CRISPR/Cas9‐mediated genomic editing of Brachytic2 creates semi‐dwarf mutant alleles for tailored maize breeding

Maize, now ranking #1 in world cereal production (accounting for ~42% of total cereal production worldwide), plays a pivotal role in securing food and feed supply globally (FAO, 2023). Historically, increasing planting density has been adopted as a key measurement to increasing maize grain yield per unit land area (Mansfield and Mumm, 2014). Plant height (PH) and ear height (EH) are key agronomic traits that determine lodging resistance and thus high-density planting tolerance of maize (Wang et al., 2020). Recently, it has been proposed that "Short corn" may represent a future avenue for maize breeding, as it stands up better to windstorms, boost yields and benefit the environment (Stokstad, 2023). Nevertheless, a major technical thwart in breeding "Short Corn" is the lack of deployable genes and elite germplasm. Brachytic2 (Br2) encodes a protein belonging to the multidrug resistant (MDR) class of P-glycoproteins harbouring two transmembrane domains (TMD1 and TMD2), and two nucleotide-binding domains (NBD1 and NBD2), and plays a role in regulating PH via mediating polar auxin transport (Multani et al., 2003). Despite its loss-of-function mutants exhibit an extremely dwarf stature, several recent studies reported that mild mutations in the last (fifth) exon of Br2 result in milder variation in PH without notable unfavourable effects on other agronomic traits (Wei et al., 2018; Xing et al., 2015). As PH and EH are complex traits regulated by a large number of quantitative loci and easily influenced by genetic backgrounds and environmental conditions, we wondered if it is possible to generate a series of br2 mutant alleles, so as to expand the portfolios of semi-dwarf maize germplasm for tailored breeding of semi-dwarf maize cultivars in different genetic backgrounds. As a proof-of-concept study, we designed a CRISPR/Cas9 vector targeting the last exon of Br2. To increase the targeting efficiency and universality in different genetic backgrounds, we first examined the genetic variation of Br2 in 45 representative inbred lines with reported high-quality genome assembly (https://www.maizegdb.org/), and designed 4 conserved targets, with three of them are completely conserved in all the 45 inbred lines, while Target3 is conserved in 38 of the 45 inbred lines (Figure 1a). The CRISPR/Cas9 knockout vector was used to transform the inbred line ZC01 according to a previously described protocol (Wang et al., 2019). We identified seven independent T1 transgenic lines (named M1 to M7) that harbour different mutations in Br2 and varying degrees of reduction in PH (Figure 1a). The M1 and M2 line had a 1-bp insertion located 48-bp upstream of the stop codon, resulting in amino acid substitution of Alanine (A1402) to Aspartic acid (D) in M1 and A1402 to Valine (V) in M2, respectively. These two lines exhibited modest reduction of PH (decreased to 89.3% and 73.7% of WT, respectively). The M3 to M5, and M7 lines harboured different mutations in the NBD2 domain, while M6 deleted the random coil and α-helix downstream of NBD2 (1372–1402 aa). M3 to M7 exhibited a dwarf phenotype (their PH decreased to 55.6%, 50.2%, 49.3%, 46.6% and 41.0% of WT, respectively). The ear weight and grain yield per plant of M1 and M2 did not notably decrease, while these traits of M3 to M7 significantly decreased (grain yield per plant decreased to 32.6%, 44.1%, 35.4%, 29.8% and 30.8% of WT, respectively, Figure 1b,c). These observations suggest that mutations in the NBD2 domain or large fragment deletions downstream of (but close to) NBD2 will cause more severe phenotypic changes, while mutations in the random coil (1401–1416 aa) near the C-terminus of BR2 likely result in mild PH change with minimal yield penalty. Detailed phenotypic analysis revealed that the reduction in PH of the above maize lines was mainly caused by shortening of the various internodes, while internode numbers (represented by total leaves number) remained largely unaltered in these br2 edited mutant plants. To better demonstrate the effects of different mutation combinations, we crossed M1 with M3 and M7 respectively, and analysed the PH and yield traits of their F1 offsprings in Hainan (18° N, 109° E) in 2023. The results showed that the PH, ear weight and grain yield per plant of the F1 offsprings were between those of the two parents (Figure 1d). To test the effects of Br2 mutations in different genetic backgrounds, we crossed the T1 lines harbouring the Br2-Cas9 cassette (as the male parent) with 28 elite inbred lines, most of them are parental lines of leading hybrids planted in China nowadays, including Zheng58, Chang7-2, Jing724 and Jing92, etc. Interestingly, all of the 28 F1 populations segregated dwarf offsprings. Six dwarf plants from four F1 populations (WIL2, WIL138, Jing724 and Jing92, Figure 1e) were further selected to verify sequence alterations in Br2. PCR-sequencing analysis revealed that all these shortened F1s contained multiallelic mutations in the 5th exon of Br2, and several new mutation types were found. These results proved the efficacy of the Br2-Cas9 cassette in generating additional allele types in different genetic backgrounds, and causing a reduction in PH. Traditional breeding for specific trait improvement is mainly based on repeated backcrossing, which was laborious and time-consuming. Previously, we have developed a Haploid-Inducer Mediated Genome Editing (IMGE) system (Wang et al., 2019), which could generate genome-edited haploids and further double haploids in elite maize backgrounds within two generations (Figure 1f). To rapidly improve the PH of elite maize inbred lines, we introgressed the Br2-Cas9 cassette into the haploid inducer line CAU5 to create an IMGE system for Br2, named CAU5Br2-Cas9. Then, we used the CAU5Br2-Cas9 lines to pollinate three elite inbred lines: WIL2, WIL18 and WW22 (2021 summer, Langfang, 39° N, 116° E). The candidate haploid seeds were planted in the field (2021 winter, Hainan). By phenotypic screening and sequencing analysis, we obtained five br2-haploids in the WIL2 background, two in the WIL18 background and two in the WW22 background (Figure 1h). The edit efficiency of WIL2, WIL18 and WW22 is 1.12% (5/447, edited haploids/total haploids), 0.85% (2/234) and 0.49% (2/404), respectively. Interestingly, three of the edited haploids (one for each background) successfully turned into edited doubled haploids through spontaneous chromosome doubling. All these doubled br2-haploids showed significant reduction in PH (decreased to 41.1%, 71.7% and 68.0% of WT, respectively, Figure 1g), further verifying the efficiency of our strategy. In summary, our results demonstrate the universality and efficacy of combining the Br2-Cas9 cassette with the IMGE technology for generating allele series of Br2 for tailored improvement of PH in different genetic backgrounds to meet customer demands, and thus should greatly facilitating molecular breeding of lodging-resistant maize cultivars adapting to high-density planting. This research was supported by grants from the National Key Research and Development Program of China (2022YFD1201200), the National Natural Science Foundation of China (32272189), the Agricultural Science and Technology Innovation Program (CAAS-CSCB-202403) and Nanfan special project (YBXM2308) of CAAS, a project from HainanYazhou Bay Seed Lab (B23YQ1507), the Natural Science Foundation of Guangdong Province (2022A1515010584) and Provincial College Student Innovation Training Program Project (202312216124). The authors declare no conflict of interests. B.W. and H.W. conceived and designed the project. B.Z. and B.W. conducted the experiments. Z.X., C.S., D.Y., Y.Z., J.C. and Y.L. participated in some experiments. B.Z. and B.W. wrote the manuscript. H.W. revised the manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.