Overexpression of ZmKL9 increases maize hybrid hundred kernel weight

Increasing maize yield remains the primary goal in maize breeding in the world. Kernel and ear morphology are two important yield‐determining traits in maize (Simmons et al., 2020). However, maize kernel shape and ear size are complex quantitative traits, for which a few functional genes with potential breeding applications have been reported. Overexpression of vacuolar protein sorting 29 (ZmVPS29) can improve the yield per plant in different maize genetic backgrounds (Chen et al., 2020). Kernel number per row 6 (KNR6) can also increase the grain yield of hybrid maize either through the introduction of useful allelic variants or by raising KNR6 transcript levels (Jia et al., 2020). Moreover, genome editing of 1‐aminocyclopropane‐1‐carboxylate oxidase 2 (ZmACO2) promoted maize ear development, resulting in an ~13.4% increase in grain yield per ear in hybrid lines (Ning et al., 2021). In a previous study, we preliminarily evaluated the potential breeding value of the quantitative trait locus kernel length 9 (qKL9), which controls kernel length and kernel weight in various populations (Gong et al., 2021). We wished to clone the causal gene responsible for the quantitative variation in maize grain yield associated with qKL9 and explore the causal polymorphisms. Our ultimate goal is to develop new functional markers and evaluate the breeding potential of the causal gene behind qKL9 to create new avenues for improving maize yield.

To further fine‐map qKL9, we developed a backcross population of about 20 000 individuals to screen for recombinants. Despite the size of the population, the low recombination rate over the candidate region harbouring qKL9 resulted in an ~736‐kb region flanked by markers IN6706 and N23, corresponding to 19 annotated genes, based on the B73 RefGen v4 genome (Figure 1a, Tables S1 and S2). An analysis of published gene expression data (Yi et al., 2019) and transcriptome deep sequencing (RNA‐seq) data of seed at 6, 9 and 14 DAP (days after pollination) of the maize inbred Mc and the BC₄F₂ line Mc ^qKL9 revealed Zm00001d046718 as the only differentially expressed gene in 6‐DAP seeds between Mc and Mc ^qKL9 . Another 13 genes were expressed at similar levels or at low levels in seeds (Figure 1b and Figure S1). Based on this result, we selected Zm00001d046718, which encodes a basic leucine zipper (bZIP) transcription factor, as the key candidate gene for qKL9, prompting us to name it ZmKL9.

Figure 1.

ZmKL9 regulates maize kernel and ear yield. (a) Fine mapping of qKL9. (b) Expression levels of the candidate genes in 6/9/14–DAP seeds in the inbred Mc and the near‐isogenic line Mc ^qKL9 . (c) Schematic diagram of the ZmKL9 locus, showing all polymorphisms and the transposable elements (TEs, shown as triangles) in the 5′ UTR and the first intron of ZmKL9. TE structures were predicted using CENSOR. (d) Genotyping results of the recombinant lines used for fine mapping for the presence of TE1 and TE2 using specific primers. (e) Local association between 123 SNPs at the ZmKL9 locus and kernel length in a set of 149 diverse maize inbred lines. (f) Distribution of kernel‐related traits in 149 diverse inbred lines as a function of the presence/absence of TE1 at ZmKL9. (g) TE1 decreases transcription from the ZmKL9 promoter in transient transfection assays in leaf protoplasts. (h, i) Kernel and ear traits in OE‐ZmKL9 and WT. (j, k) Kernel and ear traits in KO‐zmkl9 and WT. (l) Phenotypes of kernel and ear traits in F₁ hybrids between OE1‐ZmKL9 and four inbred lines. The values in (h–m) are mean ± SD. Numbers below each column indicate sample size. P‐values were obtained from two‐tailed, two‐sample Student's t‐tests.

