Identification of a novel marker and its associated laccase gene for regulating ear length in tropical and subtropical maize lines.

KEY MESSAGE: This study revealed the identification of a novel gene, Zm00001d042906, that regulates maize ear length by modulating lignin synthesis and reported a molecular marker for selecting maize lines with elongated ears. Maize ear length has garnered considerable attention due to its high correlation with yield. In this study, six maize inbred lines of significant importance in maize breeding were used as parents. The temperate maize inbred line Ye107, characterized by a short ear, was crossed with five tropical or subtropical inbred lines featuring longer ears, creating a multi-parent population displaying significant variations in ear length. Through genome-wide association studies and mutation analysis, the A/G variation at SNP_183573532 on chromosome 3 was identified as an effective site for discriminating long-ear maize. Furthermore, the associated gene Zm00001d042906 was found to correlate with maize ear length. Zm00001d042906 was functionally annotated as a laccase (Lac4), which showed activity and influenced lignin synthesis in the midsection cells of the cob, thereby regulating maize ear length. This study further reports a novel molecular marker and a new gene that can assist maize breeding programs in selecting varieties with elongated ears.

Comparative analysis of Spodoptera frugiperda (J. E. Smith) (Lepidoptera, Noctuidae) corn and rice strains microbiota revealed minor changes across life cycle and strain endosymbiont association.

Background: Spodoptera frugiperda (FAW) is a pest that poses a significant threat to corn production worldwide, causing millions of dollars in losses. The species has evolved into two strains (corn and rice) that differ in their genetics, reproductive isolation, and resistance to insecticides and Bacillus thuringiensis endotoxins. The microbiota plays an important role in insects' physiology, nutrient acquisition, and response to chemical and biological controls. Several studies have been carried out on FAW microbiota from larvae guts using laboratory or field samples and a couple of studies have analyzed the corn strain microbiota across its life cycle. This investigation reveals the first comparison between corn strain (CS) and rice strain (RS) of FAW during different developmental insect stages and, more importantly, endosymbiont detection in both strains, highlighting the importance of studying both FAW populations and samples from different stages. Methods: The composition of microbiota during the life cycle of the FAW corn and rice strains was analyzed through high-throughput sequencing of the bacterial 16S rRNA gene using the MiSeq system. Additionally, culture-dependent techniques were used to isolate gut bacteria and the Transcribed Internal Spacer-ITS, 16S rRNA, and gyrB genes were examined to enhance bacterial identification. Results: Richness, diversity, and bacterial composition changed significantly across the life cycle of FAW. Most diversity was observed in eggs and males. Differences in gut microbiota diversity between CS and RS were minor. However, Leuconostoc, A2, Klebsiella, Lachnoclostridium, Spiroplasma, and Mucispirilum were mainly associated with RS and Colidextribacter, Pelomonas, Weissella, and Arsenophonus to CS, suggesting that FAW strains differ in several genera according to the host plant. Firmicutes and Proteobacteria were the dominant phyla during FAW metamorphosis. Illeobacterium, Ralstonia, and Burkholderia exhibited similar abundancies in both strains. Enterococcus was identified as a conserved taxon across the entire FAW life cycle. Microbiota core communities mainly consisted of Enterococcus and Illeobacterium. A positive correlation was found between Spiroplasma with RS (sampled from eggs, larvae, pupae, and adults) and Arsenophonus (sampled from eggs, larvae, and adults) with CS. Enterococcus mundtii was predominant in all developmental stages. Previous studies have suggested its importance in FAW response to B. thuringensis. Our results are relevant for the characterization of FAW corn and rice strains microbiota to develop new strategies for their control. Detection of Arsenophonus in CS and Spiroplasma in RS are promising for the improvement of this pest management, as these bacteria induce male killing and larvae fitness reduction in other Lepidoptera species.

Comparative characteristics of oat doubled haploids and oat × maize addition lines: Anatomical features of the leaves, chlorophyll a fluorescence and yield parameters.

As a result of oat (Avena sativa L.) × maize (Zea mays L.) crossing, maize chromosomes may not be completely eliminated at the early stages of embryogenesis, leading to the oat × maize addition (OMA) lines development. Introgression of maize chromosomes into oat genome can cause morphological and physiological modifications. The aim of the research was to evaluate the leaves' anatomy, chlorophyll a fluorescence, and yield parameter of oat doubled haploid (DH) and OMA lines obtained by oat × maize crossing. The present study examined two DH and two disomic OMA lines and revealed that they differ significantly in the majority of studied traits, apart from: the number of cells of the outer bundle sheath; light energy absorption; excitation energy trapped in PSII reaction centers; and energy dissipated from PSII. The OMA II line was characterized by larger size of single cells in the outer bundle sheath and greater number of seeds per plant among tested lines.

