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The stomatal complex on the surface of maize leaves is closely related to photosynthesis and transpiration, and the study of maize stomatal phenotypes and the discovery of their regulatory genes are of great importance for the breeding of high-quality and high-yielding maize. In this study, rapid scanning electron microscopy was used to obtain images of the abaxial stomata of 457 maize inbred lines with extensive genetic variation, and stomata density was obtained by counting. The results of correlation showed that stomata density was significantly correlated with leaf width, and Analysis of variance found that there were significant differences (P value < 0.05) in stomata density among different leaf width and 100-grain weight. The highest stomata density was found in the inbred lines with wide and short leaves and higher 100-grain weight. Furthermore, genome-wide association analysis was performed using a mixed linear model. It showed that eight SNPs significantly associated with stomata density were obtained, which could explain 35.507% of the phenotypic variation. Among these, four SNPs on chromosome 5 were tightly linked, mainly formatting two haplotypes, CTTA (0.636) and TCCG (0.330). Twelve genes with functional annotation were identified within 100 kb upstream and downstream of the eight SNPs. One gene, GRMZM2G068277, which had been shown to be involved in plant mitotic processes and exhibited high expression at the leaf base, was therefore the most likely candidate gene for stomata density. The results presented here could provide references for further cloning of functional genes related to stomata density. [ABSTRACT FROM AUTHOR]

The rapid development of high-throughput phenotypic detection techniques makes it possible to obtain a large number of crop phenotypic information quickly, efficiently, and accurately. Among them, image-based phenotypic acquisition method has been widely used in crop phenotypic identification and characteristic research due to its characteristics of automation, non-invasive, non-destructive and high throughput. In this study, we proposed a method to define and analyze the traits related to leaf sheaths including morphology-related, color-related and biomass-related traits at V6 stage. Next, we analyzed the phenotypic variation of leaf sheaths of 418 maize inbred lines based on 87 leaf sheath-related phenotypic traits. In order to further analyze the mechanism of leaf sheath phenotype formation, 25 key traits (2 biomass-related, 19 morphology-related and 4 color-related traits) with heritability greater than 0.3 were analyzed by genome-wide association studies (GWAS). And 1816 candidate genes of 17 whole plant leaf sheath traits and 1,297 candidate genes of 8 sixth leaf sheath traits were obtained, respectively. Among them, 46 genes with clear functional descriptions were annotated by single nucleotide polymorphism (SNPs) that both Top1 and multi-method validated. Functional enrichment analysis results showed that candidate genes of leaf sheath traits were enriched into multiple pathways related to cellular component assembly and organization, cell proliferation and epidermal cell differentiation, and response to hunger, nutrition and extracellular stimulation. The results presented here are helpful to further understand phenotypic traits of maize leaf sheath and provide a reference for revealing the genetic mechanism of maize leaf sheath phenotype formation. [ABSTRACT FROM AUTHOR]

Summary: Salt stress is a major devastating abiotic factor that affects the yield and quality of maize. However, knowledge of the molecular mechanisms of the responses to salt stress in maize is limited. To elucidate the genetic basis of salt tolerance traits, a genome‐wide association study was performed on 348 maize inbred lines under normal and salt stress conditions using 557 894 single nucleotide polymorphisms (SNPs). The phenotypic data for 27 traits revealed coefficients of variation of >25%. In total, 149 significant SNPs explaining 6.6%–11.2% of the phenotypic variation for each SNP were identified. Of the 104 identified quantitative trait loci (QTLs), 83 were related to salt tolerance and 21 to normal traits. Additionally, 13 QTLs were associated with two to five traits. Eleven and six QTLs controlling salt tolerance traits and normal root growth, respectively, co‐localized with QTL intervals reported previously. Based on functional annotations, 13 candidate genes were predicted. Expression levels analysis of 12 candidate genes revealed that they were all responsive to salt stress. The CRISPR/Cas9 technology targeting three sites was applied in maize, and its editing efficiency reached 70%. By comparing the biomass of three CRISPR/Cas9 mutants of ZmCLCg and one zmpmp3 EMS mutant with their wild‐type plants under salt stress, the salt tolerance function of candidate genes ZmCLCg and ZmPMP3 were confirmed. Chloride content analysis revealed that ZmCLCg regulated chloride transport under sodium chloride stress. These results help to explain genetic variations in salt tolerance and provide novel loci for generating salt‐tolerant maize lines. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) accumulation in maize grains is detrimental to human health. Developing maize varieties with low Cd content is important for safe consumption of maize grains. However, the key genes controlling maize grain Cd accumulation have not been cloned. Here, we identified one major locus for maize grain Cd accumulation (qCd1) using a genome-wide association study (GWAS) and bulked segregant RNA-seq analysis with a biparental segregating population of Jing724 (low-Cd line) and Mo17 (high-Cd line). The candidate gene ZmHMA3 was identified by fine mapping and encodes a tonoplast-localized heavy metal P-type ATPase transporter. An ethyl methane sulfonate mutant analysis and an allelism test confirmed that ZmHMA3 influences maize grain Cd accumulation. A transposon in intron 1 of ZmHMA3 is responsible for the abnormal amino acid sequence in Mo17. Based on the natural sequence variations in the ZmHMA3 gene of diverse maize lines, four PCR-based molecular markers were developed, and these were successfully used to distinguish five haplotypes with different grain Cd contents in the GWAS panel and to predict grain Cd contents of widely used maize inbred lines and hybrids. These molecular markers can be used to breed elite maize varieties with low grain Cd contents. [ABSTRACT FROM AUTHOR]

