Artificial chromosome formation in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

We report on the construction of maize minichromosomes using shuttle vectors harboring native centromeric segments, origins of replication, selectable marker genes, and telomeric repeats. These vectors were introduced into scutellar cells of maize immature embryos by microprojectile bombardment. Several independent transformation events were identified containing minichromosomes in addition to the normal diploid complement of 20 maize chromosomes. Immunostaining indicated that the minichromosomes recruited centromeric protein C, which is a specific component of the centromere/kinetochore complex. Minichromosomes were estimated to be 15–30 Mb in size based on cytological measurements. Fluorescent in situ hybridization (FISH) showed that minichromosomes contain the centromeric, telomeric, and exogenous unique marker sequences interspersed with maize retrotransposons. Minichromosomes were detected for at least a year in actively dividing callus cultures, providing evidence for their stability through numerous cell cycles. Plants were regenerated and minichromosomes were detected in root tips, providing confirmation of their normal replication and transmission during mitosis and through organogenesis. Assembly of maize artificial chromosomes may provide a tool to study centromere function and a foundation for developing new high capacity vectors for plant functional genomics and breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Evgueni V. Ananiev, deceased Evgueni V. Ananiev and Chengcang Wu contributed equally to this work. Novel materials described in this publication may be available for noncommercial research purposes on acceptance and signing of a material transfer agreement. In some cases, such materials may contain or be derived from materials obtained from a third party. In such cases, the distribution of material will be subject to the requisite permission from any third-party owners, licensors, or controllers of all or parts of the material. Obtaining any permission will be the sole responsibility of the requestor.     

Genetic analysis and molecular mapping of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) stalk rot resistant gene<Emphasis Type="Italic"> Rfg</Emphasis>1

One single pathogen Fusarium graminearum Schw. was inoculated to maize inbred lines 1,145 (Resistant) and Y331 (Susceptive), and their progenies of F₁, F₂ and BC₁F₁ populations. Field statistical data revealed that all of the F₁ individuals were resistant to the disease and that the ratio of resistant plants to susceptive plants was 3:1 in the F₂ population, and 1:1 in the BC₁F₁ population. The results revealed that a single dominant gene controls the resistance to F. graminearum Schw.. The resistant gene to F. graminearum Schw. was denominated as Rfg1 according to the standard principle of the nomenclature of the plant disease resistant genes. RAPD (randomly amplified polymorphic DNA) combined with BSA (bulked segregant analysis) analysis was carried out in the developed F₂ and BC₁F₁ populations, respectively. Three RAPD products screened from the RAPD analysis with 820 Operon 10-mer primers showed the linkage relation with the resistant gene Rfg1. The three RAPD amplification products (OPD-20₁₀₀₀, OPA-04₁₁₀₀ and OPY-04₉₀₀) were cloned and their copy numbers were determined. The results indicated that only OPY-04₉₀₀ was a single-copy sequence. Then, OPY-04₉₀₀ was used as a probe to map the Rfg1 gene with a RIL F₇ mapping population provided by Henry Nguyen, which was developed from the cross S3×Mo17. Rfg1 was primarily mapped on chromosome 6 between the two linked markers OPY-04₉₀₀ and umc21 (Bin 6.04–6.05). In order to confirm the primary mapping result, 25 SSR (simple sequence repeat) markers and six RFLP (restriction fragment length polymorphism) markers in the Rfg1 gene-encompassing region were selected, and their linkage relation with Rfg1 was analyzed in our F₂ population. Results indicated that SSR marker mmc0241 and RFLP marker bnl3.03 are flanking the Rfg1 gene with a genetic distance of 3.0 cM and 2.0 cM, respectively. This is the first time to name and to map a single resistant gene of maize stalk rot through a single pathogen inoculation and molecular marker analysis.Communicated by H.F. Linskens     

Development of versatile gene-based SNP assays in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

