Two apple repetitive sequence elements - Springer Link

Euphytica 131: 177–187, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

177

Two apple repetitive sequence elements: characterisation and potential use as genetic markers A.M. Hadonou, J.R. Gittins1, E.R. Hiles & D.J. James Plant Breeding and Biotechnology, Horticulture Research International, East Malling, Kent ME19 6BJ, U.K.; 1 Present address: Laboratory of Plant Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi´nskiego 5A, 02-106 Warsaw, Poland Received 7 May 2002; accepted 2 December 2002

Key words: apple, fingerprinting, genetic markers, Rosaceae, transposable elements

Summary Within the 5
flanking regions of two ripening-related genes from apple (Malus pumila), two short repeat sequences were identified and named Ars1 and Ars2 (Apple repetitive sequences). Both elements were present in the promoter of the Md-ACS1-1 ACC synthase gene and Ars2 was also found in the promoter of a gene homologous to the ABG1 β-galactosidase gene. Database searches revealed other examples of each element in apple genomic sequences and a partial Ars2 element in a sequence from Asian pear (Pyrus pyrifolia). DNA dot blot analysis demonstrated that both elements are present in high numbers in the apple genome. Ars1 is thought to represent a MITE (miniature inverted-repeat transposable element) related to the MITE Alien, while the identity of Ars2, though it is undoubtedly a transposable element, remains undetermined. DNA dot blot analysis and PCR amplification using primers designed from Ars1 and Ars2 showed that elements similar to Ars1 and Ars2 are present across the Rosaceae. The amplification patterns obtained using Ars1- and Ars2-specific primers with genomic DNA from different cultivars or mapping populations suggest that these elements could be used for fingerprinting and segregation analysis in Rosaceae. Evidence from DNA database searches suggests that they may be applicable to genetic studies in a wide range of other organisms.

Introduction Transposable elements are ubiquitous mobile genetic elements that can constitute over 50% of a plant nuclear genome (Kumar & Bennetzen, 1999). They are divided into two broad classes according to their method of proliferation (Finnegan, 1992; Charlesworth et al., 1994). Class I, or retroelements, transpose by reverse transcription of an RNA intermediate, and once inserted into the genome at a new location they cannot excise. This class contains retrotransposons and potential retroviruses with long terminal repeats (LTRs) and also non-LTR elements including LINEs, SINEs (long and short interspersed nuclear elements) and processed pseudogenes. Class II elements, or DNA transposons can excise and insert and so may move from one nuclear location to another. Some encode

the factors to mediate their own transposition while others are non-autonomous, relying on the activity of a transposase encoded at a separate locus. SINEs are the predominant Class I element in mammalian genomes (eg Alu; Rowold & Herrera, 2000) although their abundance in plants is more variable. In tobacco it has been estimated that up to 50,000 SINE elements are present in the haploid genome (Yoshioka et al., 1993), while in rice and Arabidopsis they are far less abundant (Le et al., 2000; Turcotte et al., 2001). SINEs are small elements (∼100–300 bp) that are derived from genes encoding small RNAs such as 5S rRNAs, tRNAs and small nuclear and cytoplasmic RNAs. They characteristically have either a putative RNA pol III promoter with conserved A and B box motifs, a long target site duplication and/or a poly A+T tail at one terminus (Yoshioka et al., 1993;

