Identification of anther thermotolerance genes by the integration of linkage and association analysis in maize.

Maize is one of the world's most important staple crops, yet its production is increasingly threatened by the rising frequency of high-temperature stress (HTS). To investigate the genetic basis of anther thermotolerance under field conditions, we performed linkage and association analysis to identify HTS response quantitative trait loci (QTL) using three recombinant inbred line (RIL) populations and an association panel containing 375 diverse maize inbred lines. These analyses resulted in the identification of 16 co-located large QTL intervals. Among the 37 candidate genes identified in these QTL intervals, five have rice or Arabidopsis homologs known to influence pollen and filament development. Notably, one of the candidate genes, ZmDUP707, has been subject to selection pressure during breeding. Its expression is suppressed by HTS, leading to pollen abortion and barren seeds. We also identified several additional candidate genes potentially underly QTL previously reported by other researchers. Taken together, our results provide a pool of valuable candidate genes that could be employed by future breeding programs aiming at enhancing maize HTS tolerance.

A genome-wide association study identifies genes associated with cuticular wax metabolism in maize.

The plant cuticle is essential in plant defense against biotic and abiotic stresses. To systematically elucidate the genetic architecture of maize (Zea mays L.) cuticular wax metabolism, 2 cuticular wax-related traits, the chlorophyll extraction rate (CER) and water loss rate (WLR) of 389 maize inbred lines, were investigated and a genome-wide association study (GWAS) was performed using 1.25 million single nucleotide polymorphisms (SNPs). In total, 57 nonredundant quantitative trait loci (QTL) explaining 5.57% to 15.07% of the phenotypic variation for each QTL were identified. These QTLs contained 183 genes, among which 21 strong candidates were identified based on functional annotations and previous publications. Remarkably, 3 candidate genes that express differentially during cuticle development encode ß-ketoacyl-CoA synthase (KCS). While ZmKCS19 was known to be involved in cuticle wax metabolism, ZmKCS12 and ZmKCS3 functions were not reported. The association between ZmKCS12 and WLR was confirmed by resequencing 106 inbred lines, and the variation of WLR was significant between different haplotypes of ZmKCS12. In this study, the loss-of-function mutant of ZmKCS12 exhibited wrinkled leaf morphology, altered wax crystal morphology, and decreased C32 wax monomer levels, causing an increased WLR and sensitivity to drought. These results confirm that ZmKCS12 plays a vital role in maize C32 wax monomer synthesis and is critical for drought tolerance. In sum, through GWAS of 2 cuticular wax-associated traits, this study reveals comprehensively the genetic architecture in maize cuticular wax metabolism and provides a valuable reference for the genetic improvement of stress tolerance in maize.

The DEAD-box helicase RCF1 plays roles in miRNA biogenesis and RNA splicing in Arabidopsis.

RCF1 is a highly conserved DEAD-box RNA helicase found in yeast, plants, and mammals. Studies about the functions of RCF1 in plants are limited. Here, we uncovered the functions of RCF1 in Arabidopsis thaliana as a player in pri-miRNA processing and splicing, as well as in pre-mRNA splicing. A mutant with miRNA biogenesis defects was isolated, and the defect was traced to a recessive point mutation in RCF1 (rcf1-4). We show that RCF1 promotes D-body formation and facilitates the interaction between pri-miRNAs and HYL1. Finally, we show that intron-containing pri-miRNAs and pre-mRNAs exhibit a global splicing defect in rcf1-4. Together, this work uncovers roles for RCF1 in miRNA biogenesis and RNA splicing in Arabidopsis.

A P-type pentatricopeptide repeat protein ZmRF5 promotes 5' region partial cleavages of atp6c transcripts to restore the fertility of CMS-C maize by recruiting a splicing factor.

