AFLP-based mRNA fingerprinting

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 µl 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′-AGTCTGCAGTi 2 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 42 °C using 0.5 µg of the oligonucleotide mix, 1 µg mRNA and 200 U reverse transcriptase, omitting [α- 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 l (New England Biolabs). Digested cDNA was ligated with an Mse I-adapter and a biotinylated Pst l-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. Sixteen amplification products, which differed between Chinese Spring and the deletion mutant, were excised and reamplified. Purified reamplification products were labelled and hybridised with various digests of genomic DNA from Chinese Spring and the deletion mutant. Five of the products gave similar hybridisation patterns and these five probes were subsequently found to cross-hybridise with the 18S-5.8S-26S ribosomal RNA genes of wheat. The remaining 11 probes hybridised to single or low copy sequences. Two of these (M12B and M48A) detected DNA fragments that are present in Chinese Spring but absent in the deletion mutant ( Fig. 1 b). Further analysis revealed that the fragments are located on chromosome 3A (data not shown). The reproducibility of the technique was demonstrated by synthesising fresh cDNA from Chinese Spring and deletion mutant mRNA and processing this (including non-selective preamplification) as described. In the selective amplification, 10 different Mse I primers were used for direct comparison of the banding patterns obtained using the duplicate cDNA templates. There was little or no variation between the two batches of cDNA and differences between Chinese Spring and the mutant were consistent ( Fig. 1 a). We have shown that the AFLP technique can be modified to allow display of mRNAs and used to isolate sequences mapping to deleted chromosome segments in hexaploid wheat. Since our protocol used existing Mse I-adapters and primers, cDNA sequences forming an MseI recognition site (5′-TTAA-3′) at the junction of the poly A tail (∼8%) were eliminated from the analysis. We intend to modify the technique to make use of restriction enzymes with different 4 bp recognition sites. ( a ) A typical RNA fingerprinting gel produced using eight different Mse l primers with one (M02), two (M12, M15) or three (M35, M36, M37, M38, M48) selective bases. With each Msel primer, AFLP was performed using duplicate cDNA templates derived from the deletion mutant in the first two lanes and Chinese Spring in the second two. Bands M12B and M48A, which are present in Chinese Spring but absent in the mutant, are indicated. ( b ) Autoradiographs showing digests of DNA from Chinese Spring and the deletion mutant probed with reamplified M12B and M48A. Fragments absent in the mutant are indicated.