Junping Yu, Guolong Zhao, Wei Li, Ying Zhang, Peng Wang, Aigen Fu, Limei Zhao, Chunbao Zhang & Min Xu

Theoretical and Applied Genetics November 2021; vol. 134: 3661–3674

Key message

Identification and functional analysis of the male sterile gene MS6 in Glycine max.

Abstract

Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.

See https://link.springer.com/article/10.1007/s00122-021-03920-0

Figure 2; MS6 encodes an R2R3 MYB transcription factor a Manhattan plot of the SNP-index differences, ∆(SNP-index), between WT and ms6 bulks constructed with a BC₅F₂ mapping population derived from ms6 x ‘JiuB’. The red box highlights the region co-segregated with ms6. b Genetic linkage map of ms6. Genetic distances between two adjacent loci are indicated by centimorgans (cM). Chr 13, chromosome 13. c The MS6 gene structure and the mutated site in ms6. Black boxes represent the exons of MS6; arrow indicates the SNP location; nucleotide in red is the mutated site in ms6; underlined sequence is the MseI restriction site. d The GmTDF1-1 protein structure. Green blocks indicate the R2R3 MYB DNA-binding domain; arrow and amino acid in red indicate the amino acid substitution site in ms6. e Genotyping the plants with the CAPS marker based on the SNP between WT and ms6. The MseI-undigested (−) or digested ( +) PCR products are segregated on 4% agarose gel. WT, homozygous wild-type plant; Het, heterozygous (+ /ms6) plant; ms6, homozygous ms6 mutant. f Multiple sequence alignment of the R2 region from different MYB proteins. The residue corresponding to L76 in GmTDF1-1 (indicated by the red box) is conserved (color figure online).