A fast evolution within mitochondria genome(s) often generates discords between nuclear and mitochondria, which is manifested as cytoplasmic male sterility (CMS) and fertility restoration (Rf) system. The maize CMS-C trait is regulated by the chimeric mitochondrial gene, atp6c, and can be recovered by the restorer gene ZmRf5. Through positional cloning in this study, we identified the nuclear restorer gene, ZmRf5, which encodes a P-type pentatricopeptide repeat (PPR) family protein. The over-expression of ZmRf5 brought back the fertility to CMS-C plants, whereas its genomic editing by CRISPR/Cas9 induced abortive pollens in the restorer line. ZmRF5 is sorted to mitochondria, and recruited RS31A, a splicing factor, through MORF8 to form a cleaving/restoring complex, which promoted the cleaving of the CMS-associated transcripts atp6c by shifting the major cleavage site from 480th nt to 344 th nt for fast degradation, and preserved just right amount of atp6c RNA for protein translation, providing adequate ATP6C to assembly complex V, thus restoring male fertility. Interestingly, ATP6C in the sterile line CMo17A, with similar cytology and physiology changes to YU87-1A, was accumulated much less than it in NMo17B, exhibiting a contrary trend in the YU87-1 nuclear genome previously reported, and was restored to normal level in the presence of ZmRF5. Collectively these findings unveil a new molecular mechanism underlying fertility restoration by which ZmRF5 cooperates with MORF8 and RS31A to restore CMS-C fertility in maize, complemented and perfected the sterility mechanism, and enrich the perspectives on communications between nucleus and mitochondria.

zma-miR159 targets ZmMYB74 and ZmMYB138 transcription factors to regulate grain size and weight in maize.

Endosperm cell number is critical in determining grain size in maize (Zea mays). Here, zma-miR159 overexpression led to enlarged grains in independent transgenic lines, suggesting that zma-miR159 contributes positively to maize grain size. Targeting of ZmMYB74 and ZmMYB138 transcription factor genes by zma-miR159 was validated using 5' RACE and dual-luciferase assay. Lines in which ZmMYB74 was knocked out using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) presented a similar enlarged grain phenotype as those with zma-miR159 overexpression. Downstream genes regulating cell division were identified through DNA affinity purification sequencing using ZmMYB74 and ZmMYB138. Our results suggest that zma-miR159-ZmMYB modules act as an endosperm development hub, participating in the division and proliferation of endosperm cells.

Highly efficient fluorescent and hybrid white organic light-emitting diodes based on a bimolecular excited system: publisher's note.

This publisher's note contains a correction to Opt. Lett.48, 5771 (2023)10.1364/OL.506371.

First report of the maize cyst nematode Heterodera zeae in Sichuan Province of Southwest China.

