As the source of polymorphic DNA markers for map construction, we used two types of cDNA clones (callus and roots), three types of genomic clones (randomly selected clone, NotI-linking clone, YAC end-clone) and RAPD (Randomly amplified polymorphic DNA) markers, all of which were derived from the japonica rice cultivar, Niponbare. About 90 other DNA clones derived from wheat and other rice varieties were also utilized.

A NotI-linking clone is a Sau3AI digested Nipponbare genomic DNA fragment having NotI site in it. For the selection from the other fragments, NotI-linking clones were cloned with a hygromycin resistance gene at the NotI site. Therefore, genomic inserts of NotI-linking clones were divided by inserting the hygromycin resistance gene in the middle of the cloned fragment. When we map NotI-linking clone, we prepare a PCR product by special primers, named T3, T7, Nos, and 35S, or use a digested fragment of a pair of restriction enzymes.
The sequences are as follows:
T3 5'-ATTAACCCTCACTAAAG-3'
T7 5'-AATACGACTCACTATAG-3'
35S 5'-TGTGGGTTAGCATTCTTTCTG-3'
Nos 5'-TTACTAGATCGGGAATTGCCA-3'

Most of the probe sizes are the PCR product sizes by M4 and RV primers.
M4 5'-GTTTTCCCAGTCACGAC-3'
RV 5'-CAGGAAACAGCTATGAC-3'

The probe sizes of the W-markers are the original insert sizes communicated by Dr. Michael Gale, John Innes Centre, Norwich, UK.
Further analysis was performed with MAPMAKER/EXP 3.0.

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