The performance of ear length in different inbred-hybrid triplets
Ear-length heterosis in maize is a very striking phenomenon resulting from a cross of two distinct inbred lines. To explore ear-length heterosis, we selected two specific inbred lines (T121 and T126) with long and short ears, respectively. Additionally, two other inbred lines (PH4CV and PH6WC) were used to form a joint netted pattern (Fig. S1) that included six parent-hybrid triplets to adequately analyze ear-length heterosis. During maize ear differentiation, the young ear gradually elongates and becomes visible. Moreover, the elongation capability of the growth cone determines the final ear length to some extent. Here, we compared the morphologies of young ears of hybrids and their inbred parents at the 13-leaf stage when the young ears were initially apparent. The young ears of the T121 line were longer than those of the T126 line. Moreover, the F1 hybrids generated by T121 crosses (T121 × PH4CV and T121 × PH6WC) had longer young ears than those generated by corresponding T126 crosses (T126 × PH4CV and T126 × PH6WC) (Table 1, Fig. 1a). In addition, the lengths of young ears from each inbred parent were less than those of their F1 hybrids (Table 1, Fig. 1a), indicating that ear-length heterosis had already emerged.
At the maturation stage, we measured the final ear lengths of all the lines (inbred and hybrid). The T121 line, a long-ear inbred line, had an ear length that reached 19.52 ± 1.18 cm and was much longer than that of the T126 (13.52 ± 0.83 cm) line (Table 1, Fig. 1b). The six F1 hybrids, T121 × PH4CV, T121 × PH6WC, T126 × PH4CV, T126 × PH6WC, T121 × T126 and PH6WC × PH4CV, exhibited MP heterosis for ear length (Table 1). Interestingly, the hybrids produced by the T121 line (long ear), T121 × PH4CV and T121 × PH6WC, had longer ears than those produced by the short ear line T126 (Fig. 1b). However, for the MP heterosis, there was no significant difference between the other corresponding hybrids, such as T121 × PH4CV vs T126 × PH4CV (Table 1). These data indicate that the T121 line makes a superior contribution to ear length than the T126 line, but this is not a result of ear-length heterosis.
Transcriptome profiles of maize young ears among four inbred parents and six F1 hybrids
To understand the comprehensive transcriptional regulation of maize ear-length heterosis, young ears of four inbred parents and six F1 hybrids were used to perform an RNA-sequencing (RNA-seq) analysis at the 13-leaf stage. In total, 20 libraries (10 × 2) were constructed for deep Illumina NovaSeq 150-bp paired-end sequencing. When the sequencing was completed, 332,543,189 raw reads were generated, ranging from 13.87 million to 21.76 million per library (Table 2). After filtering, 320,828,384 clean reads, accounting for 96.48% of the total, were maintained (Table 2). Based on the B73 maize reference genome (Version 4), the average unique mapping rate was 85.47%, with a range from 79.03% to 87.93% (Table 2). Moreover, the two biological replicates were in close agreement (Fig. S2). Finally, 25,199 unique genes were identified that had specific expression levels in all the lines (Table S1). The RNA-seq data is available for further analyses of transcriptional regulation.
Global transcriptome changes from inbred parents to their hybrids
Variation in gene expression is closely associated with phenotypic diversity. Thus, a series of transcriptional changes should occur from two inbred parents to one hybrid. For the T121–T126–T121 × T126 triplet, 64.97% of the genes in T121 × T126 hybrid kept their expression levels within the parental range, whereas the expression levels of the remaining genes (35.03%) were out of this rang (Table 3). This data indicated that the hybrid had the sufficient potential to surpass the two parents. Using a differential expression analysis, 5,027 DEGs were identified between T121 and T126, 2,547 DEGs were identified between T121 × T126 and T121, and 2,431 DEGs were identified between T121 × T126 and T126 (Fig. 2a; Table S2). Thus, the number of DEGs between a hybrid and one parent (T121 or T126) was less than that between the two parents. Moreover, similar scenarios, including the ranges of the gene expression levels and the numbers of DEGs, were found in other parent-hybrid triplets (Table 3; Fig. 2a; Table S2). Thus, some transcriptional regulatory mechanisms appeared to be universal and common in the production of hybrids from inbred parents.
In F1 hybrids, there are two gene expression patterns: additive and non-additive. We performed a non-additive expression analysis for the six parent-hybrid triplets. In total, 1,375, 1,349, 638, 566, 712 and 1,257 non-additive genes were identified for T121–PH4CV–T121 × PH4CV, T121–PH6WC–T121 × PH6WC, T121–T126–T121 × T126, T126–PH4CV–T126 × PH4CV, T126–PH6WC–T126 × PH6WC and PH4CV–PH6WC–PH4CV × PH6WC, respectively (Fig. 2b; Table S3). A small proportion (< 6%) of non-additive patterns appeared in all the F1 hybrids (Fig. 2b), indicating that most genes displayed an additive pattern in the F1 hybrids. The additive pattern represents the expected MP level, whereas the non-additive pattern significantly deviates from the MP level. Thus, the non-additive genes in each triplet may have contributed to ear-length heterosis.
