regulation pattern of storage protein genes in Prot_highDD was increasing according to seed maturation. There is a block of gene expression pattern that raised up-regulation in Prot_lowDD, consist of genes involving protein degradation. Transcription factors were highly up-regulated in Prot_highDD at 2 WAF, including major transcription factor to regulate seed development. Gene expression of carbon precursors, protein, and oil metabolic enzymes are compared between 1 and 2 WAF in Prot_lowDD to identify initial gene expression at early maturation stage. Glycine max undergoes two rounds of whole-genome duplication

the number of genes involved in the three synthesis pathways is more than two times higher than that in Arabidopsis. Among these genes, five were conserved as single-copy genes and 44 were high copy gene families consisting of more than seven homolog members. We identified five differentially expressed genes in immature seeds aged between 1 and 2 WAF. To find out protein expression change according to storage protein difference, additional proteomic research was hired for two NIL lines. Three enzymes, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase and ketol-acid reductoisomerase showed over two times differential expression between Prot_high lines and Prot_low lines. In this research, we developed two NIL pairs of high and low seed protein content derived from different genetic backgrounds. Genomic, trasncriptomic and proteomic analysis using these NILs indicates that development regulation from transcription factors and initial carbon metabolism is important to seed storage protein synthesis.