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.
Highlights
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
We found that only one member (ZmPLD3) was expressed in pollen (Extended Data Fig. 1) and significantly upregulated in mtl/zmpla1/ nld[9], which suggested that Zea mays PHOSPHOLIPASE D3 (ZmPLD3) played a role similar to that of MTL/ZmPLA1/NLD
ZmPLD3 was grouped into the C2-phospholipase D (PLD) subfamily, which was consistent with its predicted C2 domain that binds to Ca2+ cofactors[17]
Summary
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. Further analysis of subcellular localization revealed that MTL/ZmPLA1/NLD targeted the endo-plasma membrane, a specific membrane derived from vegetative cell that surrounds the two sperm cells in pollen[14,15,16] These results implied that phospholipases highly expressed in pollen might play an important role in sexual reproduction. Mutations in ZmDMP resulted in HI with an HIR of 0.1–0.3%, and the HIR increased to 6–10% in the presence of mtl/zmpla1/nld, which suggested that more than one pathway might be involved in the high HIR observed in commercial haploid-inducer lines. Mutations in Arabidopsis orthologous genes AtDMP8 and AtDMP9 could trigger maternal haploids[26], whereas no functional MTL/ ZmPLA1/NLD orthologous genes were identified in dicots These findings implied that HI was triggered by genes in different pathways and pyramiding these causative factors together could improve the HIR sharply. Isolating new genes required for HI will contribute to breeding haploid-inducer lines with high HIR, as well as elucidating the mechanisms underlying HI
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