In hybrids, ASE frequently exists, increasing the plasticity of gene expression governed by diverse alleles from the two parents, and this may be the reason that non-additive patterns appear in F1 hybrids. Thus, we analyzed genes having ASE in all the parent-hybrid triplets and then compared them with non-additively expressed genes identified in the same triplet. Quite a number of genes having ASE were detected in the hybrids (Table S4). However, few of them were non-additively expressed (Fig. 2c–h). For instance, in the T121 × PH4CV hybrid, 1,702 genes having ASE were identified, but only 71 exhibited non-additive expression patterns (Fig. 2c). These results indicated that in F1 hybrids, ASE might have a limited contribution to the production of non-additive expression-related variation.
The major genes responsible for the ear-length variation between T121 and T126 lines
The T121 line produces longer ears than the T126 line at the 13-leaf stage. A transcriptional level analysis of young ears revealed a large number of DEGs (5,027) between the T121 and T126 lines. However, it was difficult to determine the major genes responsible for the ear-length variation. Nevertheless, compared with the T126 line, the T121 had a longer ear and might pass this advantage to its F1 hybrid. When T121 and T126 were hybridized with the other parents (PH4CV and PH6WC), the former produced F1 hybrids with longer ears compared with the latter. Consequently, we performed a differential expression analysis between the corresponding F1 hybrids, T121 × PH4CV vs T126 × PH4CV and T121 × PH6WC vs T126 × PH6WC (Table S2). In total, 890 DEGs were found to overlap between the two groups (Fig. 3a). We compared these overlapped genes with the DEGs identified between lines T121 and T126. As expected, they shared many common genes (874) (Fig. 3b), which suggested that these genes take part in the regulation of ear elongation and are mainly responsible for the ear-length variation between the T121 and T126 lines.
A gene ontology (GO) enrichment analysis was performed to identify some major terms related to ear length, as well as the key genes implicated in ear-length heterosis. A total of 1,672 GO terms were enriched for these genes in biological process (Table S5). Furthermore, the top 10 GO terms were investigated, and they revealed several terms related to development, such as GO:0048582 (regulation of post-embryonic development) and GO:0048831 (regulation of shoot system development) (Fig. 3c; Table S5). Among these terms, four genes (Fig. 3d), Zm00001d027359 (FUSCA homolog, FUS6), Zm00001d048502 (COP9 signalosome complex subunit 1, CNS1), Zm00001d052138 (E3 ubiquitin-protein ligase, COP1) and Zm00001d049958 (WD40 repeat domain family protein, CYP71), were found to also belong to GO:0048507 (meristem development), and they may make major contributions to ear-length variation.
Non-additively expressed genes contributing to ear-length heterosis
Non-additively expressed genes may be potential sources of heterosis [31]. To identify promising potential genes that contribute to ear-length heterosis derived from the T121 (or T126) line, we made multiple comparisons of non-additively expressed genes in these parent-hybrid triplets. For the T121 line, 47 non-additively expressed genes overlapped among hybrids produced by T121 × PH4CV, T121 × PH6WC and T121 × T126 (Fig. 4a). Whereas, for the T126 line, 50 common non-additively expressed genes were identified among hybrids produced by T126 × PH4CV, T126 × PH6WC and T121 × T126 (Fig. 4b). These genes should be involved in ear-length heterosis, because the ear lengths of all these F1 hybrids surpassed the MP values. Moreover, 19 genes were shared (Fig. 4c, d), and these genes displayed non-additive expression patterns in all the hybrids, suggesting that they had the potential to contribute to ear-length heterosis. The GO enrichment analysis revealed that the top 10 GO terms for the T121 and T126 lines were highly similar (Fig. 4e, f; Table S6, 7), suggesting that there are some common components of the mechanism underlying ear-length heterosis. Among the common GO terms, GO:0048506 (regulation of timing of meristematic phase transition) and GO:0048510 (regulation of timing of transition from vegetative to reproductive phase) were associated with meristem, and a shared gene, Zm00001d050649 (ZCN2), may be responsible for ear-length heterosis.
Validation of candidate gene expression by quantitative real-time PCR
The application of RNA-seq technology has greatly enhanced the global understanding of transcriptional regulatory networks. To verify the accuracy of the RNA-seq analysis, we performed a quantitative real-time PCR (qRT-PCR) analysis of five candidate genes, including four DEGs having genetic effects on ear length, Zm00001d027359, Zm00001d048502, Zm00001d052138 and Zm00001d049958, and one non-additively expressed gene having heterotic effects on ear length, Zm00001d050649. Primers were designed to specifically amplify each of the five genes (Table S8). These primers were used to conduct qRT-PCR on three biological replications of RNA from re-prepared samples. All the assayed genes showed expression patterns similar to those determined by RNA-seq (Fig. 5), verifying the reliability of our RNA-seq analysis.