Genomic basis determining root system architecture in maize.

KEY MESSAGE: A total of 389 and 344 QTLs were identified by GWAS and QTL mapping explaining accumulatively 32.2-65.0% and 23.7-63.4% of phenotypic variation for 14 shoot-borne root traits using more than 1300 individuals across multiple field trails. Efficient nutrient and water acquisition from soils depends on the root system architecture (RSA). However, the genetic determinants underlying RSA in maize remain largely unexplored. In this study, we conducted a comprehensive genetic analysis for 14 shoot-borne root traits using 513 inbred lines and 800 individuals from four recombinant inbred line (RIL) populations at the mature stage across multiple field trails. Our analysis revealed substantial phenotypic variation for these 14 root traits, with a total of 389 and 344 QTLs identified through genome-wide association analysis (GWAS) and linkage analysis, respectively. These QTLs collectively explained 32.2-65.0% and 23.7-63.4% of the trait variation within each population. Several a priori candidate genes involved in auxin and cytokinin signaling pathways, such as IAA26, ARF2, LBD37 and CKX3, were found to co-localize with these loci. In addition, a total of 69 transcription factors (TFs) from 27 TF families (MYB, NAC, bZIP, bHLH and WRKY) were found for shoot-borne root traits. A total of 19 genes including PIN3, LBD15, IAA32, IAA38 and ARR12 and 19 GWAS signals were overlapped with selective sweeps. Further, significant additive effects were found for root traits, and pyramiding the favorable alleles could enhance maize root development. These findings could contribute to understand the genetic basis of root development and evolution, and provided an important genetic resource for the genetic improvement of root traits in maize.

Testcross performance and combining ability of early-medium maturing quality protein maize inbred lines in Eastern and Southern Africa.

Limited commercial quality protein maize (QPM) varieties with low grain yield potential are currently grown in Eastern and Southern Africa (ESA). This study was conducted to (i) assess the performance of single-cross QPM hybrids that were developed from elite inbred lines using line-by-tester mating design and (ii) estimate the general (GCA) and specific (SCA) combining ability of the QPM inbred lines for grain yield, agronomic and protein quality traits. One hundred and six testcrosses and four checks were evaluated across six environments in ESA during 2015 and 2016. Significant variations (P ≤ 0.01) were observed among environments, genotypes and genotype by environment interaction (GEI) for most traits evaluated. Hybrids H80 and H104 were the highest-yielding, most desirable, and stable QPM hybrids. Combining ability analysis showed both additive and non-additive gene effects to be important in the inheritance of grain yield. Additive effects were more important for agronomic and protein quality traits. Inbred lines L19 and L20 depicted desirable GCA effects for grain yield. Various other inbred lines with favorable GCA effects for agronomic traits, endosperm modification, and protein quality traits were identified. These inbred lines could be utilized for breeding desirable QPM cultivars. The QPM hybrids identified in this study could be commercialized after on-farm verification to replace the low-yielding QPM hybrids grown in ESA.

[Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize].

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

Genome-wide identification of ZmMYC2 binding sites and target genes in maize.

BACKGROUND: Jasmonate (JA) is the important phytohormone to regulate plant growth and adaption to stress signals. MYC2, an bHLH transcription factor, is the master regulator of JA signaling. Although MYC2 in maize has been identified, its function remains to be clarified. RESULTS: To understand the function and regulatory mechanism of MYC2 in maize, the joint analysis of DAP-seq and RNA-seq is conducted to identify the binding sites and target genes of ZmMYC2. A total of 3183 genes are detected both in DAP-seq and RNA-seq data, potentially as the directly regulating genes of ZmMYC2. These genes are involved in various biological processes including plant growth and stress response. Besides the classic cis-elements like the G-box and E-box that are bound by MYC2, some new motifs are also revealed to be recognized by ZmMYC2, such as nGCATGCAnn, AAAAAAAA, CACGTGCGTGCG. The binding sites of many ZmMYC2 regulating genes are identified by IGV-sRNA. CONCLUSIONS: All together, abundant target genes of ZmMYC2 are characterized with their binding sites, providing the basis to construct the regulatory network of ZmMYC2 and better understanding for JA signaling in maize.

