Journal of Heredity Advance Access originally published online on December 15, 2004
Journal of Heredity 2005 96(1):59-65; doi:10.1093/jhered/esi013
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© 2005 The American Genetic Association
Brief Communication |
Base Composition of Coffea AFLP Sequences and Their Conservation Within the Genus
From UMR 1097 Diversité et Génome des Plantes Cultivées, Centre IRD, 911 avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France.
Address correspondence to V. Poncet at the address above, or e-mail: poncet{at}mpl.ird.fr.
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Abstract |
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Amplified fragment length polymorphism (AFLP) is often used for genetic mapping and diversity analysis, but very little information is currently available on their sequence characteristics. Species-specific sequences were analyzed from a single Coffea genome (Coffea pseudozanguebariae) associated with clustered or nonclustered AFLP loci of known genetic position. Compared with the expressed sequence tag (EST) sequence composition, their AT content exhibited a bimodal distribution with AT-poor sequences corresponding mainly to putative coding sequences. AT-rich sequences, apart from the EST distribution, were usually clustered on the genetic map and might correspond to noncoding sequences. Conversion of these AFLP markers into sequence-characterized amplified region (SCAR) anchor markers allowed us to assess sequence conservation within Coffea species with respect to species relatedness.
Polymerase chain reaction (PCR)-based amplified fragment length polymorphism (AFLP) is an efficient tool for detecting reproducible and reliable polymorphism. It has been used both to construct high-density genetic maps and to saturate specific regions in plants such as alfalfa (Medicago truncatula) (Thoquet et al. 2002), cowpea (Vigna unguiculata) (Ouedraogo et al. 2002), sunflower (Helianthus annuus) (Mokrani et al. 2002), and sorghum (Sorghum bicolor) (Menz et al. 2002).
Amplified fragment length polymorphism is based on the ligation of adapters to genomic restriction fragments followed by PCR-based amplification with adapter-specific primers. Like restriction fragment length polymorphisms (RFLPs), the majority of AFLP fragments define single loci (Qi and Lindhout 1997; Vos et al. 1995). However, AFLP markers generated with the methylation-insensitive enzyme EcoRI in combination with MseI are often not uniformly distributed over chromosomes. Strong clustering around the centromere region has been reported in tomato (Lycopersicon ssp.), through the analysis of an integrated RFLP/AFLP map (Haanstra et al. 1999), and also in barley (Hordeum vulgare) (Qi et al. 1998) and maize (Zea mays) (Vuylsteke et al. 1999). Consequently it has been suggested that the targeted regions could differ from the rest of the genome with respect to their sequence, methylation, and transcription level, A + T/C + G ratio, and/or recombination rate (Young et al. 1999).
Very little information is currently available on the sequence nature and characteristics of these AFLP bands. Indeed, for most applications, the AFLP protocol does not require preliminary genomic sequencing or cloning. Sequencing of AFLP bands has mainly been carried out in studies requiring conversion of AFLP markers into sequence-characterized amplified regions (SCARs); for example, in Brassica (Negi et al. 2000), soybean (Glycine ssp.) (Hayashi et al. 2000), carrot (Daucus carota) (Bradeen and Simon 1998), apple (Malus floribunda) (Xu et al. 2001), and willow (Salix viminalis) (Gunter et al. 2003). Most of these studies were conducted with the goal of enhancing marker-assisted selection, and involved identification and conversion of AFLP markers tightly linked to genes of interest. To our knowledge, no published studies have involved an analysis of AFLP sequences distributed all over a single genome.
The first Coffea interspecific linkage map was based on a backcross progeny [(C. pseudozanguebariae (PSE) x C. liberica var. Dewevrei (DEW)) x DEW] (Ky et al. 2000). The two parental species are diploid with the same chromosome number (2n = 2x = 22). A special strategy was applied, which involved mapping of AFLP bands specific to PSE species relative to DEW. This linkage map comprised 181 loci distributed over 14 linkage groups covering 1144 cM. Although these markers were PSE specific, they were distributed throughout the genome, which is a common feature on AFLP plant linkage maps.
In this article we analyze specific sequences of the PSE genome associated with different types of clustered and nonclustered AFLP bands. SCAR markers were derived from these PSE AFLP sequences in order to evaluate their conservation across Coffea species. The corresponding primer pairs were tested on other species to determine whether they could amplify homologous sequences.
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Materials and Methods |
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Plant DNA
Amplified fragment length polymorphism profiles were obtained for PSE and PSE x DEW F1 hybrids. Specific SCAR primers were tested on genomic DNA of Coffea arabica (ARA), Coffea canephora (CAN), Coffea congensis (CON), Coffea eugenioides (EUG), Coffea heterocalyx (HET), Coffea liberica var. Dewevrei (DEW), Coffea millotii (MIL), Coffea sp. Moloundou (MOL), C. pseudozanguebariae (PSE), and Arabidopsis thaliana (THA). All coffee trees were maintained in tropical conditions in a greenhouse in Montpellier, France. Genomic DNA was extracted from lyophilized leaves as described by Ky et al. (2000).
