RESUMO
Genome sequencing is important for discovering critical genes in crops and improving crop breeding efficiency. Generally, fresh, young leaves are used for DNA extraction from plants. However, seeds, the storage form, are more efficient because they do not require cultivation and can be ground at room temperature. Yet, only a few DNA extraction kits or methods suitable for seeds have been developed to date. In this study, we introduced an improved (IMP) Boom method that is relatively low-cost, simple to operate, and yields high-quality DNA that can withstand long-read sequencing. The method successfully extracted approximately 8 µg of DNA per gram of seed weight from soybean seeds at an average concentration of 48.3 ng/µL, approximately 40-fold higher than that extracted from seeds using a common extraction method kit. The A260/280 and A260/230 values of the DNA were 1.90 and 2.43, respectively, which exceeded the respective quality thresholds of 1.8 and 2.0. The DNA also had a DNA integrity number value (indicating the degree of DNA degradation) of 8.1, higher than that obtained using the kit and cetyltrimethylammonium bromide methods. Furthermore, the DNA showed a read length N50 of 20.96 kbp and a maximum read length of 127.8 kbp upon long-read sequencing using the Oxford Nanopore sequencer, with both values being higher than those obtained using the other methods. DNA extracted from seeds using the IMP Boom method showed an increase in the percentage of the nuclear genome with a decrease in the relative ratio of chloroplast DNA. These results suggested that the proposed IMP Boom method can extract high-quality and high-concentration DNA that can be used for long-read sequencing, which cannot be achieved from plant seeds using other conventional DNA extraction methods. The IMP Boom method could also be adapted to crop seeds other than soybeans, such as pea, okra, maize, and sunflower. This improved method is expected to improve the efficiency of various crop-breeding operations, including seed variety determination, testing of genetically modified seeds, and marker-assisted selection.
RESUMO
Induced mutation is a viable breeding strategy that is widely utilized in the development of elite plant varieties. We aimed to improve a variety of edamame by constructing novel mutant populations using the ethyl methanesulfonate in soybeans (Glycine max (L.) Merr.). In the M2 population, the flowering stage showed a considerable standard deviation compared to the wild type, confirming that the mutant populations had the expected DNA mutations. To identify the DNA mutations in the mutant populations, we used the targeting induced local lesions in genomes (TILLING) method, which is a reverse genetic method, to search for soybean flowering-related gene mutants. A total of 30 mutants from E1, E3, E4, and PhyA1 genes, which are known to be highly effective genes, or their homologous gene for flowering and maturation found in soybean quantitative trait locus analyses were isolated from our TILLING screening. Among these mutants, there were eleven nonsynonymous substitution mutants, one nonsense mutant, and two single nucleotide deletion mutants that could be expected to reduce or eliminate gene function. The e1, e3, and e4 mutants obtained in this study flowered considerably earlier than the wild type. In particular, the e1 mutant with a nonsynonymous substitution flowered approximately 1 month after sowing regardless of the sowing date, and its harvest date was approximately 1 month earlier than that of the wild type. Mutations identified using the TILLING method could not only be used as gel-based DNA markers with the same manipulation method, but the mutations could also be detected as DNA markers by the high-resolution melting method. These results indicate that mutations achieved without chromosome modification by crossbreeding are effective for early and practical improvement of superior varieties and that efficient selection of mutants by reverse genetics is an effective method for the identification of genetic modifications. The edamame mutant populations developed in this study are believed to possess various useful alleles which may be applicable in the search for mutations that lead to improved edamame yield and eating quality beyond the flowering stage.
