Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.
Plant J
; 118(6): 2296-2317, 2024 Jun.
Article
en En
| MEDLINE
| ID: mdl-38459738
ABSTRACT
Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.
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Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Reacción en Cadena de la Polimerasa
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Secuenciación de Nucleótidos de Alto Rendimiento
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Técnicas de Genotipaje
Idioma:
En
Revista:
Plant J
Asunto de la revista:
BIOLOGIA MOLECULAR
/
BOTANICA
Año:
2024
Tipo del documento:
Article