Split-gene system for hybrid wheat seed production

Abstract

Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore "linked in repulsion." Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner.

Keywords: hybrid wheat breeding; intein-mediated protein splicing; site-specific recombination; split-gene approach.

Fig. 1.

Vector constructs used in this study. (A and B) The genetic structure of the barnase expression vector (“pro-vector”) (A) and the phiC31-integrase-expression vector (B). The provector harbors attP and attB sequences that serve as targets for the site-specific recombinase phiC31. Note that only the T-DNA part of the vectors is illustrated (not drawn to scale). (C) Phage phiC31 integrase-mediated site-specific recombination results in the derivative loci A₁ and A₂. Recombination results in the hybrid products attR and attL. (D) A schematic illustration of the assembly of active barnase cytotoxin via the intein-mediated trans-splicing of two precursor molecules. Bar-N and Bar-C, N- and C-terminal gene fragments of the Bacillus amyloliquefaciens barnase gene; HPTII, hygromycin phosphotransferase; IntN and IntC, N- and C-terminal intein sequences from the DnaB gene of Synechocystis sp.; LB and RB, T-DNA left and right borders; ocs, octopine synthase terminator; NLS, SV40 T antigen nuclear localization signal (amino acids PKKKRKV); nos, nopaline synthase terminator; ocs, octopine synthase terminator; phiC31, phage phiC31 recombinase coding sequence; PSK, intron PSK7-i3 from Petunia hybrida; Ptap, tapetum-specific osg6B promoter from rice (23); Pubi, maize ubiquitin 1 promoter; S, (GGGGS)₃ flexible peptide linker; UBQ, intron UBQ10-i1 from Arabidopsis thaliana.

Fig. 2.

Experimental design for the production of hybrid seed. Genotypes that have an expected male-sterile phenotype are symbolized in blue. (A) The generation of the male-sterile parent for hybrid breeding. After transforming the provector, the primary transformants (T₀) are expected to be male-sterile as a result of barnase expression. To ensure that the phenotype is stable, and to create more target plants for recombination, the T₀ plants are backcrossed. In the event of a single-copy integration of the prolocus, the T₁ progeny are expected to segregate so that ∼50% of the plants are male-sterile (transgenic) and 50% are fertile (null-segregants). In the next step, the sterile T₁ plants are crossed with plants that express a phiC31 site-specific recombinase (encoded by pICH13130). In the developing T₂ plants, the integrase may catalyze the site-specific deletions between the attP and attB sites of the prolocus. Two alternative reactions can lead to the deletion of either the C-terminal or the N-terminal part of the locus, which results in the formation of the derivative loci A₁ or A₂. The derivative loci will naturally reside at exactly the same genetic position on two homologous chromosomes (because they originate from one prolocus). Depending on the time point at which recombination occurs, the F₂ plants may be genetic chimera that contain recombined and nonrecombined sectors. Plants carrying only A₁ or A₂ should be fertile, because no complete barnase protein can be produced. The A₁ and A₂ lines are then crossed (either as T₃ plants or T₄ plants after one round of self-pollination), and a portion of the resulting progeny is expected to carry both the A₁ and A₂ loci (25% of the progeny if both parents are hemizygous for A₁ and A₂, and 50% or 100% of the progeny if one or both parents are homozygous for A₁ or A₂). These heterozygous genetic segregants are expected to be male-sterile because of the coexpression of barnase from the A₁ and A₂ loci. (B) A strategy for maintaining the male-sterile line. Reproduction of the heterozygous male-sterile line A₁A₂ can be accomplished by crossing such lines with a homozygous fertile “maintainer” line, A₁A₁ or A₂A₂. The progeny of this cross may segregate the seeds so that 50% will have the genotype A₁A₂ (like the female parent) and, depending on the male parental line that was used, 50% will have the genotype A₁A₁ or A₂A₂. (C) Hybrid seed production. Male-sterile plants with the genotype A₁A₂ are used as the female crossing partners for hybrid seed production. The allelic position of the complementary barnase fragments enforces 100% segregation during meiosis. This results in male fertility and seed set in the hybrid progeny, because all segregants carry only an inactive barnase fragment (either A₁ or A₂). BC, backcrossing step; S, self-pollination step.

Fig. 3.

Phenotype of the female crossing partner and the hybrid F₁. (A) The morphology of the ears from the female parent that carries both isoloci 7031-A₁ and 7031-A₂. The ears display the typical “open floret” phenotype and contain no seeds. Other ears of these plants were used as pollen acceptors for the production of hybrid F₁ plants (B). All hybrid progeny plants displayed full fertility and carry either A₁ or A₂.