Qilin Tang

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.

RAPD and Internal Transcribed Spacer Sequence Analyses Reveal Zea nicaraguensis as a Section Luxuriantes Species Close to Zea luxurians

Genetic relationship of a newly discovered teosinte from Nicaragua, Zea nicaraguensis with waterlogging tolerance, was determined based on randomly amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA using 14 accessions from Zea species. RAPD analysis showed that a total of 5,303 fragments were produced by 136 random decamer primers, of which 84.86% bands were polymorphic. RAPD-based UPGMA analysis demonstrated that the genus Zea can be divided into section Luxuriantes including Zea diploperennis, Zea luxurians, Zea perennis and Zea nicaraguensis, and section Zea including Zea mays ssp. mexicana, Zea mays ssp. parviglumis, Zea mays ssp. huehuetenangensis and Zea mays ssp. mays. ITS sequence analysis showed the lengths of the entire ITS region of the 14 taxa in Zea varied from 597 to 605 bp. The average GC content was 67.8%. In addition to the insertion/deletions, 78 variable sites were recorded in the total ITS region with 47 in ITS1, 5 in 5.8S, and 26 in ITS2. Sequences of these taxa were analyzed with neighbor-joining (NJ) and maximum parsimony (MP) methods to construct the phylogenetic trees, selecting Tripsacum dactyloides L. as the outgroup. The phylogenetic relationships of Zea species inferred from the ITS sequences are highly concordant with the RAPD evidence that resolved two major subgenus clades. Both RAPD and ITS sequence analyses indicate that Zea nicaraguensis is more closely related to Zea luxurians than the other teosintes and cultivated maize, which should be regarded as a section Luxuriantes species.

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.

Expression characterization of genes for CMS-C in maize

Cytoplasmic male sterility (CMS)-C is one of the most attractive sources of male sterility in the production of hybrid maize. However, the abortion mechanism of CMS-C is currently unknown. The major aim of this work was to characterize the expression of genes and proteins during pollen abortion. The materials assayed included CMS-C line C48-2, its maintainer line N48-2, and fertile F1 (C48-2 × 18 white). A total of 20 unique genes and 25 proteins were identified by suppression subtractive hybridization and 2-D electrophoresis, respectively. Most of the genes and proteins identified are closely related to energy metabolism, stress responses, molecular chaperones, and cell death, which are generally considered to be essential to pollen development. Based on the function of these identified genes and proteins, reactive oxygen species in isolated mitochondria and DNA fragments were analyzed. The results from this study indicate that the oxidative stress which was associated with the specific expression patterns of some genes may be the physiological cause for the abortion of premature microspores in the maize CMS-C line.

The effect of different genome and cytoplasm on meiotic pairing in maize newly synthetic polyploids

Abstract Allopolyploidization plays the special role in the evolution of many crops. Moreover, the evolution in early stage of some allopolyploidization events is predicted to be effected by nuclear-cytoplasmic interactions. Maize and teosintes are well model system for study of genetic recombination in allopolyploidization. In order to investigate the effects of genome organization and cytoplasm on genome evolution in newly synthesized allopolyploids (neoallopolyploids), a series of neoallopolyploids were produced by reciprocal crosses of maize and Zea perennis. By using dual-color genomic in situ hybridization, intra- and intergenomic meiosis pairing of these polyploids were quantified and compared with regard to its genome organization and cytoplasm background. In the four neoallopolyploids, the stability of maize genome is consistently lower than that of Z. perennis genome. Additional, the stability of maize genome is affected by genome ploidy. The cytoplasm, genome composition and their interaction do have the special role in chromosome paring and the meiosis behaviors in Zea allopolyploids vary significantly and showed non-diploidization. Z. perennis cytoplasm may give a relatively relaxed environment for maize genome.

Perennial aneuploidy as a potential material for gene introgression between maize and Zea perennis

Abstract Hybridization, which allows for gene flow between crops, is difficult between maize and Zea perennis. In this study, we aim to initiate and study gene flow between maize and Z. perennis via a special aneuploid plant (MDT) derived from an interspecific hybrid of the two species. The chromosome constitution and morphological characters of MDT as well as certain backcross progenies were examined. Results from genomic in situ hybridization (GISH) indicate that aneuploid MDT consisted of nine maize chromosomes and 30 Z. perennis chromosomes. The backcross progenies of MDT×maize displayed significant diversity of vegetative and ear morphology; several unusual plants with specific chromosome constitution were founded in its progenies. Some special perennial progeny with several maize chromosomes were obtained by backcrossing MDT with Z. perennis , and the first whole chromosome introgression from maize to Z. perennis was detected in this study. With this novel material and method, a number of maize-tetraploid teosinte addition or substitution lines can be generated for further study, which has great significance to maize and Z. perennis genetic research, especially for promoting introgression and transferring desirable traits.

Study on Haploid Inducing and Its Meiotic Abnormality in Maize

Abstract The haploid-inducing line Stock 6 was used to produce haploid maize and expected to obtain maize haploid plants successfully. The detailed meiotic studies on selected haploid maize (n = x = 10) were conducted. Cytogenetic analysis revealed a high frequency of meiotic abnormality occurred in both meiosis I and meiosis II. During the prophase I, univalents were common configurations, and there were bivalents or trivalents in some pollen mother cells, however, a few cells containing five bivalents were also observed. After prophase I, chromosomes did not congregate in a single metaphase plate but they were scattered in the cytoplasm. At anaphase I, the chromosome distribution was highly irregular with almost all possible combinations. In some cells, chromosomes were grouped into the three or four masses and several spindles appeared. At the tetrad stage of meiosis II, cytokinesis splitting abnormality occurred, and a variety of diad, triad, tetrad, pentad, hexad, as well as decury microspores were easily observed. As a consequence of abnormalities of the two meiotic stages, various microspores and the pollen grains with different size were formed, and its pollen grains were almost completely sterile.

De novo assembly of Zea nicaraguensis root transcriptome identified 5 261 full-length transcripts

Abstract Zea nicaraguensis , a wild relative of cultivated maize ( Zea mays subsp. mays ), is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verification to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L −1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-redundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology (GO) annotation, 2 354 received cluster of orthologous groups (COG) terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes (KEGG) Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.