Further localization on Southern blots by looking for additional breakpoint fragments revealed the order and relative positions of restriction enzyme sites around the telomeric limit of the duplicated segment.
The resulting sequences, which were trimmed to 69 bp, were aligned to the Nipponbare sequence to determine the position of the restriction sites (DArT markers) and the position of the polymorphisms within the 69-bp sequence (SNP markers).
Therefore the position of the restriction sites is critical.
Note that the frequency of mutation observed by enrichment PCR is influenced by the position of the restriction enzyme site.
T-RFLP is a molecular biology technique developed for profiling bacterial communities based on the position of a restriction site closest to a labeled end of an amplified gene [ 29].
Unfortunately, the routine use of gDNA clones has been impeded by difficulties associated with their large size and its effect on cloning efficiency, as well as the availability and position of unique restriction enzyme sites to enable conventional cloning procedures.
However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures.
Thus, one member of the read pair should map to the exact position of the restriction digestion, while the other member should map approximately 100 200 bp away from this position as a result of the 'nick-translation' procedure (Additional file 2: Figure S4).
Another technology is optical mapping [ 12– 16], a method based on mapping the position of enzyme restriction sites along the sequence of the genome as observed by fluorescence microscopy, which was automated [ 17, 18] to achieve high-throughput solutions amenable to the analysis of complex genomes.
To obtain information about the physical positions of anchor restriction fragment length polymorphism (RFLP) markers for the mapping of QTLs, the program BlastN was used to compare the nucleotide sequences of RFLP probes against rice genome sequences.
The sequences of primers and the positions of the restriction enzymes used are illustrated in table 1.
