Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean

Abstract

The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies.

Fig 1. PCR amplification of the four genome walking libraries generated from the homozygous sterile bulk and the homozygous fertile bulk.

A) Results of the first PCR (PCR1) reaction with adaptor primer (AP1) and transposon specific primer (Trans R1). B) Results of the second PCR (PCR2) with nested adaptor primer (AP2) and nested transposon specific primer (Trans R2). M, 200 bp DNA Ladder; S, Sterile bulk (library generated from DNA of pooled 10 homozygous sterile plants); F, Fertile Bulk (library generated from DNA of pooled 10 homozygous fertile plants). Arrows indicate the bands specific to sterile plants. a, Dra1-sterile-AP2; b, EcoRV-sterile-AP2; c, StuI-sterile-AP2-1; d, StuI-sterile-AP2-2.

Fig 2. The graphical representation of Glyma.16G072300.

The gene has 28 predicted exons and the transposon Tgm9 is inserted into exon 10. The Tgm9 insertion is not drawn to scale.

Fig 3. A revertant plant showing a fertile branch.

The transposon excised out of the st8 allele in the bud that resulted in the fertile branch with multiple pods bearing viable seeds. All other branches on the MSFS plant remained sterile.

Fig 4. PCR analysis of two revertant plants.

A) Graphical representation showing locations of two PCR primers (Rev1 and Rev2) flanking the insertion site in Glyma.16G072300 (the St8 gene) and a third primer (Trans R1) located in Tgm9. With no transposon present, the primers Rev1 and Rev2 should amplify a 491 bp fragment. With Tgm9 present, the primers Rev1 and Trans R1 should amplify a 601 bp fragment. The Tgm9 insertion is not drawn to scale. B) PCR amplification of fragments from sterile and fertile branches of two revertant plants (A11-180-1 and A11-1036) using primers Rev1, Rev2 and Trans R1. In the Williams 82 sample the transposon is not present, so only 491 bp fragment is amplified. The fertile branches of A11-180-1 and A11-1036 are heterozygous, therefore both the fragments representing absence of Tgm9 (491 bp fragment) and presence of Tgm9 (601 bp fragment) were amplified. The sterile branches are homozygous for the insertion of Tgm9, so only 601 bp fragment is amplified. F, fertile branch; S, sterile branch.

Fig 5. The multiple sequence alignment of soybean, rice, Arabidopsis and yeast MER3 helicases highlighting four conserved motifs commonly found in the MER3 helicases.

There is 100% conservation of all the amino acid residues in four motifs among the four genes showing that the newly cloned Glyma.16G072300 (St8) gene is a MER3 DNA helicase. Only a part of the proteins is shown.

Fig 6. Phylogenetic tree of homologs of Glyma.16G072300 (St8).

The unrooted radiated phylogenetic tree shows the relationships among the soybean helicase, St8 and homologous proteins in soybean and other taxa. The tree was generated using the Neighbor-Joining method in the MEGA6 software. Numbers on the nodes represent percent bootstrap support in a 10,000 replicate bootstrap test, and the sum of branch lengths is 9.42170767. The evolutionary distance represented by the scale bar shows 0.2 amino acid substitutions per site.

Fig 7. Semi-quantitative RT-PCR showing expression of St8 in different soybean tissues.

Glyma.16G072300 showed an expression pattern distinct from the other DEAH/D-box helicase genes (Glyma.08g102300, Glyma.06g202500, and Glyma.01g038100) in soybean. The Elf1B gene was used to confirm equal loading of RNA for each sample.