Isolating and Cloning AFLP Fragments
Amplified fragment length polymorphism reactions, electrophoresis, and detection were performed as described in Ky et al. (2000). Selective amplification was performed using selective 3+/3+ primers (Table 1). Selected bands were excised from the dried polyacrylamide gel, eluted in 100 µl sterile ddH2O, and kept on boiling water for 5 min to release DNA from the gel. After the gel debris was spun down, the DNA-containing supernatant was transferred into a new tube and ethanol precipitated. AFLP fragments were resuspended in 50 µl sterile ddH2O and used as a template for reamplification with the appropriate preselective or selective AFLP primer combination. The standard AFLP PCR program was supplemented with an elongation step of 30 min at 72°C in order to add the 3'A overhang to the PCR products. The products were purified using E.Z.N.A. Cycle-Pure (OMEGA Bio-Tek) and cloned onto pCR4-Topo from the TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions. Plasmid DNA was extracted using the Plasmid Mini Preparation kit (Qiagen).
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The general strategy applied to select specific sequences underlying AFLP bands was based on Xu et al. (2001) and proved to be reliable and robust. In particular, after PCR amplification using the corresponding AFLP primers, inserts of five colonies were run on polyacrylamide gels along with the initial AFLP amplification to check that the cloned fragments and the original band shared the same position. The selected clones were then prepared for sequencing using the Plasmid Mini Preparation kit (Qiagen).
Sequence Analysis
The inserts were sequenced by MWG-Biotech. Sequences were edited and analyzed using the DNASTAR software package (Lasergene, Madison, WI). Similarities between sequences were tested with the DNASTAR SeqMan module using the "Assembling" option.
To compare these sequences to public database sequences, similarity searches were performed using BLAST algorithms (http://www.ncbi.nlm.nih.gov:80/BLAST/) (Altschul et al. 1997). BLASTx was used to compare sequences to protein sequences, while BLASTn was used to compare sequences to the nonredundant DNA database sequences and EST database sequences. The search was conducted in June 2004 using the default parameters. Similarity searches to C. canephora EST sequences (unpublished data) derived from fruit copy DNA (cDNA) (5706 sequences) or leaf cDNA (2709 sequences) were also performed. Similarities to known sequences were considered significant when the probability values were less than 10–4.
The base compositions of AFLP sequences were then compared to those of 103 Coffea EST sequences. The EST sequences were chosen from a C. canephora EST library derived from fruit cDNA and they showed a similarity with known protein sequences with an E-score value less than 8e-29 (BLASTx). No EST was predicted to be from plastids.
SCAR Primer Pairs
Sequence-characterized amplified region primers were designed with the public Primer 3 software package (Rozen and Skaletsky 2000) and synthesized by MWG-Biotech. When possible, primers were designed to retain the original restriction enzyme flanking site, the three selective base pairs, and the next internal sequences. The primer sizes ranged from 17 to 27 bases. One primer of every pair was 5'-tailed with the M13 sequence 5'-CACGACGTTGTAAAACGAC-3' for subsequent use of labeled M13 primer.
Each 15 µl PCR contained 25 ng of genomic DNA, 1x reaction buffer (Promega), 2.0 mM MgCl2, 200 µM of each dNTP, 0.2 µM of each primer, and 0.05 U/µl of Taq polymerase (Promega). The amplifications were performed following a "touchdown" PCR profile consisting of an initial 2 min denaturation at 94°C for 45 s, followed by five denaturation cycles at 94°C for 45 s, 1 min primer annealing at 60°C (or 55°C) with the temperature decreasing by 1°C at each cycle, and 1 min 30 s elongation at 72°C. Then 30 cycles of 45 s at 90°C, 1 min at 55°C (or 50°C), and 1 min 30 s at 72°C were performed and followed by a final 8 min elongation at 72°C. The primers and annealing temperatures are shown in Table 2.
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For visualization of the PCR product on an IR2 Automated DNA Sequencer (LI-COR, model 4200L-2, Lincoln, NE), an M13 primer coupled to an IRD700 or IRD800 infrared tag (MWG-Biotech AG) at 0.2 µM was included in the PCR and the concentration of the corresponding M13-tailed primer was decreased to 0.02 µM (Oetting et al. 1995). PCR products were electrophoresed on a 6.5% KBPlus (LI-COR, 827-05607) 25 cm gel. The gel image was processed by SAGA GT to estimate the size of the sample bands according to a 50–350 bp size standard (LI-COR, 829-05343, 829-05344).
