NPI
With the development of the polymerase chain reaction (PCR, Saiki et al., Science 230:1350-1354, 1985), it has become possible to amplify homologous regions from many different individuals and sequence them directly to compare any DNA sequence variation in the amplified regions. Inserts from clones used previously to detect RFLPs amongst maize cultivars (Helentjaris, Trends Genet. 3:217-221, 1987) were sequenced completely. Pairs of oligonucleotide primers were synthesized from each end of the inserts, approximately 500 to 1000bp apart, so that their 5' to 3' orientations were directed towards each other. Using standard conditions from the original papers, these primers were used to amplify homologous regions from 7 to 9 maize cultivars. The PCR products were then purified from agarose gels and sequenced using a Sequenase kit.
Some sample data for maize are shown in the accompanying table. It can be seen for these two regions that there are multiple DNA sequence variations between these cultivars. We estimate from our current data that there are 30 to 50 alterations per 1000bp. Both base pair changes and sequence rearrangements of various sizes are seen. Interestingly, a tandem duplication of 17bp in the 451 region of group 4 (underlined) is followed by a very similar 8bp tandem duplication less than 200bp away. In the group 5 lines neither of these sequences is duplicated. A large insertion of several hundred base pairs was seen in the 288 region in B14A (not shown) but was not found to be related to any of the transposable element sequences in the Genbank database. We are currently expanding both the amount of sequence information on specific regions and the number of individuals evaluated.
Using this type of analysis, we feel that it will now be much more practical
to compare homologous regions of numerous individuals at the DNA sequence
level. It is also interesting that in melon we have found by similar analysis
that the rate of sequence variation is about tenfold less than that seen
for maize. Since short tandem duplications and rearrangements can be generated
by transposable element activity, it is tempting to speculate that the
high level of polymorphism observed in maize is the result of transposable
element activity. To date no natural transposable elements have been identified
in tomato and melon, and both exhibit little polymorphism. It should also
be noted that the species which exhibit higher levels of RFLP variation
reproduce primarily by outcrossing whereas the less polymorphic species
are primarily self-pollinated. The different modes of reproduction may
affect the level of transposable element activity itself or it may be that
a high mutation frequency resulting from transposable element activity
or any other mechanism can not be tolerated in a self-pollinated species
and is therefore selected against. At this point we remain at the speculation
stage but the PCR method of analyzing sequences will allow us to examine
the question "Why do different species exhibit different levels of RFLP
variation?".
Return to the MNL 63 On-Line Index
Return to the Maize Newsletter Index
Return to the MaizeGDB Homepage