Method for high-throughput aflp-based polymorphism detection

Abstract

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.

1 . A kit for use in a method for detecting genetic variation in one or more members of a population, comprising: (a) an adaptor adapted for ligation to a plurality of nucleic acid fragments, and (b) a set of primers for PCR amplification having a 5′-end and a 3′-end, wherein the primers comprise one or more selective nucleotides at the 3′ end, and wherein at least one of the adaptor and the primers comprises a sample-specific identifier sequence capable of indicating sample origin of an amplification product. 
     
     
         2 . The kit according to  claim 1 , wherein the adaptor comprises the sample-specific identifier sequence. 
     
     
         3 . The kit according to  claim 1 , wherein at least one of the primers comprises the sample-specific identifier sequence. 
     
     
         4 . The kit according to  claim 1 , further comprising one or more restriction enzymes for producing the plurality of nucleic acid fragments. 
     
     
         5 . The kit according to  claim 4 , wherein the restriction enzymes comprise restriction enzymes producing blunt ended restriction fragments. 
     
     
         6 . The kit according to  claim 4 , wherein the restriction enzymes comprise restriction enzymes producing sticky ended restriction fragments. 
     
     
         7 . The kit according to  claim 6 , wherein the restriction enzymes comprise EcoRI and/or MseI. 
     
     
         8 . The kit according to  claim 1 , wherein the adaptor comprises a 3-′T overhang. 
     
     
         9 . The kit according to  claim 1 , wherein the adaptor comprises a PCR primer-binding sequence for hybridizing to a PCR primer. 
     
     
         10 . The kit according to  claim 1 , wherein the adaptor comprises a sequencing primer-binding sequence for hybridizing to a sequencing primer. 
     
     
         11 . The kit according to  claim 1 , wherein the adaptor comprises sequences complementary to sequences attached to a solid support for annealing the adaptor to a solid support. 
     
     
         12 . The kit according to  claim 1 , wherein the adaptor comprises sequences complementary to sequences attached to a bead for annealing the adaptor to a bead. 
     
     
         13 . The kit according to  claim 1 , wherein the adapter is composed of two synthetic oligonucleotides which have nucleotide sequences which are partially complementary to each other. 
     
     
         14 . The kit according to  claim 1 , wherein at least one of the primers is phosphorylated. 
     
     
         15 . The kit according to  claim 1 , wherein at least one of the primers comprises sequences at its 3′-end for hybridizing to a subset of nucleic acid sample fragments. 
     
     
         16 . The kit according to  claim 1 , wherein the kit further comprises a biotinylated capture oligonucleotide hybridization probe for capturing a subset of nucleic acid sample fragments. 
     
     
         17 . The kit according to  claim 1 , wherein the kit further comprises a polymerase for a polymerase chain reaction (PCR). 
     
     
         18 . The kit according to  claim 17 , wherein the polymerase substantially lacks 3′-5′ exonuclease activity.