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Results |
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Selection and Sequencing of AFLP Bands
Twenty-three AFLP loci were chosen on the reference map (Ky et al. 2000) to be distributed either inside (9) or outside (14) the clusters over the first 11 linkage groups (Table 1). A locus was considered as being in a cluster when the mean distance to its two neighboring markers was less than 4 cM. Three additional AFLP segregating markers had an unknown location, that is, they did not link to any linkage group. After amplification with the corresponding AFLP primer combination, AFLP bands were excised, reamplified, and cloned. Twenty-three were all successfully sequenced.
Similarity Searches
No similarities between PSE sequences or with Coffea EST sequences were noted. Comparison of PSE sequences to the databases maintained at GenBank and EMBL revealed significant similarity with only five sequences: 3-1, 3-4, 3-9, 4-1, and 14-8 (Table 1). In particular, some similarity (E-value = 5e-06) between AFLP sequence 4-1 and a protein from A. thaliana (accession no. BAB10716) was observed. Likewise, AFLP sequence 3-1 showed similarity (E-value = 1e-07) to an A. thaliana-expressed protein (accession no. NP_563868). AFLP sequence 3-9 was found to be similar (E-value = 1e-19) to a portion of an Escherichia coli chromosome (accession no. AE005652), but without significant similarity to any known protein.
Compositional Distribution of AFLP Sequences
The base compositions of the AFLP sequences were calculated and compared to those of Coffea EST sequences (Figure 1). The EST AT content distribution was unimodal, with a mean of 55.0%. The AFLP sequences showed an AT content distribution ranging from 49.2% to 75.2% with two modes. Taking 65% AT as the cutting point, the first mode, with a mean AT content of 58.6%, fit with the EST sequence distribution. The second mode showed significantly higher AT content, with a mean of 71.4%.
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ALFP sequences 3-1 and 4-1, which showed some similarity with A. thaliana proteins, had AT contents of 54.1% and 56.3%, respectively; that is, close to the mean AT content of EST sequences (Figure 1). When considering the nine AFLP sequences associated to clusters, seven of them had an AT content greater than 60%. AFLP sequence 3-4, which showed a similarity to repetitive DNA, had an AT content of 61.4%.
SCAR Primer Transferability Across Coffea Species
The specific primers designed from the PSE sequences were tested on genomic DNA of each coffee species (ARA, CAN, CON, EUG, HET, DEW, MIL, MOL, PSE) and A. thaliana (THA). Eleven primer pairs efficiently amplified at least one Coffea species, and SCAR primers such as Scar-3-8 or Scar-4-3 gave good amplification with the nine Coffea species tested. A. thaliana was only amplified with Scar-1-10 primers.
For PSE, the PCRs resulted in amplification of a single fragment of the expected size. For species other than EUG and ARA, the PCR also gave a single band with a size similar to that of the PSE product. When ARA was amplified with the Scar-1-10 or Scar-6-5 primer pairs, two PCR products were revealed that differed by about seven nucleotides. The same situation was observed for EUG when amplified with the Scar-6-5 primers.
In some species, blanks were found that might have indicated the presence of null alleles. However, amplification success was independent of the species considered.
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Discussion |
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GC Composition of AFLP Sequences
The distribution of the AFLP sequence composition was found to be bimodal in this study (Figure 1). One mode, CG-rich AFLP sequences, could mainly correspond to coding sequences for two reasons: (1) it fits well with the distribution of Coffea EST sequences, and (2) 3-1 and 4-1 AFLP sequences showing similarity with A. thaliana protein sequences had a GC content of 45.9% and 43.7%, respectively, included in the EST distribution.
Tested EST sequences (regulatory sequences + exons) could be considered as a good representation of Coffea coding sequences. Indeed, their compositional distribution showed a clear unimodal distribution that would correspond to the distribution reported for dicot genes, whereas Poaceae coding sequences are characterized by distributions that are at least bimodal (Carels and Bernardi 2000). Moreover, the Coffea genus, with an EST composition mean of 45.0% GC, was very similar to Solanaceae, the phylogenetically closest family (see, e.g., Fulton et al. 2002).
The other distribution mode of the AFLP sequence composition consisted of sequences with AT content greater than 65%, which is significantly different from the distribution of Coffea EST sequences. Consequently these sequences could mainly correspond to noncoding sequences. For example, the 3-4 AFLP sequence, with an AT content of 61.4%, higher than the mean AT content of EST sequences, showed a great degree of similarity to an unknown repetitive DNA fragment from the Mediterranean fruit fly (Ceratitis capitata).
Clustering of AFLP Markers
The analyzed AFLP sequences were selected according to their location on the Coffea interspecific linkage map of Ky et al. (2000), where eight EcoRI/MseI AFLP clusters had been observed. Such clustering was also noted in other plant AFLP linkage maps; for example, in potato (Solanum tuberosum) (van Eck et al. 1995), barley (Powell et al. 1997), soybean (Keim et al. 1997), Arabidopsis (Alonso-Blanco et al. 1998), and maize (Vuylsteke et al. 1999).
