A Comparative Transcriptome Analysis Reveals New Insights into Pre-Harvest Sprouting (PHS) in Wheat

Background: pre-harvest sprouting (PHS) is a significant cause of yield loss in cereal crops, and is an important topic of study for the improvement of wheat quality. Many studies have focused on PHS in wheat during the last 10 years, especially on the involvement of abscisic acid (ABA) in PHS, however, a lot remained unknown about this topic.Results: In this study, a PHS resistant line was isolated from an ethylmethane sulfonate (EMS) mutant population derived from the wheat cultivar ‘Long 13-3778’, namely ‘LQ18’. The mutant line LQ18 showed highly significant resistance to PHS compared with the wild-type. Transcriptome sequencing was conducted to determine the differences between the LQ18 mutant and the wild-type at the level of gene expression. The results showed no conclusive evidence that the ABA biosynthesis and signaling pathways contribute to the differences in PHS between the mutant and the wild-type, and some genes and their alleles associated with PHS tolerance showed differential expression between the mutant and wild-type lines. The most interesting result of this study was that the expression levels of the chitinase family genes showed significant differences between the mutant and the wild-type as determined by GO enrichment analysis, and a subsequent analysis of differential expression profiling of the chitinase genes led to the same conclusion.Conclusions: Transcriptomic analysis in this study have revealed the global transcriptome profiles of the PHS sensitive wheat cultivar ‘Long 13-3778’ and its PHS resistant mutants. Furthermore, this study has proposed a possible explanation of the connection between PHS and the chitinase family for the first time, which added to our understanding of PHS and seed dormancy in common wheat.

Easymap: a user-friendly software package for rapid mapping by sequencing of point mutations and large insertions

Mapping-by-sequencing strategies combine next-generation sequencing (NGS) with classical linkage analysis, allowing rapid identification of the causal mutations of the phenotypes exhibited by mutants isolated in a genetic screen. Computer programs that analyze NGS data obtained from a mapping population of individuals derived from a mutant of interest in order to identify a causal mutation are available; however, the installation and usage of such programs requires bioinformatic skills, modifying or combining pieces of existent software, or purchasing licenses. To ease this process, we developed Easymap, an open-source program that simplifies the data analysis workflows from raw NGS reads to candidate mutations. Easymap can perform bulked segregant mapping of point mutations induced by ethylmethane sulfonate (EMS) with DNA-seq or RNA-seq datasets, as well as tagged-sequence mapping for large insertions, such as transposons or T-DNAs. The mapping analyses implemented in Easymap have been validated with experimental and simulated datasets from different plant and animal model species. Easymap was designed to be accessible to all users regardless of their bioinformatics skills by implementing a user-friendly graphical interface, a simple universal installation script, and detailed mapping reports, including informative images and complementary data for assessment of the mapping results.

Identification and Characterization of a Newly Isolated Chitinase-Producing Strain Bacillus licheniformis SSCL-10 for Chitin Degradation

Chitinases or chitinolytic enzymes have different applications in the field of medicine, agriculture, and industry. The present study is aimed at developing an effective hyperchitinase-producing mutant strain of novel Bacillus licheniformis. A simple and rapid methodology was used for screening potential chitinolytic microbiota by chemical mutagenesis with ethylmethane sulfonate and irradiation with UV. There were 16 mutant strains exhibiting chitinase activity. Out of the chitinase-producing strains, the strain with maximum chitinase activity was selected, the protein was partially purified by SDS-PAGE, and the strain was identified as Bacillus licheniformis (SSCL-10) with the highest specific activity of 3.4 U/mL. The induced mutation model has been successfully implemented in the mutant EMS-13 (20.2 U/mL) that produces 5-6-fold higher yield of chitinase, whereas the mutant UV-11 (13.3 U/mL) has 3-4-fold greater chitinase activity compared to the wild strain. The partially purified chitinase has a molecular weight of 66 kDa. The wild strain (SSCL-10) was identified as Bacillus licheniformis using 16S rRNA sequence analysis. This study explores the potential applications of hyperchitinase-producing bacteria in recycling and processing chitin wastes from crustaceans and shrimp, thereby adding value to the crustacean industry.

