An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

An EMS mutant library for cucumber

Cucumber is an important vegetable crop and a model crop for the study of sex expression in plants. However, the genomic resources and tools for functional genomics studies in cucumber are still limited. In this paper, we conducted ethyl methyl sulfone（EMS） mutagenesis in the northern China ecotype cucumber inbred line 406 to construct a mutant library. We optimized the conditions of EMS mutagenesis on inbred line 406 which included treatment of seeds at 1.5% EMS for 12h. We obtained a number of mutant lines showing inheritable morphological changes in plant architecture, leaves, floral organs, fruits and other traits through M1, M2 and M3 generations. The F2 segregating populations were constructed and analyzed.We found that a short fruit mutant and a yellow-green fruit peel mutant were both under the control of a single recessive gene, respectively. These results provide valuable germplasm resources for the improvement of cucumber genetics and functional genomic research.

CRISPR-Cas9-mediated construction of a cotton CDPK mutant library for identification of insect-resistance genes

Calcium-dependent protein kinases(CDPKs)act as key signal transduction enzymes in plants,especially in response to diverse stresses,including herbivory.In this study,a comprehensive analysis of the CDPK gene family in upland cotton revealed that GhCPKs are widely expressed in multiple cotton tissues and respond positively to various biotic and abiotic stresses.We developed a strategy for screening insect-resistance genes from a CRISPR-Cas9 mutant library of GhCPKs.The library was created using 246 single-guide RNAs targeting the GhCPK gene family to generate 518 independent T0 plants.The average target-gene coverage was 86.18%,the genome editing rate was 89.49%,and the editing heritability was 82%.An insect bioassay in the field led to identification of 14 GhCPK mutants that are resistant or susceptible to insects.The mutant that showed the clearest insect resistance,cpk33/74(in which the homologous genes GhCPK33 and GhCPK74 were knocked out),was selected for further study.Oral secretions from Spodoptera litura induced a rapid influx of Ca2+in cpk33/74 leaves,resulting in a significant increase in jasmonic acid content.S-adenosylmethionine synthase is an important protein involved in plant stress response,and protein interaction experiments provided evidence for interactions of GhCPK33 and GhCPK74 with GhSAMS1 and GhSAM2.In addition,virus-induced gene silencing of GhSAMS1 and GhSAM2 in cotton impaired defense against S.litura.This study demonstrates an effective strategy for constructing a mutant library of a gene family in a polyploid plant species and offers valuable insights into the role of CDPKs in the interaction between plants and herbivorous insects.

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China:Review and Perspective

With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Genetic analysis of GEFs and GDIs in rice reveals the roles of OsGEF5,OsGDI1,and OsGEF3 in the regulation of grain size and plant height

Guanine nucleotide exchange factors(GEFs)and guanine nucleotide-dissociation inhibitors(GDIs)regulate small GTPase proteins,which function as molecular switches in various signaling pathways,but their identification and functions in plants are not well understood.Using in-silico analysis and transgenic approaches,respectively,we dissected the evolutionary relationships and functions of all GEF and GDI genes in rice.Intron-exon distribution and phylogenetic analyses identified 30 GEF and 10 GDI genes in rice that shared close evolutionary relationships with other eukaryotes.Tissue-specific expression and co-expression analyses revealed that phylogenetically related genes had similar expression patterns.GEF and GDI genes were highly expressed in panicles,hulls,and stamens.Co-expression network analysis identified panicle and stamen-specific modules of co-expressed genes in both families.Mapping of these genes in known protein interactomes further identified two and one small G-protein sub-networks.A mutant library of GEF and GDI families was constructed by CRISPR knockout of each gene,and their genotypes and phenotypes were confirmed.Phenotype changes occurred with the mutation of only three genes(OsGEF5,OsGDI1,and OsGEF3).OsGEF5 and OsGDI1 single mutants exhibited significantly reduced height and longer and thinner grains,whereas OsGEF3 mutants had reduced grain length compared to the wild type.Haplotype and eGWAS analyses showed that natural variations in the three genes affected gene expression in reproductive tissues that were significantly associated with the phenotypic variation.BiFC assays demonstrated that GDI1 and GEF3 interacted with grain-size protein GS3,pointing to a role of these genes in the regulation of grain size and plant architecture connected to heterotrimeric G-proteins in rice.

Comparing Genetic Characteristics of Retrotransposon TOS17 During Different Tissue Culture Processes in the Rice Cultivars Nipponbare and Shishoubaimao

As a retrotransposon, TOS17 was a useful tool for rice genetic and functional genomic research. To ascertain the feasibility of constructing a TOS17 insertion mutation library in the rice cultivar Shishoubaimao, the genetic and expression characteristics of TOS17 were analyzed. We made solid and suspension tissue cultures and confirmed the copy numbers of TOS17 at different time points in both tissue culture processes by real-time quantitative PCR （RT-qPCR）. Three primary copies of TOS17 were detected in naturally grown Shishoubaimao. TOS17 was activated by tissue culture, and the copy numbers of TOSI7 increased along with a prolonged tissue culture time in both the Nipponbare and the Shishoubaimao cultivars. Therefore, Shishoubaimao is a potential candidate for constructing a TOS17 insertion mutant library. Compared with Nipponbare, TOS17 was more active in Shishoubaimao during tissue culture. Higher copy numbers of TOS17 were obtained with the suspension tissue culture process than with the solid tissue culture process over the same time courses. We concluded that 3-4 months of suspension tissue culture time is suitable for constructing a TOS17 insertion mutant library in Shishoubaimao.

TEAseq-based identification of 35,696 Dissociation insertional mutations facilitates functional genomic studies in maize

In plants,transposable element(TE)-triggered mutants are important resources for functional genomic studies.However,conventional approaches for genome-wide identification of TE insertion sites are costly and laborious.This study developed a novel,rapid,and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing(TEAseq).Using TEAseq,we systemically profiled the Dissociation(Ds)insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background.The Ac-containing individuals were excluded for getting rid of the potential somatic insertions.We characterized 35,696 germinal Ds insertions tagging 10,323 genes,representing approximately 23.3%of the total genes in the maize genome.The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci,and insertions occurred preferentially in gene body regions.Furthermore,we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development.We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations.Overall,our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.

An investigation of the possible methods and potential benefits of de novo cloning of Nannochloropsis oceanica genes

Species in the microalgal genus Nannochloropsis are increasingly used as models for theoretical and applied studies. Herewe attempt to generate InDei variations in the genome of Nannochloropsis oceanica, and then decipher the genetic basisof its economic and biological traits with bulked mutant analysis modified from bulked segregant analysis. In addition, wedescribe our efforts to construct site-tagged and gene-traceable mutant libraries to clone its genes through reverse geneticapproaches. Currently, more than a half of N. oceanica protein-encoding genes are annotated against databanks. However, nofunctional gene has been de novo cloned from N. oceanica and no new function has been assigned to any of its annotatablegenes. Here, we discuss the possible methods and potential benefits of de novo cloning of N. oceanica genes.