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.

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.

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.

A 2-bp insertion(c.6768insCC) in MC1R causes recessive white coat color in Bama miniature pigs

Coat color is an important characteristic of various breeds of domestic animal species.Variation in farm animal coat color is of considerable interest for concealment,communication and protection against solar radiation（Slominski et al.,2004）.It also plays an important role in the regulation of physiological processes（Miyagi and Terai,2013）.