A combination of QTL mapping and genome-wide association study revealed the key gene for husk number in maize.

KEY MESSAGE: Two key genes Zm00001d021232 and Zm00001d048138 were identified by QTL mapping and GWAS. Additionally, they were verified to be significantly associated with maize husk number (HN) using gene-based association study. As a by-product of maize production, maize husk is an important industrial raw material. Husk layer number (HN) is an important trait that affects the yield of maize husk. However, the genetic mechanism underlying HN remains unclear. Herein, a total of 13 quantitative trait loci (QTL) controlling HN were identified in an IBM Syn 10 DH population across different locations. Among these, three QTL were individually repeatedly detected in at least two environments. Meanwhile, 26 unique single nucleotide polymorphisms (SNPs) were detected to be significantly (p < 2.15 × 10-6) associated with HN in an association pool. Of these SNPs, three were simultaneously detected across multiple environments or environments and best linear unbiased prediction (BLUP). We focused on these environment-stable and population-common genetic loci for excavating the candidate genes responsible for maize HN. Finally, 173 initial candidate genes were identified, of which 22 were involved in both multicellular organism development and single-multicellular organism process and thus confirmed as the candidate genes for HN. Gene-based association analyses revealed that the variants in four genes were significantly (p < 0.01/N) correlated with HN, of which Zm00001d021232 and Zm00001d048138 were highly expressed in husks and early developing ears among different maize tissues. Our study contributes to the understanding of genetic and molecular mechanisms of maize husk yield and industrial development in the future.

RotatedStomataNet: a deep rotated object detection network for directional stomata phenotype analysis.

KEY MESSAGE: Innovatively, we consider stomatal detection as rotated object detection and provide an end-to-end, batch, rotated, real-time stomatal density and aperture size intelligent detection and identification system, RotatedeStomataNet. Stomata acts as a pathway for air and water vapor in the course of respiration, transpiration, and other gas metabolism, so the stomata phenotype is important for plant growth and development. Intelligent detection of high-throughput stoma is a key issue. Nevertheless, currently available methods usually suffer from detection errors or cumbersome operations when facing densely and unevenly arranged stomata. The proposed RotatedStomataNet innovatively regards stomata detection as rotated object detection, enabling an end-to-end, real-time, and intelligent phenotype analysis of stomata and apertures. The system is constructed based on the Arabidopsis and maize stomatal data sets acquired destructively, and the maize stomatal data set acquired in a non-destructive way, enabling the one-stop automatic collection of phenotypic, such as the location, density, length, and width of stomata and apertures without step-by-step operations. The accuracy of this system to acquire stomata and apertures has been well demonstrated in monocotyledon and dicotyledon, such as Arabidopsis, soybean, wheat, and maize. The experimental results that the prediction results of the method are consistent with those of manual labeling. The test sets, the system code, and their usage are also given ( https://github.com/AITAhenu/RotatedStomataNet ).

DArTseq-based SNP markers reveal high genetic diversity among early generation fall armyworm tolerant maize inbred lines.

Diversity analysis using molecular markers serves as a powerful tool in unravelling the intricacies of inclusivity within various populations and is an initial step in the assessment of populations and the development of inbred lines for host plant resistance in maize. This study was conducted to assess the genetic diversity and population structure of 242 newly developed S3 inbred lines using 3,305 single nucleotide polymorphism (SNP) markers and to also assess the level of homozygosity achieved in each of the inbred lines. A total of 1,184 SNP markers were found highly informative, with a mean polymorphic information content (PIC) of 0.23. Gene diversity was high among the inbred lines, ranging from 0.04 to 0.50, with an average of 0.27. The residual heterozygosity of the 242 S3 inbred lines averaged 8.8%, indicating moderately low heterozygosity levels among the inbred lines. Eighty-four percent of the 58,322 pairwise kinship coefficients among the inbred lines were near zero (0.00-0.05), with only 0.3% of them above 0.50. These results revealed that many of the inbred lines were distantly related, but none were redundant, suggesting each inbred line had a unique genetic makeup with great potential to provide novel alleles for maize improvement. The admixture-based structure analysis, principal coordinate analysis, and neighbour-joining clustering were concordant in dividing the 242 inbred lines into three subgroups based on the pedigree and selection history of the inbred lines. These findings could guide the effective use of the newly developed inbred lines and their evaluation in quantitative genetics and molecular studies to identify candidate lines for breeding locally adapted fall armyworm tolerant varieties in Ghana and other countries in West and Central Africa.

Modeling the economic impact for Chile of an import ban on genetically modified maize.

