Soybean (Glycine max) cultivars adapted to high latitudes have a weakened or absent sensitivity to photoperiod. The purposes of this study were to determine the molecular basis for photoperiod insensitivity in various soybean accessions, focusing on the sequence diversity of the E4 (GmphyA2) gene, which encodes a phytochrome A (phyA) protein, and its homoeolog (GmphyA1), and to disclose the evolutionary consequences of two phyA homoeologs after gene duplication. We detected four new single-base deletions in the exons of E4, all of which result in prematurely truncated proteins. A survey of 191 cultivated accessions sourced from various regions of East Asia with allele-specific molecular markers reliably determined that the accessions with dysfunctional alleles were limited to small geographical regions, suggesting the alleles’ recent and independent origins from functional E4 alleles. Comparison of nucleotide diversity values revealed lower nucleotide diversity at non-synonymous sites in GmphyA1 than in E4, although both have accumulated mutations at almost the same rate in synonymous and non-coding regions. Natural mutations have repeatedly generated loss-of-function alleles at the E4 locus, and these have accumulated in local populations. The E4 locus is a key player in the adaptation of soybean to high-latitude environments under diverse cropping systems.

Purple-blue flower of soybean (Glycine max [L.] Merr.) is controlled by the W2 locus. Previous studies revealed that a MYB transcription factor gene GmMYB-G20-1 was located at a position similar to the W2 gene and that a base substitution generated a stop codon in the MYB domains of 2 soybean lines with purple-blue flowers. This study was conducted to confirm the relationship between GmMYB-G20-1 and the W2 gene. Cleaved amplified polymorphic sequence analysis to detect the base substitution suggested that a similar mutation occurred in 2 other soybean lines having purple-blue flowers, 037-E-8, and Yogetsu 1-blue. Thus, all genotypes having purple-blue flowers had identical base substitutions. To verify the function of GmMYB-G20-1, apple latent spherical virus (ALSV) vectors were constructed to perform virus-induced gene silencing of GmMYB-G20-1. A cultivar Harosoy with purple flowers (W2W2) was infected by the empty ALSV vector (wtALSV) or the GmMYB-G20-1-ALSV vector containing a fragment (nucleotide position 685–885) of GmMYB-G20-1. Plants infected by empty vectors had only purple flowers. In contrast, most flowers of plants infected with GmMYB-G20-1-ALSV had irregular gray/blue sectors in flower petals and some of the flowers had almost gray/blue petals. These results strongly suggest that silencing of GmMYB-G20-1 can alter flower color and that it may correspond to the W2 gene.