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In addition to the ten
linear DAF primers utilized in the present study as above, ten
additional mini-hairpin primers (Hirao et al. 1992; Caetano-Anolles
and Gresshoff 1994) were also evaluated for their utility in
detecting polymorphism. Mini-hairpin primers form a loop of 3-4
nucleotides, a stem of only two nucleotides and a core arbitrary
sequence of variable length (1-8 nucleotides) at the 3' terminal end.
This loop-stem region closes during early ramping (Caetano-Anolles
and Gresshoff 1994; Gresshoff et al. 1997). In the present study,
mini-hairpin primers are each made up of a core arbitrary sequence of
3 bases (in earlier studies, a core sequence of 3 bases was found
most appropriate) at the 3' terminus and a loop stem of 8 bases. Each
of the ten mini-hairpin primers listed in Table
2 could be tried,
each only on a single pair of genotypes of bread wheat differing for
one quality trait. Four of these primers (HPC 23, HPC 26, HPC 29 and
HPC 44) gave excellent DAF profiles with distinct scorable amplified
products (Fig.
2; Lanes 1, 2, 5
and 6). The remaining six mini-hairpin primers, however, gave only a
few faint bands superimposed on high background smear, suggesting
their limited utility in detecting polymorphism in a crop like bread
wheat (Fig.
2; Lanes 3 and
4).
The mini-hairpin primers were also examined for their utility in
relation to their GC/AT ratio as done above for linear DAF primers.
In this case also, higher GC content in the core sequence seems to
give a smear rather than distinct scorable DAF profile
(Fig.
2; Lanes 3 and
4). However, the presence of A or T nucleotide at the 3' end. of the
core sequence of the mini-hairpin primer, despite high GC content
(GC/AT=2: 1) gave excellent scorable DAF profiles (Fig.
2; Lanes 5 and
6). The number of amplification products (total number of bands of
<2kb) obtained with mini-hairpin primers, was almost double to
that obtained with a linear primer even at an annealing temperature
of 55C (Table
1 and
Table
2). This may be
due to a smaller core sequence (3-mer) of the primer involved in
annealing, which is provided with greater thermostability by the
hairpin loop during annealing (Caetano-Anolles and Gresshoff 1994;
Gresshoff et al. 1997).
While making an overall assessment of the utility of DAF primers in
bread wheat in terms of producing polymorphic bands, during the
present study, we found that neither the average number, nor the
proportion of polymorphic DAF products obtained with the mini-hairpin
primers was greater than those obtained with linear primers in bread
wheat (Table
1 and
Table
2). These results
thus do not support the earlier contention, that for detecting
polymorphism, the mini-hairpin primers are superior over the linear
primers. Since these earlier results pertain to smaller genome of
centipedegrass (Caetano-Anolles and Gresshoff 1994), bermudagrass
(Caetano-Anolles et al. 1995) and soybean (Caetano-Anolles and
Gresshoff 1996), it is possible that the relative utility of
mini-hairpin primers differs in different plant systems.
The genotypes of bread wheat utilized in the present study were also
earlier analyzed by us using RAPDs and microsatellite primed PCR
(MP-PCR), both of which failed to detect reproducible polymorphism
among the above genotypes. DAF, on the other hand, revealed
polymorphism between pairs of genotypes differing for individual
traits e.g. protein content, grain size, preharvest sprouting
tolerance and, leaf rust resistance (Table
1 and
Table
2). Similar
conclusions were earlier drawn by Prabhu and Gresshoff (1994), while
examining two species of Glycine. Using DAF they obtained
three fold increase in polymorphism per primer between G. max
and G. soja over the polymorphism revealed using RAPDs on
the same Glycine species (Williams et al. 1990). We therefore,
conclude that DAF technology may become a useful tool for detecting
polymorphism and for developing molecular markers for tagging genes
for molecular marker aided selection in plant breeding even for a
difficult crop like bread wheat.
Acknowledgments
We gratefully acknowledge the financial support from the
Department of Biotechnology, Government of India, for carrying out
this study. Thanks are also due to Drs. G. Caetano-Anolles and PM
Gresshoff for supplying DAF primers, and Drs. Harjit Singh and H S
Dhaliwal for seed materials.
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