We estimate producer and consumer surplus changes due to a possible GM maize import ban in Chile, which produces only non-GM grains for internal use. Without foreign non-GM sources, the ban's effect on domestic maize prices would be so significant as to induce Chile to switch from net exporter to net importer of animal products. Fixed factor owners in farm production would benefit significantly, although non-GM maize imports would moderate gains. Total social welfare measures would decline considerably, requiring large offsetting noneconomic benefits for a ban's political viability. Without non-GM imports, internal maize prices would likely eliminate domestic animal product industries; with possible imports, industries and final consumers would suffer, but much less. Currently, the country is a net importer of grain and a net exporter of pork and poultry, and so most welfare losses on the demand side of the market for maize would be in terms of the economic rents generated by the pork and poultry sectors. International competition would protect final consumers to the extent that animal product imports based on GM feed were permitted.

NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.

Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.

Induction of genetic variability of maize genotypes through radiation revealed mutants resistant to maize streak disease.

The absence of genetic variability among crop genotypes is an impediment to breeding progress, hence mutagenesis could serve as a useful tool to create genetic variation to obtain desirable traits of interest. In this study, four maize genotypes, Obatampa, Dapango, Pann 54 and Honampa which were susceptible to maize streak disease (MSD) were acutely irradiated at 254.3 Gy, using a cobalt 60 (60Co) at a rate of 300 Gy/hr. The irradiated seeds were planted with their parental controls at streak disease highly endemic environment. Field trials for the selected maize genotypes were conducted from the M1 to M4 generations to screen for MSD resistance and improved grain yield. Sixteen putative mutants and four individual parental controls were selected across the four maize genotypes at the end of the M4 generation based on disease severity score and yield indices. Detailed morphological screening and field evaluation of putative mutants showing improved plant architecture, increased grain yield and resistance to maize streak disease were tagged and selected. Obatanpa-induced-genotype was the best mutant identified with a grain yield of 6.8 t ha-1. Data on days to 50% flowering indicated that all 16 putative mutants were maturing plants.

Maize green leaf area index dynamics: genetic basis of a new secondary trait for grain yield in optimal and drought conditions.

KEY MESSAGE: Green Leaf Area Index dynamics is a promising secondary trait for grain yield and drought tolerance. Multivariate GWAS is particularly well suited to identify the genetic determinants of the green leaf area index dynamics. Improvement of maize grain yield is impeded by important genotype-environment interactions, especially under drought conditions. The use of secondary traits, that are correlated with yield, more heritable and less prone to genotype-environment interactions, can increase breeding efficiency. Here, we studied the genetic basis of a new secondary trait: the green leaf area index (GLAI) dynamics over the maize life cycle. For this, we used an unmanned aerial vehicle to characterize the GLAI dynamics of a diverse panel in well-watered and water-deficient trials in two years. From the dynamics, we derived 24 traits (slopes, durations, areas under the curve), and showed that six of them were heritable traits representative of the panel diversity. To identify the genetic determinants of GLAI, we compared two genome-wide association approaches: a univariate (single-trait) method and a multivariate (multi-trait) method combining GLAI traits, grain yield, and precocity. The explicit modeling of correlation structure between secondary traits and grain yield in the multivariate mixed model led to 2.5 times more associations detected. A total of 475 quantitative trait loci (QTLs) were detected. The genetic architecture of GLAI traits appears less complex than that of yield with stronger-effect QTLs that are more stable between environments. We also showed that a subset of GLAI QTLs explains nearly one fifth of yield variability across a larger environmental network of 11 water-deficient trials. GLAI dynamics is a promising grain yield secondary trait in optimal and drought conditions, and the detected QTLs could help to increase breeding efficiency through a marker-assisted approach.

Cytoplasmic genome contributions to domestication and improvement of modern maize.