Dry matter accumulation and partitioning during the early phases of development could significantly affect crop growth and productivity. In this study, the aboveground dry matter (DM), the DM of different organs, and partition coefficients of a maize association mapping panel of 412 inbred lines were evaluated at the third and sixth leaf stages (V3 and V6). Further, the properties of these phenotypic traits were analyzed. Genome-wide association studies (GWAS) were conducted on the total aboveground biomass and the DM of different organs. Analysis of GWAS results identified a total of 1,103 unique candidate genes annotated by 678 significant SNPs (P value < 1.28e–6). A total of 224 genes annotated by SNPs at the top five of each GWAS method and detected by multiple GWAS methods were regarded as having high reliability. Pathway enrichment analysis was also performed to explore the biological significance and functions of these candidate genes. Several biological pathways related to the regulation of seed growth, gibberellin-mediated signaling pathway, and long-day photoperiodism were enriched. The results of our study could provide new perspectives on breeding high-yielding maize varieties. [ABSTRACT FROM AUTHOR]

Southern corn rust (SCR) caused by Puccinia polysora Underw. is a major disease causing severe yield losses during maize production. Here, we identified and mapped the SCR resistance gene RppM from the near-isogenic line Kangxiujing2416 (Jing2416K), which harbors RppM in the genetic background of the susceptible inbred line Jing2416. In this study, the inheritance of SCR resistance was investigated in F2 and F3 populations derived from a cross between Jing2416K and Jing2416. The observed 3:1 segregation ratio of resistant to susceptible plants indicated that the SCR resistance is controlled by a single dominant gene. Using an F2 population, we performed bulked segregant analysis (BSA) sequencing and mapped RppM to a 3.69-Mb region on chromosome arm 10S. To further narrow down the region harboring RppM , we developed 13 insertion/deletion (InDel) markers based on the sequencing data. Finally, RppM was mapped to a region spanning 110-kb using susceptible individuals from a large F2 population. Two genes (Zm00001d023265 and Zm00001d023267) encoding putative CC-NBS-LRR (coiled-coiled, nucleotide-binding site, and leucine-rich repeat) proteins, a common characteristic of R genes, were located in this region (B73 RefGen_v4 reference genome). Sequencing and comparison of the two genes cloned from Jing2416K and Jing2416 revealed sequence variations in their coding regions. The relative expression levels of these two genes in Jing2416K were found to be significantly higher than those in Jing2416. Zm00001d023265 and Zm00001d023267 are thus potential RppM candidates. [ABSTRACT FROM AUTHOR]

In maize, high grain moisture ( GM) at harvest causes problems in harvesting, threshing, artificial drying, storage, transportation and processing. Understanding the genetic basis of GM will be useful for breeding low- GM varieties. A quantitative genetics approach was used to identify quantitative trait loci ( QTL) related to GM at harvest in field-grown maize. The GM of a double haploid population consisting of 240 lines derived from Xianyu335 was evaluated in three planting seasons and a high-density genetic linkage map covering 1546.4 cM was constructed. The broad-sense heritability of GM at harvest was 71.0%. Using composite interval mapping, six QTL for GM at harvest were identified on five chromosomes (Chr). Two QTL located on Chr1, qgm1-1 and qgm1-2, explained 5.0% and 10.8% of the phenotypic variation in GM at harvest, respectively. The QTL qgm2, qgm3, qgm4 and qgm5 accounted for 3.3%, 8.3%, 5.4% and 11.0% of the mean phenotypic variation, respectively. Because of their consistent detection over multiple planting seasons, the detected QTL appear to be robust and reliable for the breeding of low- GM varieties. [ABSTRACT FROM AUTHOR]

