Powerful combination: a genome editing system to improve efficiency of breeding inducer and haploid sorting in maize

Double haploid (DH) technology can be used to rapidly develop homozygous lines (Geiger and Gordillo, 2009). As the fundamental component of DH technology, the traditional inducer lines were developed through a process of recurrent selection over multiple generations, a method that was inherently time-consuming. The advent of gene editing technology has facilitated the creation of inducer lines in an efficient manner (Kelliher et al., 2017; Zhong et al., 2019). However, these inducer lines lack sort markers for sorting haploid, and the introduction of genetic markers is achieved through hybridization (Yu and Birchler, 2016). Though anthocyanin marker or oil content has been primarily used for sorting haploid (Qu et al., 2021), there is a notable discrepancy in the false discrimination rate for manual or automated sorting due to the influence of anthocyanin expression. The NMR system can enhance the haploid correct discrimination rate (CDR), but the equipment is expensive. Fluorescent markers represent another type of genetic markers for the sorting of haploids; however, the fluorescent is not visible to the naked eyes (Dong et al., 2018). Consequently, the current genetic markers exhibit delayed coloration (Chen et al., 2022; Wang et al., 2023), which limits the application of DH technology.

In this study, we developed a Cas9 system for breeding inducer and sorting haploid in maize, with three advantages: (i) we innovatively employed a promoter pOsBBM1 to drive Cas9 in maize, which does not yield new edits in haploid progeny, (ii) this technique integrates the promoters pOsBBM1, DsRed2 and elements capable of targeted editing of two induction genes (ZmPLA1 + ZmDMP) at the same time into the same vector. This approach facilitates the efficient generation of inducer lines without Cas9 and with the DsRed2 marker through a single genetic transformation step. Furthermore, it improves the breeding efficiency of haploid inducer lines in different maize backgrounds and reduces cost and (iii) the DsRed2 protein exhibits specific expression in the embryo which is visible to the naked eye. This allows for the efficient sorting of haploid at various stages of seed development, which is independent of the genetic background.

We selected a promoter to drive Cas9 expression highly only in rice callus (Figure S1), while the embryo-specific promoter pZmESP was utilized to drive maize codon-optimized DsRed2 (MoDsRed2) expression, supplemented with the CaMV35S enhancer for visible to the naked eye in natural light (Xu et al., 2021). During the experimental process, we observed that the promoter pOsBBM1 activity in the callus tissue exclusively (Figure 1k). Therefore, we proposed to use this vector to generate an inducer line with red fluorescent tags through a single transformation.

Figure 1

Open in figure viewerPowerPoint

A genome editing system for improving the efficiency of breeding inducers and haploid sorting. (a–c) Obtaining transgenic knockout material using a single-gene dual-target vector. (d) Comparison of agronomic traits with KN5585. (e, f) Haploid identification was performed by morphology and flow cytometry. (g) Screening of two genetic markers for different backgrounds of recipient material. (h) Comparison of HIR of different inducer lines. (i) Red fluorescence was observed in excitation light and natural light. (j) Comparison of haploid CDR of different markers. (k) Cas9 expression level in various tissues of maize. (l–n) High-throughput automated seed sorting equipment, including overall appearance, working principle and module components. (o) The technical advantages and potential application scenarios.

Ultimately, ZmPLA1 and ZmDMP single-gene mutations were obtained in KN5585 background (Figure 1b,c). The homozygous events were named KN5585-PLA1 and KN5585-DMP, respectively. Subsequently, we investigated the agronomic traits and haploid induction rate (HIR). The results demonstrated that, with the exception of plant height, no significant differences were observed in the agronomic traits when compared to the wild type (Figure 1d, Figure S2, Table S1).

For HIR evaluation, the hybrid Zhengdan958, inbred lines Zheng58 and Chang7-2 were used as maternal materials for in vivo haploid induction. To ensure the accuracy of haploid sorting, the MoDsRed2, R1-nj and flow cytometry were employed (Figure 1d–f). The results indicated that the HIR for KN5585-PLA1 and KN5585-DMP was 5.37% and 1.71%, respectively (Figure 1h). Furthermore, we also conducted sequencing and comparative analysis of the haploid induction genes in 859 haploid progenies. No mutations were identified in the haploid progenies. Meanwhile, the expression of Cas9 was measured in immature embryos of inducer line, as well as in haploid and diploid immature embryos, and the results demonstrated that Cas9 was ineffective in dividing vigorous tissues (Figure S3).

We next compared the timing differences in haploid identification utilizing MoDsRed2 and R1-nj. Observations commenced 3 days post-pollination, followed by assessments at 4–5 days interval until grain maturity. The anthocyanins were observed to be expressed in the endosperm aleurone layer and embryo at 23–35 and 33 days after pollination, respectively (Figure S4). However, the red fluorescence can be observed under external excitation light, enabling precise identification of haploid embryos from 7 to 11 days after pollination (Figure 1i), with a CDR as high as 100%.

Moreover, at the mature grain stage, under different genetic backgrounds, the haploid CDR exceeded 99% in various materials through the MoDsRed2 marker under both excitation light and natural light conditions (Figure 1g,j). Conversely, the anthocyanin marker was impeded by genetic background variations in tropical germplasm, resulting in an accuracy of only 83.3% for materials from different backgrounds (Table S2).

To enable high throughput for sorting haploid kernels, we evaluated the efficiency of an automatic sorting equipment (Figure 1l). Initially, the equipment employed the R1-nj for sorting and underwent testing on different materials. The results demonstrated that the haploid CDR was 94.9% and a diploid correct rejection rate (CRR) was 98.2% (Table S3). Subsequently, the MoDsRed2 marker was utilized to conduct a sorting test on Zheng58, the results of which demonstrated a haploid CDR of 99.7% and a diploid CRR of 99.8% (Table S4).

In conclusion, we have devised a genetic editing system that accelerates the breeding of inducer lines, which can be utilized in a variety of crops. The naked eye-visible embryo colour marker facilitates rapid and precise sorting of haploid at various stages of seed development, which benefits early embryo identification in tissue culture doubling techniques, and enables the studies on haploid formation mechanisms (Figure 1o). The implementation of automated sorting equipment has resulted in a significant increase in sorting efficiency, from 20 000 kernels per day to 20 000 kernels per hour. The portable fluorescence excitation light equipment was employed in a darkroom screening for sorting haploid, with a haploid CDR of 99.7%. Upon completion of the hardware development, the CDR is expected to reach 100%, with an anticipated sorting efficiency over 24 000 grains per hour.