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Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

ABSTRACT: Doubled haploid technology has been widely applied to multiple plant species and is recognized as one of the most important technologies for improving crop breeding efficiency. Although mutations in MATRILINEAL/Zea mays PHOSPHOLIPASE A1/NOT LIKE DAD (MTL/ZmPLA1/NLD) and Zea mays DOMAIN OF UNKNOWN FUNCTION 679 MEMBRANE PROTEIN (ZmDMP) have been shown to generate haploids in maize, knowledge of the genetic basis of haploid induction (HI) remains incomplete. Therefore, cloning of new genes underlying HI is important for further elucidating its genetic architecture. Here, we found that loss-of-function mutations of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), one of the members from the phospholipase D subfamily, could trigger maternal HI in maize. ZmPLD3 was identified through a reverse genetic strategy based on analysis of pollen-specifically expressed phospholipases, followed by validation through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. Mutations of ZmPLD3 resulted in a haploid induction rate (HIR) similar to that of mtl/zmpla1/nld and showed synergistic effects rather than functional redundancy on tripling the HIR (from 1.19% to 4.13%) in the presence of mtl/zmpla1/nld. RNA-seq profiling of mature pollen indicated that a large number of pollen-specific differentially expressed genes were enriched in processes related to gametogenesis development, such as pollen tube development and cell communication, during the double-fertilization process. In addition, ZmPLD3 is highly conserved among cereals, highlighting the potential application of these in vivo haploid-inducer lines for other important crop plant species. Collectively, our discovery identifies a novel gene underlying in vivo maternal HI and provides possibility of breeding haploid inducers with further improved HIR.

SUBMITTER: Li Y

PROVIDER: S-EPMC8677622 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIn 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.ResultsWe 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.ConclusionThese 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.

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Loss-of-function alleles of ZmPLD3 cause haploid induction in maize.

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