Haploids and doubled haploid (DH) inbred lines have become an invaluable tool for maize genetic research and hybrid breeding, but the genetic basis of in vivo induction of maternal haploids is still unknown. This is the first study reporting comparative quantitative trait locus (QTL) analyses of this trait in maize. We determined haploid induction rates (HIR) in testcrosses of a total of 1061 progenies of four segregating populations involving two temperate haploid inducers, UH400 (HIR = 8%) and CAUHOI (HIR = 2%), one temperate and two tropical inbreds with HIR = 0%, and up to three generations per population. Mean HIR of the populations ranged from 0.6 to 5.2% and strongly deviated from the midparent values. One QTL (qhir1) explaining up to p^=66% of the genetic variance was detected in bin 1.04 in the three populations involving a noninducer parent and the HIR-enhancing allele was contributed by UH400. Segregation ratios of loci in bin 1.04 were highly distorted against the UH400 allele in these three populations, suggesting that transmission failure of the inducer gamete and haploid induction ability are related phenomena. In the CAUHOI × UH400 population, seven QTL were identified on five chromosomes, with qhir8 on chromosome 9 having p^>20% in three generations of this cross. The large-effect QTL qhir1 and qhir8 will likely become fixed quickly during inducer development due to strong selection pressure applied for high HIR. Hence, marker-based pyramiding of small-effect and/or modifier QTL influencing qhir1 and qhir8 may help to further increase HIR in maize. We propose a conceptual genetic framework for inheritance of haploid induction ability, which is also applicable to other dichotomous traits requiring progeny testing, and discuss the implications of our results for haploid inducer development.

Doubled haploid (DH) technology substantially accelerates crop breeding process. Wheat haploid production through interspecific hybridization requires embryo rescue and is dependent on genetic background. In vivo haploid induction (HI) in maize has been widely used and demonstrated to be independent of genetic background and to produce haploids efficiently. Recent studies revealed that loss of function of the gene MTL/ZmPLA1/NLD triggers HI (Gilles et al., 2017; Kelliher et al., 2017; Liu et al., 2017). In addition to producing homozygous DH lines, HI system has also been used for gene editing in different genetic backgrounds without introducing the genome of the male parent (Kelliher et al., 2019; Wang et al., 2019).

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.