Guangtang Pan

Identification of candidate genes for drought tolerance by whole-genome resequencing in maize

BackgroundDrought stress is one of the major limiting factors for maize production. With the availability of maize B73 reference genome and whole-genome resequencing of 15 maize inbreds, common variants (CV) and clustering analyses were applied to identify non-synonymous SNPs (nsSNPs) and corresponding candidate genes for drought tolerance.ResultsA total of 524 nsSNPs that were associated with 271 candidate genes involved in plant hormone regulation, carbohydrate and sugar metabolism, signaling molecules regulation, redox reaction and acclimation of photosynthesis to environment were detected by CV and cluster analyses. Most of the nsSNPs identified were clustered in bin 1.07 region that harbored six previously reported QTL with relatively high phenotypic variation explained for drought tolerance. Genes Ontology (GO) analysis of candidate genes revealed that there were 35 GO terms related to biotic stimulus and membrane-bounded organelle, showing significant differences between the candidate genes and the reference B73 background. Changes of expression level in these candidate genes for drought tolerance were detected using RNA sequencing for fertilized ovary, basal leaf meristem tissue and roots collected under drought stressed and well-watered conditions. The results indicated that 70% of candidate genes showed significantly expression changes under two water treatments and our strategies for mining candidate genes are feasible and relatively efficient.ConclusionsOur results successfully revealed candidate nsSNPs and associated genes for drought tolerance by comparative sequence analysis of 16 maize inbred lines. Both methods we applied were proved to be efficient for identifying candidate genes for complex traits through the next-generation sequencing technologies (NGS). These selected genes will not only facilitate understanding of genetic basis of drought stress response, but also accelerate genetic improvement through marker-assisted selection in maize.

Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing

BackgroundIn plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages.ResultsWe used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed.ConclusionsThis study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.

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.

Genome expression profile analysis reveals important transcripts in maize roots responding to the stress of heavy metal Pb

Lead (Pb) has become one of the most abundant heavy metal pollutants of the environment. With its large biomass, maize could be an important object for studying the phytoremediation of Pb-contaminated soil. In our previous research, we screened 19 inbred lines of maize for Pb concentration, and line 178 was identified to be a hyperaccumulator for Pb in both the roots and aboveground parts. To identify important genes and metabolic pathways related to Pb accumulation and tolerance, line 178 was underwent genome expression profile under Pb stress and a control (CK). A total of approximately 11 million cDNA tags were sequenced and 4 665 539 and 4 936 038 clean tags were obtained from the libraries of the test and CK, respectively. In comparison to CK, 2379 and 1832 genes were identified up- or downregulated, respectively, more than fivefolds under Pb stress. Interestingly, all the genes were related to cellular processes and signaling, information storage and processing or metabolism functions. Particularly, the genes involved in posttranslational modification, protein turnover and chaperones; signal transduction, carbohydrate transport and metabolism; and lipid transport and metabolism significantly changed under the treatment. In addition, seven pathways including ribosome, photosynthesis, and carbon fixation were affected significantly, with 118, 12, 34, 21, 18, 72 and 43 differentially expressed genes involved. The significant upregulation of the ribosome pathway may reveal an important secret for Pb tolerance of line 178. And the sharp increase of laccase transcripts and metal ion transporters were suggested to account in part for Pb hyperaccumulation in the line.

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.

Comparative QTL Mapping of Resistance to Gray Leaf Spot in Maize Based on Bioinformatics

