ZmELF3.1 integrates the RA2-TSH4 module to repress maize tassel branching.

Tassel branch number (TBN) is a key agronomic trait for adapting to high-density planting and grain yield in maize. However, the molecular regulatory mechanisms underlying tassel branching are still largely unknown. Here, we used molecular and genetic studies together to show that ZmELF3.1 plays a critical role in regulating TBN in maize. Previous studies showed that ZmELF3.1 forms the evening complex through interacting with ZmELF4 and ZmLUX to regulate flowering in maize and that RA2 and TSH4 (ZmSBP2) suppresses and promotes TBN in maize, respectively. In this study, we show that loss-of-function mutants of ZmELF3.1 exhibit a significant increase of TBN. We also show that RA2 directly binds to the promoter of TSH4 and represses its expression, thus leading to reduced TBN. We further demonstrate that ZmELF3.1 directly interacts with both RA2 and ZmELF4.2 to form tri-protein complexes that further enhance the binding of RA2 to the promoter of TSH4, leading to suppressed TSH4 expression and consequently decreased TBN. Our combined results establish a novel functional link between the ELF3-ELF4-RA2 complex and miR156-SPL regulatory module in regulating tassel branching and provide a valuable target for genetic improvement of tassel branching in maize.

Deploying QTL-seq rapid identification and separation of the major QTLs of tassel branch number for fine-mapping in advanced maize populations.

The tassel competes with the ear for nutrients and shields the upper leaves, thereby reducing the yield of grain. The tassel branch number (TBN) is a pivotal determinant of tassel size, wherein the reduced TBN has the potential to enhance the transmission of light and reduce the consumption of nutrients, which should ultimately result in increased yield. Consequently, the TBN has emerged as a vital target trait in contemporary breeding programs that focus on compact maize varieties. In this study, QTL-seq technology and advanced population mapping were used to rapidly identify and dissect the major effects of the TBN on QTL. Advanced mapping populations (BC4F2 and BC4F3) were derived from the inbred lines 18-599 (8-11 TBN) and 3237 (0-1 TBN) through phenotypic recurrent selection. First, 13 genomic regions associated with the TBN were detected using quantitative trait locus (QTL)-seq and were located on chromosomes 2 and 5. Subsequently, validated loci within these regions were identified by QTL-seq. Three QTLs for TBN were identified in the BC4F2 populations by traditional QTL mapping, with each QTL explaining the phenotypic variation of 6.13-18.17%. In addition, for the major QTL (qTBN2-2 and qTBN5-1), residual heterozygous lines (RHLs) were developed from the BC4F2 population. These two major QTLs were verified in the RHLs by QTL mapping, with the phenotypic variation explained (PVE) of 21.57% and 30.75%, respectively. Near-isogenic lines (NILs) of qTBN2-2 and qTBN5-1 were constructed. There were significant differences between the NILs in TBN. These results will enhance our understanding of the genetic basis of TBN and provide a solid foundation for the fine-mapping of TBN. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01431-y.

Genetic mapping and functional analysis of a classical tassel branch number mutant Tp2 in maize.

Tassel branch number is a key trait that contributes greatly to grain yield in maize (Zea mays). We obtained a classical mutant from maize genetics cooperation stock center, Teopod2 (Tp2), which exhibits severely decreased tassel branch. We conducted a comprehensive study, including phenotypic investigation, genetic mapping, transcriptome analysis, overexpression and CRISPR knock-out, and tsCUT&Tag of Tp2 gene for the molecular dissection of Tp2 mutant. Phenotypic investigation showed that it is a pleiotropic dominant mutant, which is mapped to an interval of approximately 139-kb on Chromosome 10 harboring two genes Zm00001d025786 and zma-miR156h. Transcriptome analysis showed that the relative expression level of zma-miR156h was significantly increased in mutants. Meanwhile, overexpression of zma-miR156h and knockout materials of ZmSBP13 exhibited significantly decreased tassel branch number, a similar phenotype with Tp2 mutant, suggesting that zma-miR156h is the causal gene of Tp2 and targets ZmSBP13 gene. Besides, the potential downstream genes of ZmSBP13 were uncovered and showed that it may target multiple proteins to regulate inflorescence structure. Overall, we characterized and cloned Tp2 mutant, and proposed a zma-miR156h-ZmSBP13 model functioning in regulating tassel branch development in maize, which is an essential measure to satisfy the increasing demands of cereals.