BACKGROUND: Studies on maize evolution and domestication are largely limited to the nuclear genomes, and the contribution of cytoplasmic genomes to selection and domestication of modern maize remains elusive. Maize cytoplasmic genomes have been classified into fertile (NA and NB) and cytoplasmic-nuclear male-sterility (CMS-S, CMS-C, and CMS-T) groups, but their contributions to modern maize breeding have not been systematically investigated. RESULTS: Here we report co-selection and convergent evolution between nuclear and cytoplasmic genomes by analyzing whole genome sequencing data of 630 maize accessions modern maize and its relatives, including 24 fully assembled mitochondrial and chloroplast genomes. We show that the NB cytotype is associated with the expansion of modern maize to North America, gradually replaces the fertile NA cytotype probably through unequal division, and predominates in over 90% of modern elite inbred lines. The mode of cytoplasmic evolution is increased nucleotypic diversity among the genes involved in photosynthesis and energy metabolism, which are driven by selection and domestication. Furthermore, genome-wide association study reveals correlation of cytoplasmic nucleotypic variation with key agronomic and reproductive traits accompanied with the diversification of the nuclear genomes. CONCLUSIONS: Our results indicate convergent evolution between cytoplasmic and nuclear genomes during maize domestication and breeding. These new insights into the important roles of mitochondrial and chloroplast genomes in maize domestication and improvement should help select elite inbred lines to improve yield stability and crop resilience of maize hybrids.

A Scalable and Robust Chloroplast Genotyping Solution: Development and Application of SNP and InDel Markers in the Maize Chloroplast Genome.

Maize(Zea mays. L) is a globally important crop, and understanding its genetic diversity is crucial for plant breeding phylogenetic analyses and comparative genetics. While nuclear markers have been extensively used for mapping agriculturally important genes, they are limited in recognizing characteristics, such as cytoplasmic male sterility and reciprocal cross hybrids. In this study, we performed next-generation sequencing of 176samples, and the maize cultivars represented five distinct groups. A total of 89 single nucleotide polymorphisms (SNPs) and 11 insertion/deletion polymorphisms (InDels) were identified. To enable high-throughput detection, we successfully amplified and confirmed 49 SNP and InDel markers, which were defined as a Varietal Chloroplast Panel (VCP) using the Kompetitive Allele Specific PCR (KASP). The specific markers provided a valuable tool for identifying chloroplast groups. The verification experiment, focusing on the identification of reciprocal cross hybrids and cytoplasmic male sterility hybrids, demonstrated the significant advantages of VCP markers in maternal inheritance characterization. Furthermore, only a small subset of these markers is needed to provide useful information, showcasing the effectiveness of these markers in elucidating the artificial selection process of elite maize lines.

Phenotypic Investigation and RNA-seq of KN1 Involved in Leaf Angle Formation in Maize (Zea mays L.).

Leaf angle (LA) is one of the core agronomic traits of maize, which controls maize yield by affecting planting density. Previous studies have shown that the KN1 gene is closely related to the formation of maize LA, but its specific mechanism has not been fully studied. In this study, phenotype investigation and transcriptomic sequencing were combined to explore the mechanism of LA changes in wild type maize B73 and mutant kn1 under exogenous auxin (IAA) and abscisic acid (ABA) treatment. The results showed that the effect of exogenous phytohormones had a greater impact on the LA of kn1 compared to B73. Transcriptome sequencing showed that genes involved in IAA, gibberellins (GAs) and brassinosteroids (BRs) showed different differential expression patterns in kn1 and B73. This study provides new insights into the mechanism of KN1 involved in the formation of maize LA, and provides a theoretical basis for breeding maize varieties with suitable LA.

ZmSMR10 Increases the Level of Endoreplication of Plants through Its Interactions with ZmPCNA2 and ZmCSN5B.

As a plant-specific endoreplication regulator, the SIAMESE-RELATED (SMR) family (a cyclin-dependent kinase inhibitor) plays an important role in plant growth and development and resistance to stress. Although the genes of the maize (Zea mays) SMR family have been studied extensively, the ZmSMR10 (Zm00001eb231280) gene has not been reported. In this study, the function of this gene was characterized by overexpression and silencing. Compared with the control, the transgenic plants exhibited the phenotypes of early maturation, dwarfing, and drought resistance. Expression of the protein in prokaryotes demonstrates that ZmSMR10 is a small protein, and the results of subcellular localization suggest that it travels functionally in the nucleus. Unlike ZmSMR4, yeast two-hybrid experiments demonstrated that ZmSMR10 does not interact strongly with with some cell cycle protein-dependent protein kinase (CDK) family members ZmCDKA;1/ZmCDKA;3/ZmCDKB1;1. Instead, it interacts strongly with ZmPCNA2 and ZmCSN5B. Based on these results, we concluded that ZmSMR10 is involved in the regulation of endoreplication through the interaction of ZmPCNA2 and ZmCSN5B. These findings provide a theoretical basis to understand the mechanism of the regulation of endoreplication and improve the yield of maize through the use of molecular techniques.

Genome-Wide Association Studies on the Kernel Row Number in a Multi-Parent Maize Population.