178 Gilbert & Labuda, 1999). SINEs lack coding capacity and are thought to rely on functions encoded by the host genome and LINEs for their proliferation (Gilbert & Labuda, 1999). MITEs (miniature inverted-repeat transposable elements) are a large family of small transposable elements recently recognised in plants (Wessler et al., 1995), which are also found in the genomes of diverse non-plant species (Zhang et al., 2000). They are characterised by their small size (∼80–500 bp), low G+C content, the absence of any coding capacity, high copy number (∼1,000–15,000 per haploid genome), target site preference (A + T rich, 2–3 nt) and terminal inverted repeats which, in some cases, are long enough to form stable stem-loop secondary structures (Wessler et al., 1995). MITEs are believed to represent non-autonomous Class II transposable elements which have been derived from larger autonomous elements (Zhang et al., 2001). It has recently been reported (Jiang et al., 2003) that Mite actively transposes with the assistance of related transposase encoding elements. In plants, MITEs are frequently associated with genes (Wessler, 1998; Zhang et al., 2000). There are a number of examples where MITEs contain recognised cis-acting elements or polyA addition sequences and so they may have played (and are still playing) an important role in gene regulation (Bureau & Wessler, 1994; Wessler et al., 1995; Bureau et al., 1996). However, because most identified MITEs are present at a particular locus in all members of a species, their evolutionary impact has been hard to assess. Few transposable elements have been described in apple. A putative SINE was identified in the promoter region of an allele of the ACC synthase gene (MdACS1-2; Sunako et al., 1999). This insertion apparently prevents transcription of this gene and fruit from cultivars which are homozygous for this allele produce little ethylene and have a long storage life. Recently, the insertion of a large LTR-type retrotransposon into introns of the apple pistillata gene homologue (MdPI) has been reported to be the cause of the parthenocarpic phenotype in a number of cultivars (Yao et al., 2001). James et al. (2001) have isolated and sequenced the 5
flanking regions for the apple genes encoding Md-ACS1-1 ACC synthase (Md-ACS1-1; 5.4 kb) and an ABG1-related β-galactosidase (ABG1r; 2.8 kb). Following analysis of these DNA sequences, we identified two short elements which are thought to represent a MITE and another small transposable element. Here we describe the properties and distribution of

Table 1. Apple, cherry and strawberry varieties used for fingerprinting Species

varieties

Malus pumila

Cox Falstaff Fiesta McIntosh Prima Wijcik Charger Colney Early Rivers Merton Late Napoleon Stella Van Chiraffa Emily Florence Maras des Bois Onda Star Toyonoka

Prunus avium

Fragaria ananassa

these elements and their potential as tools for genetic analysis. Materials and methods Plant material For the extraction of genomic DNA for dot blot analysis, leaf material was collected from the following species Malus pumila ‘McIntosh’, Crataegus oxyacantha ‘Crimson Cloud’, Fragaria ananassa ‘Bolero’, Fragaria vesca A, Maddenia hypoleuca, Nevusia alabamensis, Physocarpus opulifolius, Prinsepia sinensis, Prunus domestica ‘Avalon’ and Sorbus aucuparia ‘Faestigiata’. For fingerprinting, DNA was also extracted from leaves of Arabidopsis thaliana, Malus cv Baskatong and a number of cultivars of Malus pumila, Fragaria ananassa and Prunus avium (Table 1). Leaf material from members of an interspecific progeny (Prunus avium ‘Napoleon’ × P. nipponica) and an intraspecific progeny (Fragaria vesca ‘Yellow Wonder’ × F. vesca ‘Pawtuckaway’) was used to prepare DNA for segregation analysis. For each progeny, a subset of six seedlings was sampled along with the parents.

179 Promoter isolation

Launcher (http://searchlauncher.bcm.tmc.edu/). Multiple alignments were drawn using BOXSHADE (http: //www.ch.embnet.org/) with manual editing.

The promoters of the ripening-related genes MdACS1-1 ACC synthase and ABG1r β-galactosidase were isolated by screening an apple genomic DNA library (Watillon et al., 1992). The amplified λ library was plated, plaques lifted onto nylon membrane (Magna, Micron Separations inc., USA) and probed using digoxygenin (DIG)-labelled DNA fragments in HYBSOL buffer (Yang et al., 1993) following the protocol supplied with the DIG kit (Boehringer Mannheim). Probe fragments were derived from the cloned cDNAs encoding the Md-ACS1 ACC synthase (Dong et al., 1991) and ABG1 β-galactosidase (Ross et al., 1994). Single positive plaques were isolated and the phage purified using standard procedures (Sambrook et al., 1989). DNA was isolated from the two clones, named λACS and λABG, using a Wizard Lambda Preps DNA Purification kit (Promega). Two large DNA sub-fragments produced by digestion with endonucleases Sac I (7.0 kb; Md-ACS1-1) and Sph I (5.5 kb; ABG1r) carried the promoter and 5
coding regions of the two genes. These were sub-cloned into vector pGEM3Z (Promega) and, following further characterisation by restriction mapping and sequencing, promoter + 5
UTR fragments were isolated for the Md-ACS1-1 (5.4 kb) and ABG1r (2.8 kb) genes.