To identify the polymorphic sites in ZmKL9, we determined the genomic sequence of ZmKL9 in Mc and Mc ^qKL9 , which revealed single nucleotide polymorphisms (SNPs) and insertion/deletions (InDels) in the promoter and coding region, as well as the insertion of a 288‐ and 557‐bp transposable element (TE) in the 5′ untranslated region (5′ UTR) and the first intron, respectively, named TE1 and TE2, in Mc relative to Mc ^qKL9 (Figure 1c, Table S3). Moreover, we genotyped the recombinants (Figure 1a) for the presence of TE1 and TE2 and established that their presence cosegregates with the kernel length phenotype (Figure 1d). We also sequenced ZmKL9 in a set of 149 diverse maize inbred lines and determined that TE1 is significantly associated with kernel length by association mapping (Figure 1e). Indeed, inbred lines harbouring TE1 (TE+) were characterized by shorter kernels and lower hundred kernel weight (HKW), on average than the lines lacking TE1 (TE–), although kernel width was not affected (Figure 1f). We investigated the possible effect of the presence of TE1 on ZmKL9 expression by performing a transient luciferase (LUC) transcriptional assay in maize protoplasts. Accordingly, we placed the firefly LUC reporter gene under the control of the ZmKL9 promoter from Mc (with TE1) or Mc ^qKL9 (without TE1) and detected lower LUC activity derived from the construct containing TE1 (TE+ construct) relative to the construct lacking the TE (TE− construct; Figure 1g). We concluded that the TE1 in the ZmKL9 5′ UTR is a key polymorphism that influences the transcriptional output of ZmKL9.

To elucidate the biological function of ZmKL9, we generated two overexpression lines (OE‐ZmKL9‐1 and OE‐ZmKL9‐2) and two ZmKL9 knockout lines (CR‐zmkl9‐1 and CR‐zmkl9‐2) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) genome editing (Figures S2 and S3). Compared with their respective nontransgenic control lines, the two ZmKL9 overexpression lines (OE‐ZmKL9‐1 and OE‐ZmKL9‐2) had longer kernels, higher HKW, and longer and heavier ears (Figure 1h,i). Conversely, the two knockout lines CR‐zmkl9‐1 and CR‐zmkl9‐2 produced shorter kernels and ears, and had a lower HKW and ear weight than the corresponding wild‐type lines (Figure 1j,k). These results strongly supported the notion that ZmKL9 is the causal gene underlying qKL9 and positively controls kernel and ear yield in maize.

To evaluate the ability of ZmKL9 to control maize yield in different genetic backgrounds, we crossed one ZmKL9‐overexpressing line and its nontransgenic control line (OE‐ZmKL9‐1 and WT1) to four different elite inbred lines (Chang7‐2, Zheng58, PH6WC, and PH4CV). We confirmed that ZmKL9 expression is higher in F₁ hybrids between each inbred and the OE line relative to the nontransgenic F₁ hybrids. We also established that kernel length and HKW of the F₁ hybrids derived from the OE line displayed greater values, while kernel width was not affected, compared with F₁ hybrids derived from the wild‐type control lines (Figure 1l). Moreover, in addition to kernel‐related traits, multiple ear‐related traits increased significantly in F₁ hybrids derived from the OE lines, including ear length, kernel numbers per ear, and ear weight relative to the F₁ hybrids derived from the wild‐type control line (Figure 1m). We concluded that the overexpression of the ZmKL9 can positively affect the grain and ear yield of maize in different genetic backgrounds.

Here, we report that the cloning of ZmKL9 controls the quantitative variation of kernel and ear length in maize. We demonstrate that the presence of a transposon element in the 5′ UTR of ZmKL9 is an important functional site that may be used as a molecular marker for marker‐assisted selection. Furthermore, we propose that the introduction of a transgene, overexpressing ZmKL9, into different maize inbred lines will have great potential for maize yield improvement. Additional details of the research are described in Appendix S1.

Conflicts of interest

The authors declare no conflicts of interest.

Author contributions

FQ and DG designed the project. DG conducted the experiments. YW, HZ, KL, and QS participated in some experiments and conducted the field trials. DG analysed the data and wrote the manuscript. FQ revised the manuscript.

Supporting information

Appendix S1 Supplemental Methods.

Figure S1 Expression pattern of candidate genes in the qKL9 region.

Figure S2 Overexpression of ZmKL9.

Figure S3 Sequences of two homozygous CRISPR‐Cas9 knockout lines with deletions in the target site (CR‐zmkl9‐1 and CR‐zmkl9‐2).

Table S1 Putative genes in the 736‐kb qKL9 region.

Table S2 Primers for the markers for further fine mapping qKL9.

Table S3 Primers for the markers for the amplification of ZmKL9.

Table S4 Primers for the markers for LUC activity assay.