AFLP, a robust and rapid technique for displaying large numbers of DNA
polymorphisms, is being used extensively for genetic mapping and fingerprinting
in plants (
1
). Use of this technique avoids problems which may be encountered with
reproducibility and optimisation of reaction conditions when using arbitrarily
primed PCR. Since the primer sets are readily available (Life Technologies and
Perkin Elmer), we have explored whether AFLP could be applied to generating
mRNA fingerprints in polyploid crop plants. In polyploid species, mutant stocks
can be created by the deletion of short chromosome segments on one of the
constituent homoeologous genomes. Messenger RNA fingerprints would be useful
for identifying genes located within these deleted segments. However, the
expression of monomorphic mRNAs from homoeologous genes in polyploids would
make fingerprints indistinguishable when comparing normal and deletion stocks. If the homoeologous gene sequences were polymorphic, e.g. for a
restriction enzyme site, then their AFLP fingerprints would be easily
distinguishable and the expressed sequences could be genetically mapped. To
test the sensitivity of this approach we used hexaploid wheat (2n = 6x arranged
in seven groups of three homoeologous chromosome pairs) and one of its deletion
mutants.
Messenger RNA was extracted from immature spikes taken from wheat cv Chinese
Spring and a mutant of this variety with deletions on chromosomes 3A and 5B (
2
,
3
). We used an mRNA Quickprep kit (Pharmacia) following the manufacturer's instructions, but including treatment with DNase I before final precipitation. The mRNA was dissolved in 100 [mu]l dH
2
O and purified using an RNeasy kit (Qiagen).
Synthesis of double-stranded cDNA was performed according to instructions supplied with
Superscript reverse transcriptase (Life Technologies). An equimolar mixture of three oligonucleotides with the sequence 5'-AGTCTGCAGT
12
V-3' (where V denotes A, C or G) was used to prime first strand cDNA
synthesis. As this primer contains a recognition sequence for
Pst
I, the double-stranded cDNA can be cut with this restriction enzyme. This dual-purpose primer was designed to enable us to make use of available
AFLP adapter and primer stocks without having to modify the PCR conditions used
in the technique. The variable 3' nucleotide adjacent to the T
12
tract ensures that synthesis begins at the junction between the poly A tail and
the sequence at the 3' end of the mRNA (
4
,
5
). First strand cDNA synthesis was performed at 42oC using 0.5 [mu]g of the oligonucleotide mix, 1 [mu]g mRNA and 200 U reverse transcriptase, omitting [[alpha]-
32
P]dCTP but including RNAguard RNase inhibitor (Pharmacia). The cDNA was purified
using a QiaQuick column (Qiagen).
After second strand synthesis, cDNA was digested with 5.0 U each of
Pst
I (NBL) and
Mse
I (New England Biolabs). Digested cDNA was ligated with an
Mse
I-adapter and a biotinylated
Pst
I-adapter [patent application, Zabeau and Vos (1993), EP 0534858] and
affinity-purified using streptavidin-linked paramagnetic beads (Dynal). Preamplification was carried out
using non-selective primers to conserve purified cDNA stocks. All subsequent steps
were performed as previously reported for genomic DNA AFLP (
1
). Labelled selective amplification products were run on standard 6% acrylamide
sequencing gels and visualised by exposure to Kodak BioMax-MR film for ~50 h.
RNA fingerprints were generated from Chinese Spring and the deletion mutant cDNA
templates using 49
Mse
I-primers with two or three selective bases. Amplification products ranged
in size from <100 to >600 bp. Examples of the fingerprints obtained are shown in Figure
1
a.
REFERENCES
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