Genome-Wide Association Study and Genomic Prediction on Plant Architecture Traits in Sweet Corn and Waxy Corn.

Sweet corn and waxy corn has a better taste and higher accumulated nutritional value than regular maize, and is widely planted and popularly consumed throughout the world. Plant height (PH), ear height (EH), and tassel branch number (TBN) are key plant architecture traits, which play an important role in improving grain yield in maize. In this study, a genome-wide association study (GWAS) and genomic prediction analysis were conducted on plant architecture traits of PH, EH, and TBN in a fresh edible maize population consisting of 190 sweet corn inbred lines and 287 waxy corn inbred lines. Phenotypic data from two locations showed high heritability for all three traits, with significant differences observed between sweet corn and waxy corn for both PH and EH. The differences between the three subgroups of sweet corn were not obvious for all three traits. Population structure and PCA analysis results divided the whole population into three subgroups, i.e., sweet corn, waxy corn, and the subgroup mixed with sweet and waxy corn. Analysis of GWAS was conducted with 278,592 SNPs obtained from resequencing data; 184, 45, and 68 significantly associated SNPs were detected for PH, EH, and TBN, respectively. The phenotypic variance explained (PVE) values of these significant SNPs ranged from 3.50% to 7.0%. The results of this study lay the foundation for further understanding the genetic basis of plant architecture traits in sweet corn and waxy corn. Genomic selection (GS) is a new approach for improving quantitative traits in large plant breeding populations that uses whole-genome molecular markers. The marker number and marker quality are essential for the application of GS in maize breeding. GWAS can choose the most related markers with the traits, so it can be used to improve the predictive accuracy of GS.

Co-expression pan-network reveals genes involved in complex traits within maize pan-genome.

BACKGROUND: With the advances in the high throughput next generation sequencing technologies, genome-wide association studies (GWAS) have identified a large set of variants associated with complex phenotypic traits at a very fine scale. Despite the progress in GWAS, identification of genotype-phenotype relationship remains challenging in maize due to its nature with dozens of variants controlling the same trait. As the causal variations results in the change in expression, gene expression analyses carry a pivotal role in unraveling the transcriptional regulatory mechanisms behind the phenotypes. RESULTS: To address these challenges, we incorporated the gene expression and GWAS-driven traits to extend the knowledge of genotype-phenotype relationships and transcriptional regulatory mechanisms behind the phenotypes. We constructed a large collection of gene co-expression networks and identified more than 2 million co-expressing gene pairs in the GWAS-driven pan-network which contains all the gene-pairs in individual genomes of the nested association mapping (NAM) population. We defined four sub-categories for the pan-network: (1) core-network contains the highest represented ~ 1% of the gene-pairs, (2) near-core network contains the next highest represented 1-5% of the gene-pairs, (3) private-network contains ~ 50% of the gene pairs that are unique to individual genomes, and (4) the dispensable-network contains the remaining 50-95% of the gene-pairs in the maize pan-genome. Strikingly, the private-network contained almost all the genes in the pan-network but lacked half of the interactions. We performed gene ontology (GO) enrichment analysis for the pan-, core-, and private- networks and compared the contributions of variants overlapping with genes and promoters to the GWAS-driven pan-network. CONCLUSIONS: Gene co-expression networks revealed meaningful information about groups of co-regulated genes that play a central role in regulatory processes. Pan-network approach enabled us to visualize the global view of the gene regulatory network for the studied system that could not be well inferred by the core-network alone.

Genetic Variation in ZmPAT7 Contributes to Tassel Branch Number in Maize.