Maize is the largest crop planted in China. Nine species of cyst nematodes have been reported to affect maize production. Heterodera zeae, H. avenae and Punctodera chalcoensis can cause significant maize yield losses annually (Luc et al. 2005). In 1971, the maize cyst nematode H. zeae was first detected in Rajasthan, India (Koshy et al. 1971). Subsequently, it has been reported in many other countries such as the United States, Greece, Pakistan, and Egypt. In China, H. zeae was first identified in the maize fields of Laibin City, Guangxi Zhuang Autonomous Region (Wu et al., 2017). Cui et al. (2020) identified H. zeae in a maize field of Yuzhou City, Henan Province of Central China in 2018. From 2018 to 2022, a survey of cyst-forming nematodes was conducted in Southwest China. Fifteen soil samples of about 500 g each were collected from Luding County, Ganzi Prefecture of Sichuan Province. No major aboveground symptoms were shown on maize, but a few females were observed on the roots of maize in one field. The cysts and second-stage juveniles (J2s) were collected from each soil sample using Cobb's screening gravity method. A total of 8.50±2.0 cysts per 100 ml of soil on the average were observed in the field. A thin subcrystalline layer was discernible only in young cysts. Morphological and molecular studies of cysts and J2s indicated that the nematodes were identified to be H. zeae in a maize-field. Morphologically, the cysts were in a lemon shape, light brown or pearly white in color. The vulval cone was prominent. Fenestra ambifenestrate, and semifenestra were separated by a fairly wide vulval bridge, fenestral length and width were variable, and the cyst wall was shown in a zigzag pattern. The J2s' body was in a vermiform, tapering at both ends, with a hyaline tail. Stylet was strongly developed with round or slightly anteriorly directed knobs. Morphological measurements of the cysts (n = 9) determined that the mean body length was 417.2 µm (403.6 to 439.4 µm), body width was 429.7 µm (397.6 to 456.9µm); length-width ratio was 1.4 (0.75 to 3); fenestra length was 525.3 µm (498.5 to 570.7 µm); and the mean semifenestra width was 458.6 µm (403.6 to 546.3 µm). Morphometric measurements of second-stage juveniles (n = 20) showed a body length of 419.7µm (355.8 to 492.5 µm); a stylet length of 20.8 µm (19.51 to 23.3µm); a tail length of 41.5 µm (20 to 49.4 µm); and a hyaline tail length of 20.7 µm (16.6 to 24 µm). The main morphological characteristics and measured values were basically consistent with those described by Cui et al. (2022), and all of which were similar to those of H. zeae. Amplification of DNA from random single cysts (n = 5) was conducted using the protocol described by Cui et al. (2022). The rDNA-internal transcribed spacer (ITS) was amplified and sequenced using a pair of universal primers TW81 (5'-GTTTCCGTAGGTGAA CCTGC-3') and AB28 (5'-ATATGCTTAAGTTCAGCGGGT-3'). The ITS sequences were deposited at GenBank with the accession number OR811029.1. Alignments of sequences showed an identity of 98% with H. zeae sequences from China (OP692769.2, MW785772.1) and the USA (GU145616.1), which were confirmed using a pair of species-specific primers HzF1 (5'-GGGGAGGTGAATGTGGG-3') and HzR1 (5'-CCTTTGGCAATCGGTGA-3') of H. zeae with a targeted PCR fragment of 393 bp (Cui et al. 2022). Pathogenicity was conducted and confirmed by infection and reproduction on maize. Seeds (cv. Zhengda 619) were sown in three pots that contained 150 ml of a sterile soil mixture (loamy soil: sand=1:1), and 5 cysts (103 eggs/cyst on the average) were inoculated in each pot at 25/30°C, under a 12-h dark/12-h light condition (Cui et al. 2023). Fifteen days after sowing, third- and fourth-stage juveniles were observed in the rootstained with acid fuchsin, and a total of 32 cysts per maize plant on the average were collected at 40 days after sowing. The new cysts' morphological and molecular characteristics were identical to the cysts from the original soil samples. To the best of our knowledge, this is the first report of H. zeae as a pathogen on maize in Sichuan Province, Southwest China. Our findings will be useful for management and further research of maize cyst nematodes.

Advancements and Prospects of Genome-Wide Association Studies (GWAS) in Maize.

Genome-wide association studies (GWAS) have emerged as a powerful tool for unraveling intricate genotype-phenotype association across various species. Maize (Zea mays L.), renowned for its extensive genetic diversity and rapid linkage disequilibrium (LD), stands as an exemplary candidate for GWAS. In maize, GWAS has made significant advancements by pinpointing numerous genetic loci and potential genes associated with complex traits, including responses to both abiotic and biotic stress. These discoveries hold the promise of enhancing adaptability and yield through effective breeding strategies. Nevertheless, the impact of environmental stress on crop growth and yield is evident in various agronomic traits. Therefore, understanding the complex genetic basis of these traits becomes paramount. This review delves into current and future prospectives aimed at yield, quality, and environmental stress resilience in maize and also addresses the challenges encountered during genomic selection and molecular breeding, all facilitated by the utilization of GWAS. Furthermore, the integration of omics, including genomics, transcriptomics, proteomics, metabolomics, epigenomics, and phenomics has enriched our understanding of intricate traits in maize, thereby enhancing environmental stress tolerance and boosting maize production. Collectively, these insights not only advance our understanding of the genetic mechanism regulating complex traits but also propel the utilization of marker-assisted selection in maize molecular breeding programs, where GWAS plays a pivotal role. Therefore, GWAS provides robust support for delving into the genetic mechanism underlying complex traits in maize and enhancing breeding strategies.

Knockdown of microRNA390 Enhances Maize Brace Root Growth.

