Hailan Liu

Genome expression profile analysis of the immature maize embryo during dedifferentiation.

Maize is one of the most important cereal crops worldwide and one of the primary targets of genetic manipulation, which provides an excellent way to promote its production. However, the obvious difference of the dedifferentiation frequency of immature maize embryo among various genotypes indicates that its genetic transformation is dependence on genotype and immature embryo-derived undifferentiated cells. To identify important genes and metabolic pathways involved in forming of embryo-derived embryonic calli, in this study, DGE (differential gene expression) analysis was performed on stages I, II, and III of maize inbred line 18-599R and corresponding control during the process of immature embryo dedifferentiation. A total of ∼21 million cDNA tags were sequenced, and 4,849,453, 5,076,030, 4,931,339, and 5,130,573 clean tags were obtained in the libraries of the samples and the control, respectively. In comparison with the control, 251, 324 and 313 differentially expressed genes (DEGs) were identified in the three stages with more than five folds, respectively. Interestingly, it is revealed that all the DEGs are related to metabolism, cellular process, and signaling and information storage and processing functions. Particularly, the genes involved in amino acid and carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis and signal transduction mechanism have been significantly changed during the dedifferentiation. To our best knowledge, this study is the first genome-wide effort to investigate the transcriptional changes in dedifferentiation immature maize embryos and the identified DEGs can serve as a basis for further functional characterization.

Validation of Potential Reference Genes for qPCR in Maize across Abiotic Stresses, Hormone Treatments, and Tissue Types

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.

Cytoplasmic male sterility-regulated novel microRNAs from maize

In higher plants, microRNA (miRNA) is involved in regulation of developmental processes, including sexual organ development. Seven novel miRNA families with one known miRNA were isolated by constructing a small RNA library from a mixture of anther from a cytoplasmic male sterile line and its maintainer. Two miRNAs are conserved in plant species. A total of 18 potential targets were identified for the eight miRNA families, including 15 proteins annotated with function and three unknown proteins. The known proteins include several proteins relevant to cell structure and stress response, transcription factors, and enzymes associated with metabolic and signaling pathways, playing important roles in microspore development. Quantitative real-time PCR assay revealed different expression patterns of the miRNAs between the cytoplasmic male sterile line and its maintainer. Each of the miRNAs tended to be down-regulated after the tetrad stage in a fertile line. However, most of the miRNAs in the cytoplasmic male sterile line were shown to be up-regulated from the tetrad to mononuclear stage, displaying special expression patterns differing from the ones in fertile line. We conclude that additional inactive miRNA pathways are essential during pollen development for a fertile line to ensure male fertility. Contrarily, miRNAs are up-regulated during the period from the tetrad to mononuclear stage, which contributes to pollen abortion for a cytoplasmic male sterile line.

Genome-scale identification of resistance gene analogs and the development of their intron length polymorphism markers in maize

As introns are vulnerable to changes such as insertions and deletions when exposed to various evolutionary forces, they constitute a repository for developing genetic markers based on intron length polymorphisms (ILP). This study developed a set of genetic markers that use the potential intron length polymorphism in resistance gene analogs (RGAs) in Zea mays. By searching the genome of Zea mays B73 for the homologs of 73 R genes which have already been identified in plants, we found 861 RGAs, 632 of which have at least one intron that can serve as putative markers targeting the intron length polymorphism in RGAs (RGA-ILP). We developed 1972 candidate markers via electronic PCR (e-PCR) with primer pairs designed in each pair of exonic regions that flank an intron. Furthermore, the performance of RGA-ILP among four maize inbred lines (Huangzao4, B73, Mo17, and Dan340) was evaluated with 69 pairs of randomly selected primers. Of them, 46.4% showed bands that had discriminating length polymorphism, and between any two of the inbred lines the proportion of polymorphism ranged from 23.2 to 31.9%. To make it convenient to use these markers for those interested in molecular breeding of disease-resistant maize, we provide all related information in a web-based database named MaizeRGA, which is available at http://www.sicau.edu.cn/web/yms/rga/maizeRGA.html.

