Abstract
Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3−∕− and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3−∕− lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.
Highlights
Doubled haploid (DH) generations facilitate production of pure homozygous seed in one generation, enabling breeders to bypass the seven or eight seasons of self-pollination that are normally required to produce inbred germplasm (Wêdzony et al, 2009)
While many crops in the cereals, brassicas and cucurbits are bred through DH generations, several important crop species remain recalcitrant to standard haploid induction techniques (Kasha and Maluszynski, 2003)
Two tail-altered CENTROMERIC HISTONE3 (CENH3) transgenes, AcGREEN-CENH3 and AcGREEN-tailswap-CENH3, were each tested with two native gene complementation strategies (Figure 1)
Summary
Doubled haploid (DH) generations facilitate production of pure homozygous seed in one generation, enabling breeders to bypass the seven or eight seasons of self-pollination that are normally required to produce inbred germplasm (Wêdzony et al, 2009). Ravi and Chan produced Arabidopsis cenh3/cenh lines complemented by CENH3-tailswap transgenes in which the Nterminal tail was swapped with the shorter H3 tail These lines produced little pollen and set predominantly diploid seed after self-pollination. The authors reasoned that centromeres constructed with tail-altered CENH3 proteins function normally until they are forced to compete with wild-type centromeres for centromere loading with kinetochore components in the hybrid zygote and early embryo This causes reduced spindle attachment of the inducer genome, leading to elimination of those chromosomes via fragmentation, and micronuclei formation during mitosis. It has remained unclear why haploids were found more frequently when the altered CENH3 was on the female side of the backcross (∼30%) than the male (4%), but incomplete penetrance is a common feature of genome elimination in other induction systems (Zhao et al, 2013). While many of the strategies led to modest levels of HI, one had a strong effect, which may provide guidance for future endeavors in CENH3 engineering
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