Excision of an Active CACTA-Like Transposable Element From DFR2 Causes Variegated Flowers in Soybean [Glycine max (L.) Merr.]

Abstract

Active endogenous transposable elements, useful tools for gene isolation, have not been reported from any legume species. An active transposable element was suggested to reside in the W4 locus that governs flower color in soybean. Through biochemical and molecular analyses of several revertants of the w4-m allele, we have shown that the W4 locus encodes dihydroflavonol-4-reductase 2 (DFR2). w4-m has arisen through insertion of Tgm9, a 20,548-bp CACTA-like transposable element, into the second intron of DFR2. Tgm9 showed high nucleic acid sequence identity to Tgmt *. Its 59 and 39 terminal inverted repeats start with conserved CACTA sequence. The 39 subterminal region is highly repetitive. Tgm9 carries TNP1-and TNP2-like transposase genes that are expressed in the mutable line, T322 (w4-m). The element excises at a high frequency from both somatic and germinal tissues. Following excision, reinsertions of Tgm9 into the DFR2 promoter generated novel stable alleles, w4-dp (dilute purple flowers) and w4-p (pale flowers). We hypothesize that the element is fractured during transposition, and truncated versions of the element in new insertion sites cause stable mutations. The highly active endogenous transposon, Tgm9, should facilitate genomics studies specifically that relate to legume biology. Figure 1) and purple hypocotyls. Mutations at the W4 locus in the W1_ background result in altered pigment accumulation patterns in petals and reduced levels of purple pigments in flowers and hypocotyls. Four mutant alleles, w4, w4-m, w4-dp, and w4-p have been mapped to this locus. The w4 allele represents a spontaneous mutation, which produces near-white flowers (Figure 1) and green hypocotyls (Hartwig and Hinson 1962;Groose and Palmer 1991). The w4-m allele was identified from a cross between two experimental breeding lines with white and purple flowers, respectively (Palmer et al. 1989;Weigelt et al. 1990). w4-m is characterized by variegated flowers (Figure 1) and green hypocotyls with purple sectors (Groose et al. 1988).w4-m has been proposed to harbor a class II transposable element (Palmer et al. 1989). Presumably, somatic excision of the putative transposable element results in the variegated (Groose et al. 1988) and germinal excision wild-type phenotypes, purple flowers and purple pigments on hypocotyls (Palmer et al. 1989;Groose et al. 1990). The mutable line carrying w4-m undergoes germinal reversion at a very high frequency, about 6% per generation ). Approximately 1% of the progeny derived from germinal revertants contain new mutations in unlinked loci, presumably resulting from reinsertion of the element (Palmer et al. 1989). For example, female partial-sterile 1 (Fsp1), female partial-sterile 2 (Fsp2), female partialsterile 3 (Fsp3), and female partial-sterile 4 (Fsp4) were isolated from progenies of germinal revertants with purple flowers and were mapped to molecular linkage groups (MLG) C2, A2, F, and G, respectively (Kato and Palmer 2004). Similarly, 36 male-sterile, female-sterile mutants mapped to t...

Allelic variation of soybean flower color gene W4 encoding dihydroflavonol 4-reductase 2

“…Previous studies revealed that the W4 gene was mutated in flower color variants, Clark- w4 , T321 and T369 [3,5]. In this study, complementation tests revealed that the flower color of 222-A-3 and E30-D-1, and that of a G. soja accession, kw4, was also controlled by this gene.…”

Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.)

“…The Ac element is one of the representative hAT superfamily DNA transposons. In another case, insertion of an active CACTA-like DNA transposon was identified in the soybean DFR2 gene, while excision and reinsertion of this element affected diverse phenotypes, showing potential usage of the element in gene isolation (Xu et al 2010). …”

Molecular mapping of three male-sterile, female-fertile mutants and generation of a comprehensive map of all known male sterility genes in soybean

“…The ms7 gene was identified from the progeny of a mutable line (w4-m) harboring an endogenous transposable element, Tgm9 (Chen and Palmer 1996;Xu et al 2010). Anthers from the ms7 mutant plants were devoid of pollen grains, and dehisced anthers failed to release any aborted pollen grains or remnants of pollen grains (Palmer 2000).…”