A large number of maize single nucleotide polymorphism (SNP) candidate sequences have been generated and deposited in public databases. However, very little work has been done to date to comprehensively characterize those SNPs and identify a set of markers, which potentially would have high impact in molecular genetics research and breeding programs. Here we describe a multi-step process to identify highly polymorphic gene-based SNPs among ~130,000 public markers. A set of 695 highly polymorphic SNPs (minor allele frequency value >0.3), identified within exons, 5′ and 3′ untranslated regions of genes, were converted into four of the most popular high-throughput genotyping assays that include Illumina’s GoldenGate and Infinium chemistries, Life Technologies’ TaqMan assay and KBioSciences’ KASPar assay. The term “versatile” was applied to 162 gene-based SNPs that were successfully converted into all four chemistries and had perfect genotypic clustering patterns. This subset of discovered versatile SNP markers represents a universal tool for application in various molecular genetics and breeding projects in maize, where genotyping is based on one of the four above-mentioned chemistries. This study demonstrated that despite the availability of millions of discovered SNPs in maize, only a very small portion of those polymorphisms could be utilized for the development of robust, versatile assays, and has real practical value in marker-assisted selection.     

Two novel meiotic restitution mechanisms in haploid maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

Cloning,characterization, and transformation of the phosphoethanolamine <Emphasis Type="Italic">N</Emphasis>-methyltransferase gene (<Emphasis Type="Italic">ZmPEAMT1</Emphasis>) in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-l-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering. Note: Nucleotide sequence data are available in GenBank under the following accession numbers: maize (Zea mays, ZmPEAMT1, AY626156; ZmPEAMT2, AY103779); rice (Oryza sativa, OsPEAMT1/Os01g50030, NM_192178; OsPEAMT2/Os05g47540, XM_475841); wheat (Triticum aestivum, TaPEAMT, AY065971); Arabidopsis (Arabidopsis thaliana, AtNMT1/At3g18000, AY091683; AtNMT2/At1g48600, NM_202264; AtNMT3/At1g73600, NM_106018); oilseed rape (Brassica napus, BnPEAMT, AY319479), tomato (Lycopersicon esculentum, AF328858), spinach (Spinacia oleracea, AF237633).     

A leaf-based regeneration and transformation system for maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Efficient methods for in vitro propagation, regeneration, and transformation of plants are of pivotal importance to both basic and applied research. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. We have developed a novel tissue culture and plant regeneration system that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. We report here: (i) a protocol for the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) a protocol for inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants.     

Genetic diversity of starch synthesis genes of Chinese maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) with SNAPs

Analysis of genetic diversity in maize populations is a very important step for understanding genetic structure and subsequently for genetic manipulations in maize breeding. Sh2, Bt2, Sh1, Wx1, Ae1 and Su1 involved in starch biosynthesis are important genes associated with yield and quality traits in maize breeding programs. In this study, genetic diversity of these six genes in 67 Chinese elite maize inbred lines was measured using single-nucleotide amplified polymorphisms (SNAPs). The results indicated that the number of haplotypes of each gene and population was far less than theoretically expected 2 ⁿ (n = the number of the SNAPs). Phenetic clustering analysis showed that the kernel phonetic (semi-) dent and (semi-) flint lines were belong to distinct subclusters based on haplotypes of SNAPs, with a few exceptions. In addition, the genetic origin of these maize inbred lines was associated with the clustered subgroups. Intragenic linkage disequilibrium (LD) was observed in some of the SNAPs in Bt2, Sh1 and Ae1, while intergenic LD was observed in some of the SNAPs in Bt2, Sh1 and Su1. Association study of kernel phenotypes and SNAP haplotypes showed that the (semi-) dent and (semi-) flint lines had the common haplotype of TA and CC at two SNAP sites in Bt2 (Bt2-2 and Bt2-5), respectively. Two haplotypes of ATGT and GTGC at four SNAP sites in Sh1 (Sh1-2, Sh1-3, Sh1-4 and Sh1-5) were associated with temperature and tropical origin of the maize inbred lines, respectively.     

Expression and functional analysis of <Emphasis Type="Italic">ZmDWF4</Emphasis>, an ortholog of <Emphasis Type="Italic">Arabidopsis DWF4</Emphasis> from maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518 bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6 kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of phenylpropanoid pathway genes within European maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbreds

Background  

Forage quality of maize is influenced by both the content and structure of lignins in the cell wall. Biosynthesis of monolignols, constituting the complex structure of lignins, is catalyzed by enzymes in the phenylpropanoid pathway.     