Brace root architecture is a critical determinant of maize's stalk anchorage and nutrition uptake, influencing root lodging resistance, stress tolerance, and plant growth. To identify the key microRNAs (miRNAs) in control of maize brace root growth, we performed small RNA sequencing using brace root samples at emergence and growth stages. We focused on the genetic modulation of brace root development in maize through manipulation of miR390 and its downstream regulated auxin response factors (ARFs). In the present study, miR167, miR166, miR172, and miR390 were identified to be involved in maize brace root growth in inbred line B73. Utilizing short tandem target mimic (STTM) technology, we further developed maize lines with reduced miR390 expression and analyzed their root architecture compared to wild-type controls. Our findings show that STTM390 maize lines exhibit enhanced brace root length and increased whorl numbers. Gene expression analyses revealed that the suppression of miR390 leads to upregulation of its downstream regulated ARF genes, specifically ZmARF11 and ZmARF26, which may significantly alter root architecture. Additionally, loss-of-function mutants for ZmARF11 and ZmARF26 were characterized to further confirm the role of these genes in brace root growth. These results demonstrate that miR390, ZmARF11, and ZmARF26 play crucial roles in regulating maize brace root growth; the involved complicated molecular mechanisms need to be further explored. This study provides a genetic basis for breeding maize varieties with improved lodging resistance and adaptability to diverse agricultural environments.

Genetic dissection of maize (Zea mays L.) chlorophyll content using multi-locus genome-wide association studies.

BACKGROUND: The chlorophyll content (CC) is a key factor affecting maize photosynthetic efficiency and the final yield. However, its genetic basis remains unclear. The development of statistical methods has enabled researchers to design and apply various GWAS models, including MLM, MLMM, SUPER, FarmCPU, BLINK and 3VmrMLM. Comparative analysis of their results can lead to more effective mining of key genes. RESULTS: The heritability of CC was 0.86. Six statistical models (MLM, BLINK, MLMM, FarmCPU, SUPER, and 3VmrMLM) and 1.25 million SNPs were used for the GWAS. A total of 140 quantitative trait nucleotides (QTNs) were detected, with 3VmrMLM and MLM detecting the most (118) and fewest (3) QTNs, respectively. The QTNs were associated with 481 genes and explained 0.29-10.28% of the phenotypic variation. Additionally, 10 co-located QTNs were detected by at least two different models or methods, three co-located QTNs were identified in at least two different environments, and six co-located QTNs were detected by different models or methods in different environments. Moreover, 69 candidate genes within or near these stable QTNs were screened based on the B73 (RefGen_v2) genome. GRMZM2G110408 (ZmCCS3) was identified by multiple models and in multiple environments. The functional characterization of this gene indicated the encoded protein likely contributes to chlorophyll biosynthesis. In addition, the CC differed significantly between the haplotypes of the significant QTN in this gene, and CC was higher for haplotype 1. CONCLUSION: This study's results broaden our understanding of the genetic basis of CC, mining key genes related to CC and may be relevant for the ideotype-based breeding of new maize varieties with high photosynthetic efficiency.

Multi-omic characterization of the maize GPI synthesis mutant gwt1 with defects in kernel development.

BACKGROUND: Glycosylphosphatidylinositol (GPI) and GPI-anchored proteins (GAPs) are important for cell wall formation and reproductive development in Arabidopsis. However, monocot counterparts that function in kernel endosperm development have yet to be discovered. Here, we performed a multi-omic analysis to explore the function of GPI related genes on kernel development in maize. RESULTS: In maize, 48 counterparts of human GPI synthesis and lipid remodeling genes were identified, in which null mutation of the glucosaminyl-phosphatidylinositol O-acyltransferase1 gene, ZmGWT1, caused a kernel mutant (named gwt1) with defects in the basal endosperm transport layer (BETL). We performed plasma membrane (PM) proteomics to characterize the potential GAPs involved in kernel development. In total, 4,981 proteins were successfully identified in 10-DAP gwt1 kernels of mutant and wild-type (WT), including 1,638 membrane-anchored proteins with different posttranslational modifications. Forty-seven of the 256 predicted GAPs were differentially accumulated between gwt1 and WT. Two predicted BETL-specific GAPs (Zm00001d018837 and Zm00001d049834), which kept similar abundance at general proteome but with significantly decreased abundance at membrane proteome in gwt1 were highlighted. CONCLUSIONS: Our results show the importance of GPI and GAPs for endosperm development and provide candidate genes for further investigation of the regulatory network in which ZmGWT1 participates.