Isolation of genomic DNA from young leaf tissue, was carried out using a standard protocol (Dellaporta et al., 1984) or by the use of a Nucleon Phytopure kit (Amersham Pharmacia Biotech.). Aliquots of 0.1µg and 1µg of genomic DNA from Rosaceae species were denatured and applied to a nylon membrane. Similarly, aliquots of 0.1 ng (∼107 copies), 1 ng (∼108 copies) and 10 ng (∼109 copies) of plasmid DNA containing the cloned Ars1 or Ars2 elements were denatured and applied to the membrane. After neutralisation and fixing, the membranes were hybridised with the DIG-labelled Ars1 and Ars2 probe fragments. The membranes were washed at low stringency (20 mM Na2 HPO4 pH 7.2, 1% SDS, 1 mM EDTA at 37 ◦ C). Conditions for hybridisation, washing, and detection were those recommended by the DIG kit supplier (Boehringer Mannheim). Hybridisation of the probe DNAs to the blots was detected by chemiluminescence and recorded on blue-sensitive X-ray film (GRI Ltd., Dunmow, UK).

DNA sequencing and computer analysis

PCR amplifications

The Md-ACS1-1 and ABG1r promoter fragments were sequenced to patent quality on both strands using dideoxy chain termination sequencing reactions analysed on denaturing polyacrylamide gels by Lark Technologies Inc. (Houston, USA). Additional sequencing during promoter isolation was performed by Sequiserve (Vaterstetten, Germany) using dyelabelled primers and an automatic fluorescence sequencer (James et al., 2001). The initial identification of the repeat sequences was facilitated by a similarity search of public sequence databases using FASTA (Pearson, 1990) and BLAST (Altschul et al., 1990) accessed via the Internet. Internal repeats were identified using the RepEater application accessed via the BIOCCELERATOR server at the Weizmann Institute of Science (http://sgbcd.weizmann.ac.il/). Alignments of DNA sequences were performed using ALIGN accessed via the GENESTREAM Bioinformatics Resource Server (http://www2.igh.cnrs.fr/) and ClustalW1.8 (Higgins et al., 1996) at the Baylor College of Medicine Search

PCR amplifications were performed following standard procedures using oligonucleotide primers designed from consensus sequences of the Ars1 and Ars2 element termini. Because the Ars1 element has large terminal inverted repeats, it was possible to use a single forward terminus primer to amplify within elements, Ars1-INTRA (5
-CTCGTTTGGAAGTGTTTTTAA-3
) and a single reverse terminus primer to amplify between adjacent elements, Ars1-INTER (5
-CAAAAGTGTTTTCAGTCATTT-3
). Designed initially to amplify the Ars2 element, the primers Ars2F (5
-CGAAAAATTCCCGGTACTGTT-3
) and Ars2R (5
-CTAATGAAAAGGGTTTGAAAACTT-3
) were subsequently used separately to amplify between adjacent elements. Amplified PCR products were analysed by gel electrophoresis on 1.5% agarose gels in TAE buffer.

Quantitative DNA dot blot analysis

180

Figure 1. Diagrammatic representation of the 5.4 kb Md-ACS1-1 (GenBank AY062129) and 2.8 kb ABG1r (GenBank AY062128) promoter fragments showing major repeat elements. A scale in bp is drawn relative to the start codon positions. Ars1: Apple repetitive sequence 1; Ars2: apple repetitive sequence 2; dr: direct repeat. ir: inverted repeat; tr: tandem repeat; ∗ : multiple tandem repeat of AATGTTRYTT. Ars2a and Ars2b: duplication of Ars2.

Results and discussion Isolation of two apple ripening-related promoters and identification of two novel repeat sequences Screening of an apple genomic DNA library with homologous cDNA probes was used to identify clones carrying the 5
flanking sequences for the ripening-related Md-ACS1 ACC synthase and ABG1 β-galactosidase genes. Sequencing the termini of subclones during the isolation procedure confirmed the identity of the genes (data not shown). The MdACS1 ACC synthase promoter sequence (5.4 kb) was practically identical to two other GenBank accessions (U89156 and AB010102), both genomic clones of the Md-ACS1 ACC synthase gene from cultivar ‘Golden Delicious’ (Harada et al., 1997; Sunako et al., 1999). Accession AB010102 contains a putative SINE insertion, and so this has been classified as a distinct allele, Md-ACS1-2 (Sunako et al., 1999). This insertion is absent from the sequence we obtained, which confirms that our sequence is derived from the fully func-