Identification, and Functional and Expression Analyses of the CorA/MRS2/MGT-Type Magnesium Transporter Family in Maize.

Magnesium (Mg(2+)) is an essential macronutrient for plant growth and development, and the CorA/MRS2/MGT-type Mg(2+) transporters play important roles in maintaining Mg(2+) homeostasis in plants. Although the MRS2/MGT genes have been identified in two model plant species, Arabidopsis and rice, a comprehensive analysis of the MRS2/MGT gene family in other plants is lacking. In this work, 12 putative MRS2/MGT genes (ZmMGT1- ZmMGT12) were identified in maize and all of them were classified into five distinct subfamilies by phylogenetic analysis. A complementation assay in the Salmonella typhimurium MM281 strain showed that five representatives of the 12 members possess Mg(2+) transport abilities. Inhibition of ZmMGT protein activity using the hexaamminecobalt (III) (Co-Hex) inhibitor indicated that the ZmMGT protein mediated both low-affinity and high-affinity Mg(2+) transport in maize. A semi-quantitative reverse transcription-PCR (RT-PCR) analysis revealed that eight genes were constitutively expressed in all of the detected tissues, with one being specifically expressed in roots and three having no detectable expression signals. A quantitative RT-PCR analysis showed that some ZmMGT members displayed differential responses to Mg(2+) deficiency and aluminum (Al) stress. Furthermore, root growth inhibition and Mg(2+) accumulation analyses in two maize inbred lines, which conferred different levels of Al tolerance, revealed that ZmMGT proteins contributed to the Al resistance of the Al tolerance genotype. We hypothesize that ZmMGT family members function as Mg(2+) transporters and may play a role in linking Mg(2+) deficiency and Al stress responses. Our results will be valuable in a further analysis of the important biological functions of ZmMGT members in maize.

Genome-wide identification of microRNAs responding to early stages of phosphate deficiency in maize.

Phosphorus (P) is an essential element involved in numerous biochemical reactions. In plants, stress responses, such as the expression of microRNAs (miRNAs), are induced to help them adapt to low phosphate (Pi) concentrations. In this study, deep sequencing was performed using the roots and leaves of maize seedlings grown under low Pi concentrations to identify miRNAs that are differentially expressed during the early stages of Pi deficiency. Eight small RNA libraries were constructed, and 159 known miRNAs representing 32 miRNA families and 10 novel miRNAs. Members of the miR396 family were extremely abundant. Further, 28 Pi-responsive miRNAs were identified (27 known and 1 novel) of which 8 and 7 were significantly expressed exclusively in leaf and root tissues, respectively. The analysis of Pi-responsive miRNAs target genes suggested that most target genes functioning as transcription factors were involved in root and leaf development. The expression profiles of selected Pi-responsive miRNAs and target genes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, we discuss the significance of the differences in expression patterns of these miRNAs during the early and later stages of Pi starvation. This study provides useful information concerning the role of miRNAs in response to Pi starvation and will further our understanding of the mechanisms governing Pi homeostasis in maize.

Genome-wide analysis of Gro/Tup1 family corepressors and their responses to hormones and abiotic stresses in maize

Gro/Tup1 proteins act as negative transcriptional regulators and play crucial roles in many growth and developmental processes in a wide range of organisms. However, our understanding of Gro/Tup1 protein functions in plants is confined to the model plant Arabidopsis. Here, 11 Gro/Tup1 genes, which were characterized by the typical LisH and WD40 repeat domains, were identified in maize through a genome-wide survey. A phylogenetic analysis revealed that maize Gro/Tup1 proteins could be divided into three subfamilies, in which members shared similar protein and gene structures. The predicted maize Gro/Tup1 genes were distributed on seven chromosomes and segmental duplication contributed to their expansion. Many predicted cis-elements associated with hormones, biotic- or abioticstress responses, meristem and seed development, and circadian rhythms, were found in their putative promoter regions. A potential associated protein analysis identified a large number of candidates, including transcription factors, chromatin-modifying enzymes, protein kinases, and ubiquitinconjugating enzymes. An expression profile derived from the RNA-seq data indicated that Gro/Tup1 genes in maize were widely expressed in various organs and tissues. Quantitative real-time PCR revealed that these genes responded to at least one hormone or abiotic stress, either in roots or in shoots. Our study provides useful information on the Gro/Tup1 genes in maize and will facilitate the further functional validation of these genes in growth and development, hormone responses, and biotic- or abiotic-stress resistance.

