Cargando…

Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds

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 roo...

Descripción completa

Detalles Bibliográficos
Autores principales: Shioya, Naohiro, Ogiso-Tanaka, Eri, Watanabe, Masanori, Anai, Toyoaki, Hoshino, Tomoki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457885/
https://www.ncbi.nlm.nih.gov/pubmed/37631182
http://dx.doi.org/10.3390/plants12162971
_version_ 1785097031856947200
author Shioya, Naohiro
Ogiso-Tanaka, Eri
Watanabe, Masanori
Anai, Toyoaki
Hoshino, Tomoki
author_facet Shioya, Naohiro
Ogiso-Tanaka, Eri
Watanabe, Masanori
Anai, Toyoaki
Hoshino, Tomoki
author_sort Shioya, Naohiro
collection PubMed
description 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 A(260/280) and A(260/230) 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 N(50) 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.
format Online
Article
Text
id pubmed-10457885
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-104578852023-08-27 Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds Shioya, Naohiro Ogiso-Tanaka, Eri Watanabe, Masanori Anai, Toyoaki Hoshino, Tomoki Plants (Basel) Article 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 A(260/280) and A(260/230) 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 N(50) 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. MDPI 2023-08-17 /pmc/articles/PMC10457885/ /pubmed/37631182 http://dx.doi.org/10.3390/plants12162971 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Shioya, Naohiro
Ogiso-Tanaka, Eri
Watanabe, Masanori
Anai, Toyoaki
Hoshino, Tomoki
Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds
title Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds
title_full Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds
title_fullStr Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds
title_full_unstemmed Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds
title_short Development of a High-Quality/Yield Long-Read Sequencing-Adaptable DNA Extraction Method for Crop Seeds
title_sort development of a high-quality/yield long-read sequencing-adaptable dna extraction method for crop seeds
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10457885/
https://www.ncbi.nlm.nih.gov/pubmed/37631182
http://dx.doi.org/10.3390/plants12162971
work_keys_str_mv AT shioyanaohiro developmentofahighqualityyieldlongreadsequencingadaptablednaextractionmethodforcropseeds
AT ogisotanakaeri developmentofahighqualityyieldlongreadsequencingadaptablednaextractionmethodforcropseeds
AT watanabemasanori developmentofahighqualityyieldlongreadsequencingadaptablednaextractionmethodforcropseeds
AT anaitoyoaki developmentofahighqualityyieldlongreadsequencingadaptablednaextractionmethodforcropseeds
AT hoshinotomoki developmentofahighqualityyieldlongreadsequencingadaptablednaextractionmethodforcropseeds