Screening of Leaf Shape Mutants Induced by EMS and Analysis of Agronomic Traits in Azuki Bean (Vigna angularis)

[Objective] M3 progenies of Jingnong 6 variety induced by EMS chemical mutagenesis were screened and identified for obtaining valuable mutation material.[Method] Azuki bean cultivar Jingnong 6 was treated with EMS.The mutation rate,mutation types,agronomic traits and yield components of the leaf mutants were analyzed.[Result] The results showed that there is the most abundant mutational type of leaf shape and the highest mutation frequency treated with 0.9% EMS for 24 hours.Comprehensive analysis on agronom...

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.

A simple and efficient method for CRISPR/Cas9-induced mutant screening

The clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated protein 9（Cas9） system provides a technological breakthrough in mutant generation. Several methods such as the polymerase chain reaction（PCR）/restriction enzyme（RE） assay, T7 endonuclease I（T7EI） assay, Surveyor nuclease assay, PAGE-based genotyping assay, and high-resolution melting（HRM） analysis-based assay have been developed for screening CRISPR/Cas9-induced mutants. However, these methods are timeand labour-intensive and may also be sequence-limited or require very expensive equipment. Here, we described a cost-effective and sensitive screening technique based on conventional PCR, annealing at critical temperature PCR（ACT-PCR）, for identifying mutants. ACT-PCR requires only a single PCR step followed by agarose gel electrophoresis. We demonstrated that ACT-PCR accurately distinguished CRISPR/Cas9-induced mutants from wild type in both rice and zebrafish. Moreover, the method can be adapted for accurately determining mutation frequency in cultured cells. The simplicity of ACT-PCR makes it particularly suitable for rapid, large-scale screening of CRISPR/Cas9-induced mutants in both plants and animals.

A High-Throughput Method for Screening Arabidopsis Mutants with Disordered Abiotic Stress-Induced Calcium Signal

It is established that different stresses cause signal-specific changes in cellular Ca2 ~ level, which function as messengers in modulating diverse physiological processes. These calcium signals are important for stress adaptation. Though numbers of downstream components of calcium signal cascades have been identified, upstream events in calcium signal remain elusive, specifically components required l＇~~r calcium signal generation due to the lack of high-throughput genetic assay. Here, we report the development of an easy and efficient method in a forward genetic screen for Ca2＋ signals-deficient mutants in Arahidopsis thaliana. Using this method, 121 mutants with disordered NaCI- and H=O2-induced Ca2＋ signals are isolated.