Genetic analysis of leaf morphology underlying the plant density response by QTL mapping in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Maize yield increase has been strongly linked to plant population densities over time with changes in plant architecture, but the genetic basis for the plant architecture response to plant density is unknown, as is its stability across environments. To elucidate the genetic basis of the plant architecture response to density in maize, we mapped quantitative trait loci (QTLs) for leaf morphology-related traits in four sets of recombinant inbred line (RIL) populations under two plant density conditions. Forty-five QTLs for six traits were detected in both high and low plant density conditions. Thirty-seven QTLs were only detected when grown under high plant density, and 34 QTLs were only detected when grown under low plant density. Twenty-two meta-QTLs (mQTLs) were identified by meta-analysis, and mQTL1-1, mQTL3-2 and mQTL8 were identified when grown under high and low plant densities, with R ² of some initial QTLs > 10 %, suggesting the mQTLs might be hot spots of the important QTLs for the related traits under planting density stress conditions. The results presented here provide useful information for further research and the marker-assisted selection of varieties targeting increased plant density and will help to reveal the molecular mechanisms related to leaf morphology in response to density.     

Genome-wide identification,systematic analysis and characterization of <Emphasis Type="Italic">SRO</Emphasis> family genes in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

SIMILAR TO RCD ONE (SRO) is a small plant-specific gene family, which play essential roles in plant growth and development as well as in abiotic stresses. However, the function of SROs in maize is still unknown. In our study, six putative SRO genes were isolated from the maize genome. A systematic analysis was performed to characterize the ZmSRO gene family. The ZmSRO gene family was divided into two groups according to the motif and intron/exon analysis. Phylogenetic analysis of them with other plants showed that the clades of SROs along with the divergence of monocot and dicot and ZmSROs were more closely with OsSROs. Many abiotic stress response and hormone-induced cis-regulatory elements were identified from the promoter region of ZmSROs. Furthermore, RNA-seq analysis indicated that SRO genes were widely expressed in different tissues and development stages in maize, and the expression divergence was also obviously observed. Analyses of expression in response to PEG6000 and NaCl treatment, in addition to exogenous application of ABA and GA hormones showed that the majority of the members display stress-induced expression patterns. Taken together, our results provide valuable reference for further functional analysis of the SRO gene family in maize, especially in abiotic stress responses.     

Brassinosteroids affect ethylene production in the primary roots of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

To better understand the physiological roles of brassinosteroids (BRs) in the primary roots of maize, we examined their effect on ethylene production. Exogenously applied brassinolide (BL; 10^-9 to 10^-7 M) incrementally increased the level ethylene in a dose-dependent manner. This BL-induced production was enhanced in the presence of IAA, thereby implying a synergistic effect between BR and IAA. At 10^-7 M BL, the level of free ACC was increased, but that of conjugated ACC was diminished. Moreover, greater concentrations of BL proportionally increased ACC oxidase activity. In contrast, higher levels of IAA increased the endogenous content of conjugated ACC as well as ACC synthase activity. Based on these results, we conclude that BR activates ethylene production mainly via ACC oxidase, and interacts with IAA to produce ethylene. However, the functional site for ethylene production is different for each hormone.     

Changes of photosynthetic activities of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) seedlings in response to cadmium stress

Maize (Zea mays L.) seedlings were grown in nutrient solution culture containing 0, 5, and 20 μM cadmium (Cd) and the effects on various aspects of photosynthesis were investigated after 24, 48, 96 and 168 h of Cd treatments. Photosynthetic rate (P _N) decreased after 48 h of 20 μM Cd and 96 h of 5μM Cd addition, respectively. Chl a and total Chl content in leaves declined under 48 h of Cd exposure. Chl b content decreased on extending the period of Cd exposure to 96 h. The maximum quantum efficiency and potential photosynthetic capacity of PSII, indicated by F_v/F_m and F_v/F_o, respectively, were depressed after 96 h onset of Cd exposure. After 48 h of 5μM Cd and 24 h of 20 μM Cd treatments, the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in the leaves started to decrease, respectively. We found that the limitation of photosynthetic capacity in Cd stressed maize leaves was associated with Cd toxicity on the light and the dark stages. However, Cd stress initially reduced the activities of Rubisco and PEPC and subsequently affected the PSII electron transfer, suggesting that the Calvin cycle reactions in maize plants are the primary target of the Cd toxic effect rather than PSII.     