Dynamic patterns of gene expression and regulatory variation in the maize seed coat.

BACKGROUND: Seed size is an important factor contributing to maize yield, but its molecular mechanism remains unclear. The seed coat, which serves as one of the three components of the maize grain, determines seed size to a certain extent. The seed coat also shares the maternal genotype and is an ideal material for studying heterosis. RESULTS: In this study, the self-pollinated seeds of the maize hybrid Yudan888 and its parental lines were continuously collected from 0 day after pollination (DAP) to 15 DAP for phenotyping, cytological observation and RNA-seq. The phenotypic data showed that 3 DAP and 8 DAP are the best time points to study maize seed coat heterosis. Cytological observations indicated that maize seed coat heterosis might be the result of the coordination between cell number and cell size. Furthermore, the RNA-seq results showed that the nonadditive genes changed significantly between 3 and 8 DAP. However, the number of genes expressed additively was not significantly different. Our findings suggest that seed coat heterosis in hybrid is the result of nonadditive expression caused by dynamic changes in genes at different time points during seed expansion and seed coat development. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated that genes related to DNA replication, cell cycle regulation, circadian rhythms and metabolite accumulation contributed significantly to hybrid seed coat heterosis. CONCLUSION: Maize seed coat phenotyping allowed us to infer that 3 DAP and 8 DAP are important time points in the study of seed coat heterosis. Our findings provide evidence for genes involved in DNA replication, cell cycle regulation, circadian rhythms and metabolite accumulation in hybrid with high or low parental expression as major contributors to hybrid seed coat heterosis.

Single-cell RNA sequencing profiles reveal cell type-specific transcriptional regulation networks conditioning fungal invasion in maize roots.

Stalk rot caused by Fusarium verticillioides (Fv) is one of the most destructive diseases in maize production. The defence response of root system to Fv invasion is important for plant growth and development. Dissection of root cell type-specific response to Fv infection and its underlying transcription regulatory networks will aid in understanding the defence mechanism of maize roots to Fv invasion. Here, we reported the transcriptomes of 29 217 single cells derived from root tips of two maize inbred lines inoculated with Fv and mock condition, and identified seven major cell types with 21 transcriptionally distinct cell clusters. Through the weighted gene co-expression network analysis, we identified 12 Fv-responsive regulatory modules from 4049 differentially expressed genes (DEGs) that were activated or repressed by Fv infection in these seven cell types. Using a machining-learning approach, we constructed six cell type-specific immune regulatory networks by integrating Fv-induced DEGs from the cell type-specific transcriptomes, 16 known maize disease-resistant genes, five experimentally validated genes (ZmWOX5b, ZmPIN1a, ZmPAL6, ZmCCoAOMT2, and ZmCOMT), and 42 QTL or QTN predicted genes that are associated with Fv resistance. Taken together, this study provides not only a global view of maize cell fate determination during root development but also insights into the immune regulatory networks in major cell types of maize root tips at single-cell resolution, thus laying the foundation for dissecting molecular mechanisms underlying disease resistance in maize.

Maize tubulin folding cofactor B is required for cell division and cell growth through modulating microtubule homeostasis.

Tubulin folding cofactors (TFCs) are required for tubulin folding, α/ß tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.

Highly efficient fluorescent and hybrid white organic light-emitting diodes based on a bimolecular excited system.

A bimolecular excited system is considered as a promising candidate for developing white organic light-emitting diodes (WOLEDs) with reduced phosphorescent components. However, for actualizing high-performance WOLED, little attention has been paid to electromers compared to exciplexes. Herein, we construct the bimolecular excited system to prepare fluorescent WOLEDs by combining the electromer emission with the exciplex emission, achieving a maximum power efficiency of 11.8 lm/W with a color rendering index (CRI) of over 80. Furthermore, phosphorescent dopants are doped into an exciplex host to construct hybrid WOLEDs. The fabricated complementary-color and three-color devices achieve maximum efficiencies of 55.3 cd/A (46.8 lm/W) and 34.1 cd/A (26.8 lm/W), respectively. The spectral coverages of WOLEDs are broadened by the bimolecular excited system, and CRIs are further improved at high luminance. Our strategy may bring light to the future development of highly efficient WOLEDs with economy and sustainability.