The integration QTL map for gray leaf spot resistance in maize was constructed by compiling a total of 57 QTLs available with genetic map IBM2 2005 neighbors as reference. Twenty-six ”real QTLs” and seven consensus QTLs were identified by refining these 57 QTLs using overview and meta-analysis approaches. Seven consensus QTLs were found on chromosomes 1.06, 2.06, 3.04, 4.06, 4.08, 5.03, and 8.06, and the map coordinates were 552.53, 425.72, 279.20, 368.97, 583.21, 308.68 and 446.14 cM, respectively. Using a synteny conservation approach based on comparative mapping between the maize genetic map and rice physical map, a total of 69 rice and maize resistance genes collected from websites Gramene and MaizeGDB were projected onto the maize genetic map IBM2 2005 neighbors, and 2 (Rgene32, ht1), 4 (Rgene5, rp3, scmv2, wsm2), and 4 (ht2, Rgene6, Rgene8 and Rgene7) positional candidate genes were found in three consensus QTLs on chromosomes 2.06, 3.04, and 8.06, respectively. The results suggested that the combination of meta-analysis of gray leaf spot in maize and sequence homologous comparison between maize and rice could be an efficient strategy for identifying major QTLs and corresponding candidate genes for the gray leaf spot.

Combined small RNA and degradome sequencing reveals microRNA regulation during immature maize embryo dedifferentiation

Genetic transformation of maize is highly dependent on the development of embryonic calli from the dedifferentiated immature embryo. To better understand the regulatory mechanism of immature embryo dedifferentiation, we generated four small RNA and degradome libraries from samples representing the major stages of dedifferentiation. More than 186 million raw reads of small RNA and degradome sequence data were generated. We detected 102 known miRNAs belonging to 23 miRNA families. In total, we identified 51, 70 and 63 differentially expressed miRNAs (DEMs) in the stage I, II, III samples, respectively, compared to the control. However, only 6 miRNAs were continually up-regulated by more than fivefold throughout the process of dedifferentiation. A total of 87 genes were identified as the targets of 21 DEM families. This group of targets was enriched in members of four significant pathways including plant hormone signal transduction, antigen processing and presentation, ECM-receptor interaction, and alpha-linolenic acid metabolism. The hormone signal transduction pathway appeared to be particularly significant, involving 21 of the targets. While the targets of the most significant DEMs have been proved to play essential roles in cell dedifferentiation. Our results provide important information regarding the regulatory networks that control immature embryo dedifferentiation in maize.

The genetic architecture of flowering time and photoperiod sensitivity in maize as revealed by QTL review and meta analysis.

The control of flowering is not only important for reproduction, but also plays a key role in the processes of domestication and adaptation. To reveal the genetic architecture for flowering time and photoperiod sensitivity, a comprehensive evaluation of the relevant literature was performed and followed by meta analysis. A total of 25 synthetic consensus quantitative trait loci (QTL) and four hot-spot genomic regions were identified for photoperiod sensitivity including 11 genes related to photoperiod response or flower morphogenesis and development. Besides, a comparative analysis of the QTL for flowering time and photoperiod sensitivity highlighted the regions containing shared and unique QTL for the two traits. Candidate genes associated with maize flowering were identified through integrated analysis of the homologous genes for flowering time in plants and the consensus QTL regions for photoperiod sensitivity in maize (Zea mays L.). Our results suggest that the combination of literature review, meta-analysis and homologous blast is an efficient approach to identify new candidate genes and create a global view of the genetic architecture for maize photoperiodic flowering. Sequences of candidate genes can be used to develop molecular markers for various models of marker-assisted selection, such as marker-assisted recurrent selection and genomic selection that can contribute significantly to crop environmental adaptation.

Applications and development trends in biopesticides

Biopesticides are very effective in the agricultural pest control without causing serious harm to ecological chain or worsening environmental pollution. The research and development of practical applications in the field of biopesticides greatly mitigate environmental pollution caused by chemical pesticide residues and promotes sustainable development of agriculture. Since the advent of biopesticides, a large number of products have been released, several of which have already played dominant roles in the market. The development of biopesticides stimulates modernization of agriculture and will, without doubt, gradually replace chemical pesticides. Many biopesticides are ideal substitutes for their traditional chemical counterparts in pollution-free agricultural production, but some of them display certain toxicity; this should be taken into consideration by the researchers in the field. In this paper, we discuss the current development and application of biopesticides from various categories, the problems occurring in the process of their development and proposing the introduction of various constraints. We review various studies and analyze the development trends in biopesticides in agriculture, demand, market and other fields. Key words: Biopesticides, application status, constraint, development trends.