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Method for the identification of single mutations in large genomic regions using massive parallel sequencing

Content Provider	Springer Nature Link
Author	Sturre, Marcel J. G. Shirzadian Khorramabad, Reza Schippers, Jos H. M. Chin A Woeng, Thomas F. C. Hille, Jacques Dijkwel, Paul P.
Copyright Year	2008
Abstract	Map-based cloning of mutant genes is straightforward if the genome sequence and sufficient molecular markers are available. When a mutated gene in Arabidopsis causes a clear phenotype and is located in a genomic region where sufficient meiotic recombination takes place, the gene can be identified within 6–12 months. However, mutated genes that cause weak phenotypes are difficult to map to small genomic intervals due to faulty selection of F2 plants. Here, we describe a method that allows for rapid identification of roughly mapped genes by using a massive parallel sequencing strategy. A genomic region of 150 kb was PCR amplified in 7–17 kb pieces from an EMS Arabidopsis onset of leaf death (old) mutant and its wild-type accession Landsberg erecta (Ler-0). Massive parallel sequencing and subsequent de novo assembly of the short sequences reliably identified 253 polymorphisms in a 110-kb region between the reference Col-0 and Ler-0 sequence. The analysis further revealed potential mutations in the old mutant of which one was confirmed to be present in the mutant. Thus the described method can be used for accelerating the map-based cloning of genes that cause weak phenotypes. An accompanying advantage is that the amplified fragments can be cloned and used to complement the mutant.
Starting Page	51
Ending Page	59
Page Count	9
File Format	PDF
ISSN	13803743
Journal	Molecular Breeding
Volume Number	23
Issue Number	1
e-ISSN	15729788
Language	English
Publisher	Springer Netherlands
Publisher Date	2008-08-17
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	EMS mutagenesis Mutant Map-based cloning Massive parallel sequencing SSAKE SNP Biotechnology Plant Physiology Plant Sciences Plant Pathology Plant Genetics & Genomics
Content Type	Text
Resource Type	Article
Subject	Genetics Plant Science Molecular Biology Biotechnology Agronomy and Crop Science

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