The genetic architecture of prolificacy in maize revealed by association mapping and bulk segregant analysis.

KEY MESSAGE: Here, we revealed maize prolificacy highly correlated with domestication and identified a causal gene ZmEN1 located in one novel QTL qGEN261 that regulating maize prolificacy by using multiple-mapping methods. The development of maize prolificacy (EN) is crucial for enhancing yield and breeding specialty varieties. To achieve this goal, we employed a genome-wide association study (GWAS) to analyze the genetic architecture of EN in maize. Using 492 inbred lines with a wide range of EN variability, our results demonstrated significant differences in genetic, environmental, and interaction effects. The broad-sense heritability (H2) of EN was 0.60. Through GWAS, we identified 527 significant single nucleotide polymorphisms (SNPs), involved 290 quantitative trait loci (QTL) and 806 genes. Of these SNPs, 18 and 509 were classified as major effect loci and minor loci, respectively. In addition, we performed a bulk segregant analysis (BSA) in an F2 population constructed by a few-ears line Zheng58 and a multi-ears line 647. Our BSA results identified one significant QTL, qBEN1. Importantly, combining the GWAS and BSA, four co-located QTL, involving six genes, were identified. Three of them were expressed in vegetative meristem, shoot tip, internode and tip of ear primordium, with ZmEN1, encodes an unknown auxin-like protein, having the highest expression level in these tissues. It suggested that ZmEN1 plays a crucial role in promoting axillary bud and tillering to encourage the formation of prolificacy. Haplotype analysis of ZmEN1 revealed significant differences between different haplotypes, with inbred lines carrying hap6 having more EN. Overall, this is the first report about using GWAS and BSA to dissect the genetic architecture of EN in maize, which can be valuable for breeding specialty maize varieties and improving maize yield.

Characterization and fine mapping of a maize lesion mimic mutant (Les8) with enhanced resistance to Curvularia leaf spot and southern leaf blight.

KEY MESSAGE: A novel light-dependent dominant lesion mimic mutant with enhanced multiple disease resistance was physiologically, biochemically, and genetically characterized; the causal gene was fine mapped to a 909 kb interval containing 38 genes. Identification of genes that confer multiple disease resistance (MDR) is crucial for the improvement of maize disease resistance. However, very limited genes are identified as MDR genes in maize. In this study, we characterized a dominant disease lesion mimics 8 (Les8) mutant that had chlorotic lesions on the leaves and showed enhanced resistance to both curvularia leaf spot and southern leaf blight. Major agronomic traits were not obviously altered, while decreased chlorophyll content was observed in the mutant, and the genetic effect of the Les8 mutation was stable in different genetic backgrounds. By BSR-seq analysis and map-based cloning, the LES8 gene was mapped into a 909 kb region containing 38 candidate genes on chromosome 9 wherein no lesion mimic or disease-resistance genes were previously reported. Using transcriptomics analysis, we found that genes involved in defense responses and secondary metabolite biosynthesis were enriched in the significantly up-regulated genes, while genes involved in photosynthesis and carbohydrate-related pathways were enriched in the significantly down-regulated genes in Les8. In addition, there was an overaccumulation of jasmonic acid and lignin but not salicylic acid in Les8. Taken together, this study revealed candidate genes and potential mechanism underlying Les8-conferred MDR in maize.

Identification of novel loci associated with starch content in maize kernels by a genome-wide association study using an enlarged SNP panel.

