In vivo haploid induction leads to increased frequency of twin-embryo and abnormal fertilization in maize

Abstract

Background: In vivo haploid induction (HI) based on Stock6-derived inducer lines has been the most prevalent means of producing haploids. Nevertheless, the biological mechanism of HI is not fully understood, the twin-embryo kernels had been found during haploid induction, which may provide potential evidence for the abnormal double fertilization during HI.

Results: We investigated twin-embryo frequency in progenies of different haploid inducers. Results reveal that increasing the HI potential significantly improved the frequency of twin-embryo kernels. Compared with the average twin-embryo kernel frequency (average frequency = 0.07%) among progenies pollinated by the haploid inducer line CAUHOI, the frequency of twin-embryo was improved to 0.16% in progenies pollinated by the haploid inducer line CAU5. This result was further confirmed by pollinating single hybrid ND5598 with four haploid inducers possessing differentiated HIRs, where twin-embryo frequency was highly correlated with HIR. Among 237 twin-embryo kernels, we identified 30 haploid twin-embryo kernels (12.66%), a frequency which was much greater than the average HI rate for three other inducer lines (frequency range 2-10%). In addition, aneuploids, occurred at high frequency (8 in 41 twin plants). This level of aneuploidy provides new insight into the abnormal double fertilization during HI. Moreover, we observed differences in growth rate between twin plants in the field, as 4.22% of the twin plants grew at a significantly different rate. Both simple sequence repeats markers (SSR) and 3072 SNP-chip genotyping results revealed that > 90% of the twin plants shared the same origin, and the growth difference could be attributed to aneuploidy, competition for nutrients, and possible hormone regulation.

Conclusion: These results demonstrate that an enhanced HI ability can increase twin-embryo kernel frequency, and high frequency of both haploid twin-embryo kernels and aneuploidy observed in this research give us new insights to understand the mechanism of both HI and abnormal embryogenesis.

Keywords: Flow cytometry; Haploid induction rate; In vivo haploid induction; Twin-embryo; Twin-embryo kernel rate.

Fig. 1

Classification based on the phenotype of twin-embryo kernels. A-D were type V, type Y, type II and uncertain type twin-embryo kernels. a-d were corresponding phenotype after embryo separation

Fig. 2

Field performance of twin embryo plants. A Twin plants in seedling stage. B PHD type I, II, and III twin plants in the field. C1-C3 Ear performance of three PHD types. D Variation of plant height difference between twin plants

Fig. 3

Ploidy determinations based on chromosome number and flow cytometry. A-B were diploid twin-embryo kernel and haploid twin-embryo kernel, respectively. a-b were chromosome number of corresponding diploid and haploid twin-embryo kernel. C1-C5 were flow cytometry result of diploids (C1), haploids (C2), and three kinds of aneuploids (C3, C4 and C5). Y axis is the number of cells detected, x axis represents for the fluorescent light area (FL2-A)

Fig. 4

Genotyping results (SNPs) for plants from twin-embryo kernels. Red for the number of same genotype between two twin plants and green for different genotype between twin plants. Percentage of the same genotype is inside the red bar