A novel method of multiplex SNP genotyping assay through variable fragment length allele-specific polymerase chain reaction: Multiplex VFLASP-ARMS.

Variable Fragment Length Allele-Specific Polymerase Chain Reaction (VFLASP) and Amplification Refractory Mutation System (ARMS) are reliable methods for detecting allelic variations resulting from single base changes within the genome. Due to their widespread application, allele variations caused by Single Nucleotide Polymorphisms (SNPs) can be readily detected using allele-specific primers. In the context of the current study, VFLASP was combined with ARMS method as a novel strategy to enhance the efficacy of both techniques. Clinically important base variations within SNP regions used in the study were detected by a fragment analysis method. To validate the accuracy of the developed VFLASP-ARMS method, specifically designed synthetic sequences were tested using a capillary electrophoresis system. Allele-specific primers exhibit differences solely at the 3' end based on the sequence of the SNP. Additionally, to increase the specificity of the primers, a base was intentionally added for incompatibility. Therefore, allele discrimination on fragment analysis has been made possible through the 3-6 bp differences in the amplicons. With the optimization of the system, designed synthetic sequences provided reliable and reproducible results in wild-type, heterozygous, and homozygous genotypes using the VFLASP-ARMS method. Hence, our results demonstrated that VFLASP-ARMS method, offers a novel design methodology that can be included in the content of SNP genotyping assays.

Gene-Editing Technologies and Applications in Legumes: Progress, Evolution, and Future Prospects.

Legumes are rich in protein and phytochemicals and have provided a healthy diet for human beings for thousands of years. In recognition of the important role they play in human nutrition and agricultural production, the researchers have made great efforts to gain new genetic traits in legumes such as yield, stress tolerance, and nutritional quality. In recent years, the significant increase in genomic resources for legume plants has prepared the groundwork for applying cutting-edge breeding technologies, such as transgenic technologies, genome editing, and genomic selection for crop improvement. In addition to the different genome editing technologies including the CRISPR/Cas9-based genome editing system, this review article discusses the recent advances in plant-specific gene-editing methods, as well as problems and potential benefits associated with the improvement of legume crops with important agronomic properties. The genome editing technologies have been effectively used in different legume plants including model legumes like alfalfa and lotus, as well as crops like soybean, cowpea, and chickpea. We also discussed gene-editing methods used in legumes and the improvements of agronomic traits in model and recalcitrant legumes. Despite the immense opportunities genome editing can offer to the breeding of legumes, governmental regulatory restrictions present a major concern. In this context, the comparison of the regulatory framework of genome editing strategies in the European Union and the United States of America was also discussed. Gene-editing technologies have opened up new possibilities for the improvement of significant agronomic traits in legume breeding.

Transgenic tobacco plants overexpressing a cold-adaptive nitroreductase gene exhibited enhanced 2,4-dinitrotoluene detoxification rate at low temperature.

Plants encounter many environmental factors such as low and high temperatures during phytoremediation processes. In this study, our aim was to produce the transgenic tobacco plants by using a newly characterized bacterial nitroreductase, Ntr, which was active at a broad range temperature in order to detoxify 2,4-dinitrotoluene (2,4-DNT) at lower temperature. The presence of Ntr and its heterologous expression was verified in T1 transgenic plants and their growing ability were determined under toxic amount of 2,4-DNT (35 µM). Fresh weight and dry weight of transgenic plants were significantly higher than wild type (WT) under toxic 2,4-DNT at 22 °C, indicating higher growth capacity of the transgenics. Transgenic plants also showed a higher tolerance than WT when exposed to 2,4-DNT at 15 °C. Moreover, transformation rate of 2,4-DNT was gradually decreased through decreasing temperatures in WT media, however, it was increased through decreasing temperatures in transgenic plant TR3-25 media and it had the highest transformation rate (54%) of 2,4-DNT at 4 °C. Correlatively, 2,4-DNT treatment at 4 °C led to a significant decrease in H2O2 level in transgenic plants. Thus, transgenic plants overexpressing nitroreductase might have an important advantage for phytoremediation of toxic nitroaromatic compounds in field applications at low temperatures.