tional Md-ACS1-1 allele. Both published sequences are considerably shorter than the one presented here, extending only to 2211 (MDU89156) and 2363 bp (AB010102) upstream of the ATG start codon. The genomic DNA sequence isolated by library screening with the ABG1 cDNA probe is highly similar to ABG1 but not identical, and so it has been denoted an ABG1-related gene (data not shown). The base substitutions observed may be due to the use of a different apple cultivar as the source of the DNA. Alternatively, this could be a separate allele of ABG1 or possibly another member of the highly homologous β-galactosidase gene family in apple (Ross et al., 1994). No other sequences with extensive homology to the ABG1r promoter sequence (2.8 kb) have previously been reported. The Md-ACS1-1 and ABG1r promoter sequences described here have been deposited at GenBank as accessions AY062129 and AY062128, respectively. The promoter sequences were examined using text searches for features which may be related to ripening-

181 specific expression. Numerous small sequence elements with homology to cis-elements implicated in ethylene or ripening-related expression were identified, although their function awaits confirmation (J.R. Gittins, unpublished observation). BLAST and FASTA searches for small regions of high homology and a screen for repetitive elements identified a number of direct, inverted and tandem repeats (Figure 1). The Md-ACS1-1 promoter fragment, in particular, contains a large number of repeats, constituting 23% of the whole sequence. Two of the larger repeat elements were highly similar to sequences in the non-coding regions of a number of genes from apple and one from a related species. The first sequence, an inverted repeat named Ars1 (Apple repetitive sequence 1) found in the MdACS1-1 promoter (Figure 1) is also present in the promoters of apple ACO2 ACC oxidase (AF015787) and KNAP1 Knotted-like homeobox (Z71981) genes. A direct repeat named Ars2 was found in the MdACS1-1 promoter (Figure 1) and this duplication is also present in the promoter sequence of the MdACS1-2 allele (AB010102). A single copy of Ars2 was found in the reverse orientation in the ABG1r promoter sequence (Figure 1). Single copies of this element are also located in the promoters of the Ypr10∗ a major apple allergen Mal d 1 gene (AF020542), the Ypr10∗ c ribonuclease-like PR-10c gene (AY026910) and in intron 4 of the MdPI apple pistillata gene homologue (AJ291491). A sequence with homology to the 3
region of Ars2 is also present in the intron of the Pyrus pyrifolia S2-RNase gene (AB014073). Characterisation of Ars1 – an element related to the Alien MITE family The three identified Ars1 elements have sizes of 232 bp (Md-ACS1-1), 222 bp (KNAP1) and 177 bp (ACO2) without any apparent coding capacity. The A+T contents of these sequences are 67, 72 and 79%, respectively. Each element contains terminal inverted repeats of more than 50 nucleotides and sub-terminal repeats, which would permit single strands to anneal to form stable stem-loop structures (data not shown). These repeats are the regions of highest homology between the elements (Figure 2A). Variability in the central region is responsible for the smaller size of the elements in the KNAP1 and ACO2 promoters. The overall sequence identity values are between 58 and 69%. Examination of the flanking regions demonstrates that each element has imperfect 4 bp terminal direct

repeats which are likely to represent the insertion site duplication, although there appears to be no clear insertion site consensus (Figure 2A). Dot blot analysis using an Ars1 DNA fragment, amplified using the primer Ars1-INTRA as the probe showed that this element is present at approximately 200 to 500 copies per haploid genome in apple (Figure 3A). The high copy number, lack of coding capacity, the high A+T content and the ability to form stable stem-loop structures make it likely that Ars1 elements represent MITE genomic repeat sequences, of which we believe this to be the first report in a woody perennial species. Close examination of the Ars1 termini demonstrates homology of the termini to the Alien element first described in Capsicum annuum and found to be ubiquitous in the genomes of higher plants (Pozueta-Romero et al., 1996). The consensus sequence for the terminal nucleotides of Ars1 is 5
GGACTCGTTTGGAAGTGT-3
compared to that of Alien, 5
-AGGAGTCGTTTGGTAGAGT-3
. There is little homology between the internal sequences of Ars1 and Alien; therefore these elements probably represent distinct subfamilies of the same family of MITE elements. A similar situation has previously been observed within other MITE families. In MITEs of the Tourist family in cereal grasses, only the terminal inverted repeats show significant conservation between the different sub-families (Bureau & Wessler, 1994; Chang et al., 2001). The Emigrant MITE of Arabidopsis has 23 nt terminal inverted repeats with high homology to those of the Wujin element from the yellow fever mosquito (Casacuberta et al., 1998). These homologies within terminal sequences indicate a common ancestor for the elements but may also suggest the use of a common integration machinery. Characterisation of Ars2 – a small transposable element The Ars2 elements identified thus far in apple range in size between 119 and 153 bp with A+T contents of between 70 and 81%, and no apparent coding capacity. A multiple alignment of the eight apple elements and one partial element from Pyrus pyrifolia demonstrates high levels of homology between the sequences (Figure 2B), with identity values of 95% and above for the paired ACS1 allele elements and up to 85% for other combinations. At the termini, there are imperfect 17 nt inverted repeats (Figure 2B) and within the elements there are many short sub-terminal repeats which give the sequences the potential to form elab-