The Effect of Single and Combined Use of Gamma Radiation and Ethylmethane Sulfonate on Early Growth Parameters in Sorghum

Success in inducing genetic variation through mutagenic agents is dependent on the source and dose of application. The objective of this study was to determine the optimum doses of a single and combined use of gamma radiation and ethylmethane sulfonate (EMS) for effective mutation breeding in sorghum. The study involved two concurrent experiments as follows: in experiment I, the seeds of four sorghum genotypes (‘Parbhani Moti’, ‘Parbhani Shakti’, ‘ICSV 15013′, and ‘Macia’) were treated using gamma radiation (0, 300, 400, 500 and 600 Gy), EMS (0, 0.5 and 1.0%), and gamma radiation followed by EMS (0 and 300 Gy and 0.1% EMS; 400 Gy and 0.05% EMS). In experiment II, the seeds of two genotypes (‘Macia’ and ‘Red sorghum’) were treated with seven doses of gamma radiation only (0, 100, 200, 300, 400, 500 and 600 Gy). Overall, the combined applied doses of gamma radiation and EMS are not recommended due to poor seedling emergence and seedling survival rate below LD50. The best dosage of gamma radiation for genotypes Red sorghum, Parbhani Moti, Macia, ICSV 15013 and Parbhani Shakti ranged between 392 and 419 Gy, 311 and 354 Gy, 256 and 355 Gy, 273 and 304 Gy, and 266 and 297 Gy, respectively. The EMS optimum dosage ranges for genotypes Parbhani Shakti, ICSV 15013, Parbhani Moti and Macia were between 0.41% and 0.60%, 0.48% and 0.58%, 0.46% and 0.51%, and 0.36% and 0.45%, respectively. The above dose rates are useful to induce genetic variation in the tested sorghum genotypes for greater mutation events in sorghum breeding programs.

A conserved but plant-specific CDK-mediated regulation of DNA replication protein A2 in the precise control of stomatal terminal division

The R2R3-MYB transcription factor FOUR LIPS (FLP) controls the stomatal terminal division through transcriptional repression of the cell cycle genes CYCLIN-DEPENDENT KINASE (CDK) B1s (CDKB1s), CDKA;1, and CYCLIN A2s (CYCA2s). We mutagenized the weak mutant allele flp-1 seeds with ethylmethane sulfonate and screened out a flp-1 suppressor 1 (fsp1) that suppressed the flp-1 stomatal cluster phenotype. FSP1 encodes RPA2a subunit of Replication Protein A (RPA) complexes that play important roles in DNA replication, recombination, and repair. Here, we show that FSP1/RPA2a functions together with CDKB1s and CYCA2s in restricting stomatal precursor proliferation, ensuring the stomatal terminal division and maintaining a normal guard-cell size and DNA content. Furthermore, we provide direct evidence for the existence of an evolutionarily conserved, but plant-specific, CDK-mediated RPA regulatory pathway. Serine-11 and Serine-21 at the N terminus of RPA2a are CDK phosphorylation target residues. The expression of the phosphorylation-mimic variant RPA2aS11,21/D partially complemented the defective cell division and DNA damage hypersensitivity in cdkb1;1 1;2 mutants. Thus, our study provides a mechanistic understanding of the CDK-mediated phosphorylation of RPA in the precise control of cell cycle and DNA repair in plants.

Characterization and Gene Mapping of non-open hull 1 (noh1) Mutant in Rice (Oryza sativa L.)

Hull opening is a key physiological process during reproductive development, strongly affecting the subsequent fertilization and seed development in rice. In this study, we characterized a rice mutant, non-open hull 1 (noh1), which was derived from ethylmethane-sulfonate (EMS)-treated Xinong 1B (Oryza sativa L.). All the spikelets of noh1 developed elongated and thin lodicules, which caused the failure of hull opening and the cleistogamy. In some spikelets of the noh1, sterile lemmas transformed into hull-like organs. qPCR analysis indicated that the expression of A- and E-function genes was significantly upregulated, while the expression of some B-function genes was downregulated in the lodicules of noh1. In addition, the expression of A-function genes was significantly upregulated, while the expression of some sterile-lemma maker genes was downregulated in the sterile lemma of noh1. These data suggested that the lodicule and sterile lemma in noh1 mutant gained glume-like and lemma-like identity, respectively. Genetic analysis showed that the noh1 trait was controlled by a single recessive gene. The NOH1 gene was mapped between the molecular markers ZJ-9 and ZJ-25 on chromosome 1 with a physical region of 60 kb, which contained nine annotated genes. These results provide a foundation for the cloning and functional research of NOH1 gene.

Characterisation of a PR-1: Luciferase transgenic line deployed to uncover novel defence-related Arabidopsis mutants by luciferase imaging

In order to identify components of the defence signalling network that may contribute to the establishment of disease resistance, we generated a novel PR-1::Luciferase transgenic line which was deployed in an imaging based screen to uncover novel defence-related mutants. Approximately, 5000 ethylmethane sulfonate (EMS) lines and 30 000 activation tagged lines were generated and screened for enhanced LUC activity via ultra low light imaging.