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.

CRISPR/Cas System: Applications and Prospects for Maize Improvement

“…In addition, in the presence of zmpla1 , the HIR of zmpld3 mutant was increased by a factor of 3 (from 1.19% to 4.13%), showing a synergistic effect rather than functional redundancy. The HIR of zmpld3 (±)- zmpla1 - zmdmp was greatly improved by comparison with that of zmpld3 - zmpla1 and zmpla1 - zmdmp …”

“…The HIR of zmpld3 (±)-zmpla1-zmdmp was greatly improved by comparison with that of zmpld3-zmpla1 and zmpla1-zmdmp. 76 Making full use of DH and CRISPR/Cas9-mediated genome-editing technologies will produce a powerful effect in crop breeding, but both of them still have shortcomings in the rapid generation of pure elite lines with comprehensive beneficial traits. The haploid-inducer-mediated genome-editing (IMGE) approach 77 and HI editing (HI-Edit) technology 42 have been developed to quickly and effectively deliver edits to the elite maize inbred line, which used a maize haploid-inducer line taking along a CRISPR/Cas9 cassette that targets a favorable agronomic traits.…”

Green Revolution to Gene Revolution: Technological Advances in Agriculture to Feed the World

“…Both genes play important roles in plant immunity and genome stability [ 146 ]. In corn, CRISPR/Cas9-mediated knockout of Zea mays PHOSPHOLIPASE D3 ( ZmPLD3 ) resulted in either 1-bp insertion or a combination of 1-bp insertion and 5-bp deletion in two separate mutant lines, which triggered haploid induction [ 147 ]. In rice, relatively larger deletions of 71 and 33 bp (rather than 1-bp deletion) were necessary to effectively knockout Oryza sativa MICRORNA168a ( OsMIR168a ), which caused significant transcription profile changes.…”

Improving Yield and Yield Stability in Winter Rye by Hybrid Breeding

“…Advances in deciphering the genetic code of in vivo haploid induction [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ] and the recently published reference genome sequences for rye [ 41 , 42 ] offer the promising option to search for desired alleles in rye orthologs of the genes MTL / ZmPLA1 / NLD or cenh3. This candidate gene approach might lead to an application of DH technology in rye to reduce the time required for producing parental lines by several years, as DH technology is known to simplify logistics [ 43 ].…”