Tassel branch number (TBN) is one of the important agronomic traits that contribute to the efficiency of seed production and has been selected strongly during the modern maize breeding process. However, the genetic mechanisms of TBN in maize are not entirely clear. In this study, we used a B73 × CML247 recombination inbred lines (RILs) population to detect quantitative trait loci (QTLs) for TBN. A total of four QTLs (qTBN2a, qTBN2b, qTBN4, and qTBN6) and six candidate genes were identified through expression analysis. Further, one of the candidates (GRMZM2G010011, ZmPAT7) encoding an S-acyltransferase was selected to validate its function by CRISPR-Cas9 technology, and its loss-of-function lines showed a significant increase in TBN. A key SNP(-101) variation in the promoter of ZmPAT7 was significantly associated with TBN. A total of 17 distant eQTLs associated with the expression of ZmPAT7 were identified in expression quantitative trait loci (eQTL) analysis, and ZmNAC3 may be a major factor involved in regulating ZmPAT7. These findings of the present study promote our understanding of the genetic basis of tassel architecture and provide new gene resources for maize breeding improvement.

QDtbn1 , an F-box gene affecting maize tassel branch number by a dominant model.

Tassel branch number (TBN) is one of the important agronomic traits that directly contribute to grain yield in maize (Zea mays L.), and identification of genes precisely regulating TBN in the parental lines is important for maize hybrid breeding. In this study, a quantitative trait nucleotide (QTN), QDtbn1 , related to tassel branch number was identified using a testcrossing association mapping population through association mapping with the Indels/SNPs in the 5'-UTR (untranslated region) of Zm00001d053358, which encodes a Kelch repeat-containing F-box protein. QDtbn1 was further confirmed to be associated with TBN by a dominant model using an F2 population, and over-expressing of the candidate gene resulted in a decreasing of TBN, implying that QDtbn1 was governed by the candidate gene with a negative model. This makes QDtbn1 very useful in maize hybrid breeding. QDtbn1 could interact with a maize Skp1-like protein and a SnRK1 protein, and the SnRK1 could also interact with a SnRK2.8 protein. In addition, quantitative real-time PCR assay showed that five substrates of SnRK2 were down-regulated in the over-expressed plants. These imply that the SCF (Skp1/Cul1/F-box protein/Roc1) complex and ABA signal pathway might be involved in the modulation of TBN in maize.

Combination of multi-locus genome-wide association study and QTL mapping reveals genetic basis of tassel architecture in maize.

Maize tassel architecture is a complex quantitative trait that is significantly correlated with biomass yield and grain yield. The present study evaluated the major trait of maize tassel architecture, namely, tassel branch number (TBN), in an association population of 359 inbred lines and an IBM Syn 10 population of 273 doubled haploid lines across three environments. Approximately 43,958 high-quality single nucleotide polymorphisms were utilized to detect significant QTNs associated with TBN based on new multi-locus genome-wide association study methods. There were 30, 38, 73, 40, 47, and 53 QTNs associated with tassel architecture that were detected using the FastmrEMMA, FastmrMLM, EM-BLASSO, mrMLM, pkWMEB, and pLARmEB models, respectively. Among these QTNs, 51 were co-identified by at least two of these methods. In addition, 12 QTNs were consistently detected across multiple environments. Furthermore, 19 QTLs distributed on chromosomes 1, 2, 3, 4, 6, and 7 were detected in 3 environments and the BLUP model based on 6618 bin markers, which explained 3.64-10.96% of the observed phenotypic variations in TBN. Of these, three QTLs were co-detected in two environments. One QTN associated with TBN was localized to one QTL. Approximately 55 candidate genes were detected by common QTNs and LD criteria. One candidate gene, Zm00001d016615, was identified as a putative target of the RA1 gene. Meanwhile, RA1 was previously validated to plays an important role in tassel development. In addition, the newly identified candidate genes Zm00001d003939, Zm00001d030212, Zm00001d011189, and Zm00001d042794 have been reported to involve in a spikelet meristem identity module. The findings of the present study improve our understanding of the genetic basis of tassel architecture in maize.