Kernel row number (KRN) is a crucial trait in maize that directly influences yield; hence, understanding the mechanisms underlying KRN is vital for the development of high-yielding inbred lines and hybrids. We crossed four excellent panicle inbred lines (CML312, CML444, YML46, and YML32) with Ye107, and after eight generations of selfing, a multi-parent population was developed comprising four subpopulations, each consisting of 200 lines. KRN was accessed in five environments in Yunnan province over three years (2019, 2021, and 2022). The objectives of this study were to (1) identify quantitative trait loci and single nucleotide polymorphisms associated with KRN through linkage and genome-wide association analyses using high-quality genotypic data, (2) identify candidate genes regulating KRN by identifying co-localized QTLs and SNPs, and (3) explore the pathways involved in KRN formation and identify key candidate genes through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Our study successfully identified 277 significant Quantitative trait locus (QTLs) and 53 significant Single Nucleotide Polymorphism (SNPs) related to KRN. Based on gene expression, GO, and KEGG analyses, SNP-177304649, SNP-150393177, SNP-135283055, SNP-138554600, and SNP-120370778, which were highly likely to be associated with KRN, were identified. Seven novel candidate genes at this locus (Zm00001d022420, Zm00001d022421, Zm00001d016202, Zm00001d050984, Zm00001d050985, Zm00001d016000, and Zm00014a012929) are associated with KRN. Among these, Zm00014a012929 was identified using the reference genome Mo17. The remaining six genes were identified using the reference genome B73. To our knowledge, this is the first report on the association of these genes with KRN in maize. These findings provide a theoretical foundation and valuable insights into the genetic mechanisms underlying maize KRN and the development of high-yielding hybrids through heterosis.

Genome-wide identification and evolutionary analysis of the AP2/EREBP, COX and LTP genes in Zea mays L. under drought stress.

AP2 (APETALA2)/EREBP (ethylene-responsive element-binding protein), cytochrome c oxidase (COX) and nonspecific lipid transfer proteins (LTP) play important roles in the response to drought stress. This is the first study to identify the COX gene in Zea mays L. via genome-wide analysis. The qRT‒PCR results indicated that AP2/EREBP, COX and LTP were downregulated, with fold changes of 0.84, 0.53 and 0.31, respectively, after 12 h of drought stress. Genome-wide analysis identified 78 AP2/EREBP, 6 COX and 10 LTP genes in Z. mays L. Domain analysis confirmed the presence of the AP2 domain, Cyt_c_Oxidase_Vb domain and nsLTP1 in the AP2/EREBP, COX and LTP proteins, respectively. The AP2/EREBP protein family (AP2) includes five different domain types: the AP2/ERF domain, the EREBP-like factor (EREBP), the ethylene responsive factor (ERF), the dehydration responsive element binding protein (DREB) and the SHN SHINE. Synteny analysis of the AP2/EREBP, COX and LTP genes revealed collinearity orthologous relationships in O. sativa, H. vulgare and A. thaliana. AP2/EREBP genes were found on the 10 chromosomes of Z. mays L. COX genes were found on chromosomes 1, 3, 4, 5, 7 and 8. LTP genes were found on chromosomes 1, 3, 6, 8, 9 and 10. In the present study, the Ka/Ks ratios of the AP2/EREBP paralogous pairs indicated that the AP2/EREBP genes were influenced primarily by purifying selection, which indicated that the AP2/EREBP genes received strong environmental pressure during evolution. The Ka/Ks ratios of the COX-3/COX-4 paralogous pairs indicate that the COX-3/COX-4 genes were influenced primarily by Darwinian selection (driving change). For the LTP genes, the Ka/Ks ratios of the LTP-1/LTP-10, LTP-5/LTP-3 and LTP-4/LTP-8 paralogous pairs indicate that these genes were influenced primarily by purifying selection, while the Ka/Ks ratios of the LTP-2/LTP-6 paralogous pairs indicate that these genes were influenced primarily by Darwinian selection. The duplication time of the AP2/EREBP paralogous gene pairs in Z. mays L. ranged from approximately 9.364 to 100.935 Mya. The duplication time of the COX-3/COX-4 paralogous gene pair was approximately 5.217 Mya. The duplication time of the LTP paralogous gene pairs ranged from approximately 19.064 to 96.477 Mya. The major focus of research is to identify the genes that are responsible for drought stress tolerance to improve maize for drought stress tolerance. The results of the present study will improve the understanding of the functions of the AP2/EREBP, COX and LTP genes in response to drought stress.