Maize is one of the most important crops in the world and is also an essential raw material for the food, fuel, and fodder industries. Maize hybrids are widely used today, and gain of elite inbred lines is a crucial step for hybrid breeding. Doubled haploid (DH) technology based on in vivo haploid induction (HI) is often used to accelerate the efficiency of breeding of maize and other crops (Ishii et al., 2016). Maize is a typical diploid plant (2n = 20) with a very low rate (∼0.1%) in producing haploid (2n = 10) naturally. In vivo HI by inducer line Stock6 can lead to maternal haploid with a rate of 1%–2% when it is used as pollinator (Coe, 1959). Stock6-derived inducers have been considered as the most effective method for DH breeding in maize.

Although the phenomenon of Stock6-induced haploidy was discovered 50 years ago, the genetic and biological mechanism of HI is still unclear. Several quantitative trait loci (QTLs) affecting the haploid induction rate (HIR) have been mapped (Prigge et al., 2012, Dong et al., 2013, Hu et al., 2016), of which the qhir1 QTL located in bin 1.04 had the greatest effect on HI. Furthermore, qhir1 has been narrowed down to a 243-kb region based on the B73 reference genome, which paves the way for gene cloning (Dong et al., 2013). In addition, fine mapping of qhir1 and functional studies have revealed that, in addition to regulating HIR, qhir1 also affects embryo abortion rate, endosperm abortion rate, and segregation distortion (Dong et al., 2013, Xu et al., 2013).

In the region defined by fine mapping, we identified 13 genes in the B73 reference genome including GRMZM2G471240 and GRMZM2G062320, encoding phospholipase A (named as ZmPLA1) and thiolase, respectively. The other 11 genes are either low-confidence genes or transposable elements-related genes (Figure 1A and Supplemental Table 1). RNA sequencing of the anthers of B73 and B73-inducer (with ∼80% B73 background, HIR = 10%) was performed at the different developmental stages (meiosis, one nucleus, two nuclei, three nuclei; Figure 1B). In the mapping region of qhir1, only three transcripts were detected (fragments per kilobase of exon per million mapped reads ≥ 5) based on B73 genome including two long noncoding RNAs and ZmPLA1; however, in B73-inducer, only ZmPLA1 was expressed, and no significant gene expression difference was found between B73 and B73-inducer. Thus, ZmPLA1 is the most likely candidate gene for qhir1 (Figure 1B). ZmPLA1 was observed in anther and not in other tissues based on the public data for B73 from MaizeGDB (www.maizegdb.org), and its mRNAs were detected at the second mitosis stage (two-nuclei stage) and reached the highest level at the three-nuclei stage (Figure 1B).

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