S-type cytoplasmic male sterility (CMS-S) is the largest group among the three major types of CMS in maize. CMS-S exhibits fertility instability as a partial fertility restoration in a specific nuclear genetic background, which impedes its commercial application in hybrid breeding programs. The fertility instability phenomenon of CMS-S is controlled by several minor quantitative trait locus (QTLs), but not the major nuclear fertility restorer (Rf3). However, the gene mapping of these minor QTLs and the molecular mechanism of the genetic modifications are still unclear. Using completely sterile and partially rescued plants of fertility instable line (FIL)-B, we performed bulk segregant RNA-Seq and identified six potential associated genes in minor effect QTLs contributing to fertility instability. Analyses demonstrate that these potential associated genes may be involved in biological processes, such as floral organ differentiation and development regulation, energy metabolism and carbohydrates biosynthesis, which results in a partial anther exsertion and pollen fertility restoration in the partially rescued plants. The single nucleotide polymorphisms (SNPs) identified in two potential associated genes were validated to be related to the fertility restoration phenotype by KASP marker assays. This novel knowledge contributes to the understanding of the molecular mechanism of the partial fertility restoration of CMS-S in maize and thus helps to guide the breeding programs. [ABSTRACT FROM AUTHOR]

The RNA-binding glycine-rich protein (RB-GRP) family is characterized by the presence of a glycine-rich domain arranged in (Gly)n-X repeats and an RNA-recognition motif (RRM). RB-GRPs participate in varied physiological and biochemical processes especially in the stress response of plants. In this study, a total of 23 RB-GRPs distributed on 10 chromosomes were identified in maize ( Zea mays L.), and they were divided into four subgroups according to their conserved domain architecture. Five pairs of paralogs were identified, while none of them was located on the same chromosomal region, suggesting that segmental duplication is predominant in the duplication events of the RB-GRPs in maize. Comparative analysis of RB-GRPs in maize, Arabidopsis ( Arabidopsis thaliana L.), rice ( Oryza sativa L.), and wheat ( Triticum aestivum) revealed that two exclusive subgroups were only identified in maize. Expression of eight ZmRB-GRPs was significantly regulated by at least two kinds of stresses. In addition, cis-elements predicted in the promoter regions of the ZmRB-GRPs also indicated that these ZmRB-GRPs would be involved in stress response of maize. The preliminary genome-wide analysis of the RB- GRPs in maize would provide useful information for further study on the function of the ZmRB-GRPs. [ABSTRACT FROM AUTHOR]

• An automated maize plant type 3D phenotype analysis system has been developed. • 44 traits of maize plant architecture were defined and extracted. • 40 significant SNP/QTLs were identified for the 17 traits. • ConvexHull trait may be used as potential indicators for plant architecture. Maize (Zea mays) is one of the world's most important crops, and its abundant and stable yield is crucial for ensuring global food security. Optimizing maize plant architecture can effectively enhance canopy structure, and ensure an ample supply of assimilates, thereby constituting a crucial strategy for achieving high yields in high-density planting systems. In this study, we used the phenotyping platform MVS-Pheno to synchronously collect multi-view image data of plant architecture, and the 3D phenotype analysis algorithm was developed to batch and automatically extract traits of spatial geometric structure. Using this phenotypic acquisition and analysis platform, 44 traits of maize plant architecture were defined and extracted, including 6 categories: basic phenotype, projection area related phenotype, leaf related phenotype, plant architecture dispersion related phenotype, volume related phenotype, and color related phenotype. Based on abundant phenotypic traits of plant architecture, we analyzed the phenotypic variations among a group of 495 inbred lines and further conducted GWAS to reveal the genetic components of the plant architecture. In summary, our work demonstrates valuable advances in high-throughput identification of qualitative traits for plant architecture, which could have major implications for improving high-density tolerant maize breeding and production. [ABSTRACT FROM AUTHOR]

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5 , ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding. • We are the first to genome-widely identify monovalent cation-proton antiporter (CPA) superfamily in maize. • The 35 CPA genes include 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs) and 6 K+ efflux antiporters (ZmKEAs). • Yeast complementation experiments and maize mutant analysis validated the salt tolerance functions of ZmNHX5 and ZmKEA2. [ABSTRACT FROM AUTHOR]