METHOD FOR HIGH-THROUGHPUT AFLP-BASED POLYMORPHISM DETECTION

摘要

The invention relates to a method for the high throughput discovery, detection and genotyping of one or more genetic markers in one or more samples, comprising the steps of restriction endonuclease digest of DNA, adaptor-ligation, optional pre-amplification, selective amplification, pooling of the amplified products, sequencing the libraries with sufficient redundancy, clustering followed by identification of the genetic markers within the library and/or between libraries and determination of (co-)dominant genotypes of the genetic markers.

主权项

1. A method for genotyping genetic markers in a sample, comprising the steps of:
(a) reducing complexity of at least two nucleic acid samples using amplified fragment length polymorphism (AFLP) to produce at least two reproducible subsets of amplified adaptor-ligated restriction fragments from the at least two nucleic acid samples, wherein each reproducible subset comprises a unique identifier tag; (b) sequencing a pool of the at least two reproducible subsets of amplified adaptor-ligated restriction fragments using sequencing by synthesis on a solid support; (c) aligning the sequences of the reproducible subsets of amplified adaptor-ligated restriction fragments; (d) identifying AFLP and/or single-nucleotide polymorphism (SNP) markers within a selection of amplified adaptor-ligated restriction fragments that are sequenced with a redundancy of at least 6; and (e) determining dominant or co-dominant genotypes of the genetic markers in the selected amplified adaptor-ligated restriction fragments that are sequenced with the redundancy of at least 6.