Starch is a major component of cereals, comprising over 70% of dry weight. It serves as a primary carbon source for humans and animals. In addition, starch is an indispensable industrial raw material. While maize (Zea mays) is a key crop and the primary source of starch, the genetic basis for starch content in maize kernels remains poorly understood. In this study, using an enlarged panel, we conducted a genome-wide association study (GWAS) based on best linear unbiased prediction (BLUP) value for starch content of 261 inbred lines across three environments. Compared with previous study, we identified 14 additional significant quantitative trait loci (QTL), encompassed a total of 42 genes, and indicated that increased marker density contributes to improved statistical power. By integrating gene expression profiling, Gene Ontology (GO) enrichment and haplotype analysis, several potential target genes that may play a role in regulating starch content in maize kernels have been identified. Notably, we found that ZmAPC4, associated with the significant SNP chr4.S_175584318, which encodes a WD40 repeat-like superfamily protein and is highly expressed in maize endosperm, might be a crucial regulator of maize kernel starch synthesis. Out of the 261 inbred lines analyzed, they were categorized into four haplotypes. Remarkably, it was observed that the inbred lines harboring hap4 demonstrated the highest starch content compared to the other haplotypes. Additionally, as a significant achievement, we have developed molecular markers that effectively differentiate maize inbred lines based on their starch content. Overall, our study provides valuable insights into the genetic basis of starch content and the molecular markers can be useful in breeding programs aimed at developing maize varieties with high starch content, thereby improving breeding efficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01437-6.

Phylogeny, gene structures, and expression patterns of the auxin response factor (GhARF2) in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Auxin response factors (ARFs) are a class of transcription factors that regulate the expression of auxin-responsive genes and play important functions in plant growth and development. To understand the biological functions of the auxin response factor GhARF2 gene in upland cotton, the coding sequence (CDS) of GhARF2 gene was cloned, and its protein sequence, evolutionary relationship, subcellular localization and expression pattern were analysed. METHODS: The CDS sequence of GhARF2 gene was cloned from upland cotton variety Baimian No.1, and its protein sequence was analyzed by bioinformatics method. The subcellular localization of GhARF2 protein was detected by tobacco epidermal transient transformation system, and the tissue expression and stress expression pattern of GhARF2 were analyzed by quantitative Real­Time PCR (qRT-PCR). RESULTS: The full-length CDS of GhARF2 gene was 2583 bp, encoded 860 amino acids, and had a molecular weight and an isoelectric point of 95.46 KDa and 6.02, respectively. The GhARF2 protein had multiple phosphorylation sites, no transmembrane domain, and secondary structures dominated by random coils and alpha helix. The GhARF2 protein had 3 conserved typical domains of ARF gene family members, including the B3 DNA binding domain, the Auxin_resp domain, and the Aux/IAA domain. Phylogenetic analysis revealed that ARF2 proteins in different species were clustered in the Group A subgroup, in which GhARF2 was closely related to TcARF2 of Theobroma cacao L. (Malvaceae). The subcellular localization results showed that the GhARF2 protein was localized in the nucleus. Analysis of tissue expression pattern showed that the GhARF2 gene was expressed in all tested tissues, with the highest expression levels in sepal, followed by leaf, and the lowest expression levels in fiber. Further stress expression analysis showed that the GhARF2 gene was induced by drought, high-temperature, low-temperature and salt stress, and had different expression patterns under different stress conditions. CONCLUSION: These results established a foundation for understanding the functions of GhARF2 and breeding varieties with high-stress tolerance in cotton.

Mapping the gene of a maize leaf senescence mutant and understanding the senescence pathways by expression analysis.

KEY MESSAGES: Narrowing down to a single putative target gene behind a leaf senescence mutant and constructing the regulation network by proteomic method. Leaf senescence mutant is an important resource for exploring molecular mechanism of aging. To dig for potential modulation networks during maize leaf aging process, we delimited the gene responsible for a premature leaf senescence mutant els5 to a 1.1 Mb interval in the B73 reference genome using a BC1F1 population with 40,000 plants, and analyzed the leaf proteomics of the mutant and its near-isogenic wild type line. A total of 1355 differentially accumulated proteins (DAP) were mainly enriched in regulation pathways such as "photosynthesis", "ribosome", and "porphyrin and chlorophyll metabolism" by the KEGG pathway analysis. The interaction networks constructed by incorporation of transcriptome data showed that ZmELS5 likely repaired several key factors in the photosynthesis system. The putative candidate proteins for els5 were proposed based on DAPs in the fined QTL mapping interval. These results provide fundamental basis for cloning and functional research of the els5 gene, and new insights into the molecular mechanism of leaf senescence in maize.