Lateral roots affect the proteome of the primary root of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Lateral roots are initiated from the pericycle cells of other types of roots and remain in contact with these roots throughout their life span. Although this physical contact has the potential to permit the exchange of signals, little is known about the flow of information from the lateral roots to the primary root. To begin to study these interactions the proteome of the primary root system of the maize (Zea mays L.) lrt1 mutant, which does not initiate lateral roots, was compared with the corresponding proteome of wild-type seedlings 9 days after germination. Approximately 150 soluble root proteins were resolved by two-dimensional electrophoresis and analyzed by MALDI-ToF mass spectrometry and database searching. The 96 most abundant proteins from a pH 4–7 gradient were analyzed; 67 proteins representing 47 different Genbank accessions were identified. Interestingly, 10 (15/150) of the detected proteins were preferentially expressed in lrt1 roots that lack lateral roots. Eight of these lrt1-specific proteins were identified and four are involved in lignin metabolism. This study demonstrates for the first time the influence of lateral roots on the proteome of the primary root system. To our knowledge this is the first study to demonstrate an interaction between two plant organs (viz., lateral and primary roots) at the level of the proteome.     

Evaluation of <Emphasis Type="Italic">Hbr</Emphasis> (MITE) markers for assessment of genetic relationships among maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbred lines

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker (Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (≤0.20). Only two markers (3%) were monomorphic in all samples. Although DNA sequencing indicated that 5.5% of same-sized DNA fragments were non-homologous, this result did not affect the cluster analyses (i.e., relationships obtained from the Hbr data were congruent with those derived from pedigree information). Distance matrices generated from Hbr markers were significantly correlated (p<0.001) with those obtained from pedigree (r=0.782), RFLPs (r=0.747), and SSRs (r=0.719). Overall, these results indicated that Hbr markers could be used in conjunction with other molecular markers for genotyping and relationship studies of related maize inbred lines. Received: 26 February 2001 / Accepted: 20 April 2001     

Genome-wide mutagenesis of <Emphasis Type="Italic">Zea mays</Emphasis> L. using <Emphasis Type="Italic">RescueMu</Emphasis> transposons

Derived from the maize Mu1 transposon, RescueMu provides strategies for maize gene discovery and mutant phenotypic analysis. 9.92 Mb of gene-enriched sequences next to RescueMu insertion sites were co-assembled with expressed sequence tags and analyzed. Multiple plasmid recoveries identified probable germinal insertions and screening of RescueMu plasmid libraries identified plants containing probable germinal insertions. Although frequently recovered parental insertions and insertion hotspots reduce the efficiency of gene discovery per plasmid, RescueMu targets a large variety of genes and produces knockout mutants.     

Verification and fine mapping of <Emphasis Type="Italic">qGW1.05</Emphasis>, a major QTL for grain weight in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Grain weight, one of the important factors to determine corn yield, is a typical quantitative inheritance trait. However, the molecular genetic basis of grain weight still remains limited. In our previous researches, a major QTL associated with grain weight, qGW1.05, has been identified between SSR markers umc1601 and umc1754 at bin locus 1.05–1.06 in maize. Here, its genetic and environmental stabiliteis were verified using a BC₃F₂ population to identify the effect of qGW1.05 on grain weight. Further, qGW1.05-NILs were obtained by MAS successfully. Via a large BC₆F₂ segregation population, together with polymorphic microsatellite markers developed between the parents to screen the genotype of the recombinant plants, qGW1.05 was positioned to a 1.11 Mb genome interval. Furthermore, the progenies of 15 recombinants were tested to confirm the effect of qGW1.05 on grain weight. Combining collinearity among cereal crops and genome annotation, the several candidate genes taking part in grain development were identified in the qGW1.05 region. In this study, qGW1.05 was limited to a 1.11 Mb region on chromosome 1, which established the foundation for understanding the molecular basis underlying kernel development and improving grain weight through MAS using the tightly flanking molecular markers in maize.