Erratum to: Mining for low-nitrogen tolerance genes by integrating meta-analysis and large-scale gene expression data from maize

Abstract Nitrogen (N) is the most important macronutrient for plant growth and development. Hence, understanding genetic architectures and functional genes involved in the response to N deficiency can greatly facilitate the development of low-N-tolerant cultivars. In this study, we collected 212 quantitative trait loci (QTL) of agronomically important traits under low-N stress conditions in maize. We then identified 21 consensus QTL (cQTL) strongly induced for low-N tolerance after excluding overlapping cQTL containing QTL simultaneously identified in meta-analyses of studies performed under other environmental conditions. Among the 21 cQTL, 30 candidate maize genes were identified from maize large-scale differential expression data derived from analyses of low-N stress, and the 12 most important maize orthologs were identified using homologous BLAST analyses of genes with known functions in N use efficiency in model plants. Furthermore, maize orthologs associated with low-N tolerance and metabolism were also predicted using large-scale expression data from other model plants. The present genetic loci and candidate genes indicate the molecular mechanisms of low-N tolerance in maize and may provide information for QTL fine mapping and molecular marker-assisted selection.

Genome-Wide Expression Profile of Maize Root Response to Phosphorus Deficiency Revealed by Deep Sequencing

Phosphorus (P) is one of the three primary macronutrients that are required in large amounts for plant growth and development. To better understand molecular mechanism of maize and identify relevant genes in response to phosphorus deficiency, we used Solexa/Illuminas digital gene expression (DGE) technology to investigate six genome-wide expression profiles of seedling roots of the low-P tolerant maize inbred line 178. DGE studies were conducted at 6, 24 and 72 h under both phosphorus deficient and sufficient conditions. Approximately 3.93 million raw reads for each sample were sequenced and 6 816 genes exhibited significant levels of differential expressions in at least one of three time points in response to P starvation. The number of genes with increased expression increased over time from 6 to 24 h, whereas genes with decreased expression were more abundant at 72 h, suggesting a gradual response process for P deficiency at different stages. Gene annotations illustrated that most of differentially expressed genes (DEGs) are involved in different cellular and molecular processes such as environmental adaptation and carbohydrate metabolism. The expression of some known genes identified in other plants, such as those involved in root architecture, P metabolism and transport were found to be altered at least two folds, indicating that the mechanisms of molecular and morphological adaptation to P starvation are conserved in plants. This study provides insight into the general molecular mechanisms underlying plant adaptation to low-P stress and thus may facilitate molecular breeding for improving P utilization in maize.

A new genomic prediction method with additive-dominance effects in the least-squares framework

In our previous work, we proposed a genomic prediction method combing identical-by-state-based Haseman-Elston regression and best linear prediction with additive variance component only (HEBLP|A herein), the most essential component of genetic variation. Since the dominance effects contribute significantly in heterosis, it is desirable to incorporate the HEBLP with dominance variance component that is expected to enhance the predictive accuracy as we move to the further development: HEBLP|AD, a paralleled implementation of genomic prediction compared with genomic best linear unbiased prediction (GBLUP). The simulation results indicated that when the dominance effects contributed to a large proportion of genetic variation, HEBLP|AD and GBLUP|AD, having similar accuracy, both outperformed HEBLP|A; but when the dominance variation was none or little, HEBLP|A, HEBLP|AD, and GBLUP|AD had similar predictability. The analysis of real data from Arabidopsis thaliana F2 population also demonstrated the latter situation. In summary, HEBLP|AD performed stable whether a trait was controlled by dominance effects or not.