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Figure 2. Multiple alignment of Ars1 (A) and Ars2 (B) sequences. The sequence positions are shown relative to the start codons of the relevant genes. The orientation of the Ars1 elements for alignment was chosen according to the % similarity scores. Conserved residues are shown by white letters on a black background and hyphens indicate truncated sequences or introduced gaps. Short flanking direct repeats are underlined. →: imperfect terminal repeats; – r –: short direct repeat. ABG1r (GenBank AY062128), ACO2 (GenBank AF015787), Md-ACS1-1 (GenBank AY062129), Md-ACS1-2 (GenBank AB010102), KNAP1 (GenBank Z71981/Z71979), MdPI (GenBank AJ291491), Pyrus pyrifolia S2-RNase (GenBank AB014073), Ypr10∗ a (GenBank AF020542) and Ypr10∗ c (GenBank AY026910). Ars2 element is duplicated (a and b) in Md-ACS-1 and Md-ACS-2 promoter sequences.

183

Figure 3. Dot blot analysis of Ars elements in the Rosaceae. Dots containing 1.0 and 0.1 µg of genomic DNA were hybridised with Ars1 (A) and Ars2 element (B) probes. DNA samples: (1) Malus pumila ‘McIntosh’, (2) Crataegus oxyancatha ‘Crimson Cloud’; (3) Fragaria ananasa ‘Bolero’; (4) Fragaria vesca A, (5) Maddenia hypoleuca, (6) Nevusia alabamensis, (7) Physocarpus opuliafolius, (8) Prinsepia sinensis, (9) Prunus domestica ‘Avalon’; (10) Sorbus aucuparia ‘Faestigiata’, (11) plasmid controls containing cloned Ars elements (∼109 , ∼108 and ∼107 copies).

orate single-stranded secondary structures (data not shown). There appear to be four distinct size variants of the Ars2 element (Figure 2B). The presumed full length element of 152/153 bp is represented by the first of the sequences in the Md-ACS1 allele promoters (1a and 2a) and by the sequences in the ABG1r and Ypr10 promoters (although the 3
terminal nucleotides of the Ypr10∗ c element are not available). The next largest of 145 bp is present in the second sequence in the Md-ACS1 ACC synthase promoters (1b and 2b). This shows slight variation at the 5
terminus and has an 8 bp deletion within the central region. The third variant of 119 bp is represented by the MdPI apple pistillata homologue intron, where a truncation extends to a direct repeat of the 5
end, so that this terminus retains the same sequence. Closer examination of the sequence of the MdPI Ars2 element just outside the region of homology shows that the 5
terminus inverted repeat is almost identical (one mismatch) to those of the ABG1r and Ypr10∗ c elements. The fourth and smallest variant

of 46 bp is present in the intron of the Pyrus pyrifolia S2-RNase gene which appears to be a truncated version of Ars2, possessing only the 3
terminal region. When sequences flanking the Ars2 elements were examined, no clear insertion site consensus sequence could be identified. Rather, all sequences, other than those from the ABG1r and Ypr10∗ c promoters and the Pyrus pyrifolia S2-RNase intron sequence, have 9 or 10 nt AT-rich direct repeats of variable sequence composition. A tandem duplication of this direct repeat is found outside the 5
terminus of the full length Ars2 element in the Md-ACS1 allele 2 promoter (ACS1-2a) and adjacent to the second element in both the MdACS1 allele 1 and allele 2 promoters (ACS1-1b and ACS1-2b, Figure 2B). These direct repeats flanking the sequences and the short terminal inverted repeats within the sequences are a strong indication that Ars2 represents a transposable element or a derivative of such. To examine the abundance of Ars2, the presumed full length element from the Md-ACS1-1 ACC syn-

