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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]

Soil salinization is a worldwide problem that limits agricultural production. It is important to understand the salt stress tolerance ability of maize seedlings and explore the underlying related genetic resources. In this study, we used a high-throughput phenotyping platform with a 3D laser sensor (Planteye F500) to identify the digital biomass, plant height and normalized vegetation index under normal and saline conditions at multiple time points. The result revealed that a three-leaf period (T3) was identified as the key period for the phenotypic variation in maize seedlings under salt stress. Moreover, we mapped the salt-stress-related SNPs and identified candidate genes in the natural population via a genome-wide association study. A total of 44 candidate genes were annotated, including 26 candidate genes under normal conditions and 18 candidate genes under salt-stressed conditions. This study demonstrates the feasibility of using a high-throughput phenotyping platform to accurately, continuously quantify morphological traits of maize seedlings in different growing environments. And the phenotype and genetic information of this study provided a theoretical basis for the breeding of salt-resistant maize varieties and the study of salt-resistant genes. [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]

Summary: High‐throughput phenotyping is increasingly becoming an important tool for rapid advancement of genetic gain in breeding programmes. Manual phenotyping of vascular bundles is tedious and time‐consuming, which lags behind the rapid development of functional genomics in maize. More robust and automated techniques of phenotyping vascular bundles traits at high‐throughput are urgently needed for large crop populations. In this study, we developed a standard process for stem micro‐CT data acquisition and an automatic CT image process pipeline to obtain vascular bundle traits of stems including geometry‐related, morphology‐related and distribution‐related traits. Next, we analysed the phenotypic variation of stem vascular bundles between natural population subgroup (480 inbred lines) based on 48 comprehensively phenotypic information. Also, the first database for stem micro‐phenotypes, MaizeSPD, was established, storing 554 pieces of basic information of maize inbred lines, 523 pieces of experimental information, 1008 pieces of CT scanning images and processed images, and 24 192 pieces of phenotypic data. Combined with genome‐wide association studies (GWASs), a total of 1562 significant single nucleotide polymorphism (SNPs) were identified for 30 stem micro‐phenotypic traits, and 84 unique genes of 20 traits such as VBNum, VBAvArea and PZVBDensity were detected. Candidate genes identified by GWAS mainly encode enzymes involved in cell wall metabolism, transcription factors, protein kinase and protein related to plant signal transduction and stress response. The results presented here will advance our knowledge about phenotypic trait components of stem vascular bundles and provide useful information for understanding the genetic controls of vascular bundle formation and development. [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]

The vascular bundle in the ear-internode of maize is a key conduit for transporting photosynthetic materials between “source” and “sink”, making it critically important to examine its micro-phenotypes and genetic architecture to identify advantageous characteristics and cultivate high-yielding and high-quality varieties. Unfortunately, the limited observation methods and scope of study precludes any comprehensive and systematic investigations into the microscopic phenotypes and genetic mechanisms of vascular bundle in maize ear-internode. In this study, 47 phenotypic traits were extracted in 495 maize inbred lines using micro computed tomography (Micro-CT) scanning technology and a deep learning-based phenotype acquisition method for stem vascular bundle, which included stem slice-related, epidermis zone-related, periphery zone-related, inner zone-related and vascular bundles-related traits. Phenotypic analysis indicated that there was extensive phenotypic variation of vascular bundle traits in ear-internode, especially that in the inner zone. Of these, 30 phenotypic traits with heritability greater than 0.70 were conducted for GWAS, and a total of 4,225 significant SNPs and 416 candidate genes with detailed functional annotations were identified. Furthermore, 20 genes were highly expressed in stem-related tissues, especially in maize internodes. Functional analysis of candidate genes indicated that the pathways obtained for candidate genes of different trait groups were distinct, mainly involved in vitamin synthesis and metabolism, transport of substances, carbohydrate derivative catabolic process, protein transport and localization, and anatomical structure development. The results of this study will help to further understand the phenotypic traits of stem vascular bundles and provide a reference for revealing the genetic mechanism of maize ear-internode vascular bundles. [ABSTRACT FROM AUTHOR]