As reported by Young et al. (1999), AFLP markers derived from EcoRI + MseI selective primers are expected to form clusters in some specific genomic regions. Since EcoRI is insensitive to CpNpG methylation, AFLP markers developed with this restriction enzyme may identify polymorphism in hypermethylated regions of the genome with low recombination rates, such as in centromeres. These regions are not covered by traditional RFLP maps (Haanstra et al. 1999) and were found to correspond to centromeric regions in maize (Vuylsteke et al. 1999) and barley (Qi et al. 1998) genomic maps. In Coffea, clustering was observed on a genetic map (Ky et al. 2000) without information on the physical location of the AFLP markers. It was impossible to determine whether this resulted from a reduced recombination rate around centromeres, a higher frequency of AFLP markers in these regions, or a combination of the two.
To further explain the observed clustering, Peters et al. (2001) suggested that such polymorphism was more often encountered in centromeric regions because they are rich in noncoding DNA with theoretically less constrained sequences. In addition, repetitive sequences typical of centromeric regions may produce a number of AFLP markers representing variants of a single repeat. In the present study, we selected AFLP loci throughout the genome and none of them were in the same cluster. No sequence similarity between AFLP bands was observed, which could suggest that the AFLP sequences, even though they are located in different clusters, could correspond to repetitive sequences with a common origin.
However, AFLP fragments associated with clustered loci tended to have an AT content higher than the mean coding sequence AT content. These preliminary results are in accordance with the correspondence between centromeric AT-rich noncoding sequences and AFLP clusters.
Sequence Conservation Across Coffea Species
The 14 SCAR primer sets derived from PSE AFLP bands were assessed for their ability to amplify homologous sequences in nine Coffea species and in A. thaliana. SCAR primers would be transferable to related species, mainly when conserved sequences are used for primer design. In contrast to SCAR markers derived from coding sequences such as cDNA RFLP clones, primer binding sites are less likely to be conserved across species for SCAR markers derived from AFLP. In this context, our transferability results obtained are relatively favorable, since any Coffea species was amplified by a minimum of 58% primer pairs.
Twelve primer pairs gave amplification across Coffea species and one even amplified A. thaliana. Primer pairs amplified a single product (only one band) in all species, except Scar-6-5 in C. arabica and C. eugenioides and Scar-1-10 in C. arabica. While C. eugenioides is a diploid species, C. arabica is known to be an amphidiploid formed by hybridization between C. eugenioides and C. canephora, or ecotypes related to these diploid species (Berthou et al. 1983; Lashermes et al. 1999). In this study, the amplification patterns obtained for C. arabica were a juxtaposition of patterns of the putative diploid parental species.
The lack of amplification could have been due to mutations in the primer binding sites or to any mutational event that could prevent amplification, such as major insertion/deletion between primer sites or an absence of the locus in the genome. The initial polymorphism observed for the AFLP pattern—the presence of a band for PSE, the absence for DEW—was not conserved when we considered some of the SCAR amplifications (Table 2). Indeed, although the primers were designed to amplify the largest part of each AFLP sequence, they might not include the site of variation.
The amplification success was found to be independent of the species considered and did not reflect the between-species relatedness as defined by Lashermes et al. (1997). A similar situation has been reported for microsatellites across Coffea species (Poncet et al., in press) and is in agreement with the recent radiative speciation scenario noted within the Coffea genus (Cros et al. 1998; Leroy 1980).
Available linkage maps for Coffea species are based on random amplified polymorphic DNA (RAPD), RFLP, AFLP, and microsatellite markers (Coulibaly et al. 2003; Ky et al. 2000; Lashermes et al. 2001; Paillard et al. 1996). However, no RFLP markers were common to the different maps and only a few microsatellites have been mapped so far.
The set of SCAR primers obtained in this study might be suitable for use across the Coffea genus. During the development of SCARs, the loss of original polymorphism often leads to experimental challenges (Shan et al. 1999). Indeed, the original polymorphism was found to be mainly located at the restriction sites, and it was not always possible to design primers covering the corresponding region. Nevertheless, the amount of variation they could reveal should be further studied; for example, through enzymatic digestion of PCR products (cleaved amplified polymorphic sequences [CAPS]).
In conclusion, these SCAR primers, together with the currently developed microsatellites (Poncet et al., in press), will facilitate assembly of a panel of "anchor" PCR-based markers for comparative mapping studies in coffee trees and for marker-assisted selection.
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Acknowledgments |
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This work was partly supported by a grant from EEC ICA4-CT2001-10068.
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Footnotes |
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Corresponding Editor: J. Perry Gustafson
Received May 14, 2004
Accepted July 2, 2004
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References |
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