184 thase promoter, amplified by PCR using primers Ars2F and Ars2R, was used to probe a dot blot of genomic DNA from apple. This revealed that Ars2 is present at 5,000 to 10,000 copies per haploid genome (Figure 3B). Despite the evidence that Ars2 is a transposable element, the category into which it falls remains undecided. The three most likely possibilities for an element of this size are a MITE, a SINE or a solo LTR from a large retrotransposon. The high A+T content, high copy number and presence of terminal inverted repeats are characteristic of MITEs. However, Ars2 has long (9–10 nt) direct target site duplications which is characteristic of SINEs. Those of MITEs are generally smaller (2–3 nt) although some elements, such as MAthE3 of Arabidopsis, possess larger duplications (Surzycki & Belknap, 1999). Also in common with SINEs, Ars2 has the potential to form complex secondary structures, which suggests that it could have been derived from a small RNA gene. However, this element lacks other typical features of SINEs such as the conserved box A and B motifs of the RNA pol III promoter, and a repeated A or T rich terminal sequence. The third alternative is that the Ars2 elements represent single LTRs flanked by the target site duplication which have been produced by recombination between the LTRs or deletion of a larger retroelement. One example of a retrotransposon solo LTR is the Tat1 element of Arabidopsis (Peleman et al., 1991; Wright & Voytas, 1998). The Ars2 elements possess short terminal inverted repeats which is a feature of LTRs, although unlike Tat1 these do not show significant homology with any known transposon types. Also, the copy number of the Ars2 elements is considerably larger than Tat1 which is only present in 10 or fewer copies per haploid genome (Voytas, 1996). If Ars2 represents either a MITE or the LTR from a retrotransposon then it may be possible to find the full-length autonomous elements from which they were derived. Given the alternatives for the likely identity of Ars2, the occurrence of different size variants may either be due to loss of sequence during deletion of a large transposable element or the initiation of RNA pol III transcription from alternative start points during SINE replication. Distribution of Ars1 and Ars2 and its implications Dot blot analysis has also shown the distribution of Ars1 and Ars2 within the Rosaceae family (Figure 3). For both elements, the pattern of hybridization

was practically identical. The hybridisation signal was strong with Malus pumila, Crataegus oxyacantha, Prinsepia sinensis and Sorbus aucuparia. A weaker signal was detected with Nevusia alabamensis, Physocarpus opuliafolius and Prunus domestica; while very weak signals were seen with Fragaria ananassa, Fragaria vesca and Maddenia hypoleuca. The fact that Ars1 and Ars2 hybridise to genomic DNA of all the species suggests that both elements are present across the family Rosaceae and are not specific to a particular sub-family. Thus, their insertion and ampification may have pre-dated speciation. From the greater sequence variation seen in the Ars1 elements it is likely that the ingress of this element was a more ancient event than that of Ars2. It is noticeable that the level of hybridisation is not the same for the sub-families or for the species belonging to the same sub-family. In the sub-family Prunoideae for example, a strong hybridisation signal is detected in Prinsepia sinensis (sample 8) but not in Prunus domestica (sample 9). This result could be explained either by differences in copy numbers or by divergence of the sequences after speciation. When a number of the DNA samples were examined by Southern analysis at high stringency (20 mM Na2 HPO4 pH 7.2, 1% SDS, 1 mM EDTA at 65 ◦ C), the differences in the level of hybridisation between Prinsepia sinensis and Prunus domestica or between Sorbus aucuparia and Maddenia hypoleuca are more marked (data not shown). This indicates that sequence divergence and not low copy number is the likely reason for hybridisation signal variation seen in dot blot analyses. Ars-derived genetic markers MITEs and retroelements have previously been used to generate markers for genetic studies using a variety of PCR-based methods (Purugganan & Wessler, 1995; Flavell et al., 1998; Pearce et al., 1999; Casa et al., 2000). The methods employed range from using PCR to generate DNA species which contain restriction fragment length polymorphisms (Purugganan & Wessler, 1995), to detect insertion site polymorphisms (Flavell et al., 1998) and in AFLP-type assays (Pearce et al., 1999; Casa et al., 2000). A recently described method called IMP (Inter MITE Polymorphisms) uses primers designed from the terminal inverted repeats of MITEs to amplify the DNA fragments separating adjacent elements (Chang et al., 2001). This procedure is effective at generating polymorphic bands which may be used as markers for

185

Figure 4. Fingerprinting (A) and segregation (B) analysis by amplification using the primer Ars1-INTRA. 4A. Lanes (1), (9), (17), (25) 1kb molecular marker, lane (2) Malus cv Baskatong, (3)–(8) Malus pumila varieties (‘Cox’, ‘Falstaff’, ‘Fiesta’, ‘McIntosh’, ‘Prima’ and ‘Wijcik’), (10)–(16) Prunus avium varieties (‘Charger’, ‘Colney’, ‘Early Rivers’, ‘Merton Late’, ‘Napoleon’, ‘Stella’ and ‘Van’), (16)–(24) Fragaria ananassa varieties (‘Chiraffa’, ‘Emily’, ‘Florence’, ‘Maras des Bois’, ‘Onda’, ‘Star’ and ‘Toyonoka’). 4B. Lanes (1), (11), (20), (29) 1kb molecular marker, (2) Crataegus oxyacantha ‘Crimson Cloud’, (3) Maddenia hypoleuca, (4) Nevusia alabamensis, (5) Physocarpus opulifolius, (6) Prinsepia sinensis, (7) Prunus domestica ‘Avalon’, (8) Prunus persicae ‘Klara Meyer’, (9) Sorbus aucuparia ‘Faestigiata’, (10) Arabidopsis thaliana, (12) Prunus avium ‘Napoleon’, (13) P. nipponica, (14)–(19) F1 seedlings of P. avium ‘Napoleon’ × P. nipponica, (21) Fragaria vesca ‘Yellow Wonder’, (22) F. vesca ‘Pawtuckaway’, (23)–(28) F2 seedlings of F. vesca ‘Yellow Wonder’ × F. vesca ‘Pawtuckaway’.

molecular fingerprinting or genome mapping. Once DNA blotting had demonstrated that the Ars elements were abundant in the genomes of species other than apple, a method similar to IMP was attempted, using primers specific to the Ars termini to amplify a panel of genomic DNAs (Figure 4). The results shown in Figure 4 were obtained using primer Ars1-INTRA but all four Ars element terminus primers produced banding patterns of similar complexity when used in PCR with the same panel of genomic DNAs (data not shown). Multiple bands were produced using DNA

from members of the Rosaceae family, a number of which are polymorphic in different cultivars within the same species (Figure 4A), or within segregating progenies from two experimental crosses (Figure 4B). There were more segregating bands in the interspecific progeny (P. avium ‘Napoleon’ × P. nipponica) compared to the intraspecific progeny (F. vesca ‘Yellow Wonder’ × F. vesca ‘Pawtuckaway’) but, even in the latter progeny, an average of four segregating bands were detected for each primer. These results demonstrate that Intra MITE polymorphisms may also be

186 used to generate markers for molecular fingerprinting or genome mapping of the Rosaceae and confirm the observations of Chang et al. (2001) concerning the merits of IMP. DNA from Arabidopsis thaliana was similarly amplified in these experiments (Figure 4B, lane 10). The presence of multiple bands indicates that Arsrelated sequences are present even in a plant species distantly related to apple. Homology restricted to the termini of these elements has apparently permitted amplification using the Ars primers. This confirms the finding of Pozueta-Romero et al. (1996) that Alien-like elements are widely distributed among plant species and also indicates that Ars2-related elements also have a wide distribution. Indeed, BLAST searches (data not shown) revealed that the sequences similar to the Ars element termini are even present in Homo sapiens, Caenorhabditis elegans, Anopheles gambiae. Markers derived from these elements could therefore be transferred to a wide range of organisms without the need to develop libraries as is the case of other genetic markers such as restriction fragment length polymorphisms (RFLPs) or microsatellites.

Acknowledgements DEFRA (grant HH1104STF) and the Apple and Pear Research Council supported the work on promoter analysis. The BBSRC funded the characterisation of the Ars elements and the development of genetic markers. We are greatly indebted to Dr Bernard Watillon for the apple genomic DNA library used in this study. We are also grateful to Dr Abhaya Dandekar (UC, Davis) and Dr Gavin Ross (Hort Res, NZ) for the Md-ACS1 ACC synthase and ABG1 β-galactosidase cDNA clones, respectively. We thank our colleagues from HRI-EM (Dr Richard Walden, Kenneth Tobutt, Penny Greeves, Dan Sargent, Dr Celia James, Andy Passey and Fiona Wilson) for their assistance or their comments on the manuscript.

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