Genomic Mapping in Pinus Pinaster (Maritime Pine) Using RAPD and Protein Markers

Heredity 74 (1995) 661—668 Received 14 September 1994 Genetical Society of Great Britain

Genomic mapping in Pinus pinaster (maritime pine) using RAPD and protein markers

C. PLOMIONff, N. BAHRMANt, C.-E. DURELt & D. M. O'MALLEY tINRA, Laboratoire de Génétique et Amelioration des arbres forestiers, BP45, F-33610 Cestas, France and Forest Biotechnology Group, Department of Forestry, North Carolina State University, Box 8008 Raleigh, NC 27695, US.A.

Adetailed genomic map was constructed for one F1 individual of maritime pine, using randomly amplified polymorphic DNA (RAPD) and protein markers scored on megagametophytes of germinated seeds. Proteins allowed the localization of exclusively coding DNA in the large genome of this Pinus species, mapped with RAPD markers that essentially fail within repetitive (i.e. mostly noncoding) DNA. Dot blots experiments of 53 RAPD fragments showed that 89 per cent amplified from highly repetitive chromosomal regions. The map comprised 463 loci, including 436 RAPDs amplified from 142 10-mer oligonucleotide primers and 27 protein loci. Twelve major and one minor linkage groups were identified using a LOD score5 and a recombination fraction ® 0.30. A framework map was ordered with an interval support 4, covering 1860 cM which provided almost complete coverage of the maritime pine genome. The average distance between two framework markers was 8.3 cM; only one interval was larger than 30 cM. Protein loci were well distributed throughout the map. Their potential use as anchor points to join RAPD-based maps is discussed. Finally, the genomic maps of Arabidopsis and maritime pine were compared. Linkage groups were shown to have similar total map lengths on a chromosomal basis, despite a 57-fold difference in DNA content.

Keywords:2-Delectrophoresis, linkage map, Pinus pinaster, protein, RAPDs.

probes. The RAPD method (Williams et a!., 1990) Introduction permits identification of a large number of poly- Thefirst linkage studies of Pinus were based on morphic DNA markers distributed throughout the segregation of isozymes extracted from megagameto- genome, including both coding and noncoding regions phytes. More than 10 species were studied for about (Williams et a!., 1990). RAPDs have been used for 15 loci (reviewed by Tulsieram et a!., 1992). Conkle genomic mapping in several conifer species (Neale & (1981) located more loci but the number of markers Sederoff, 1991; Tulsieram et a!., 1992; Nelson et a!., that were resolved and analysed was still too low for 1993; Bineffi & Bucci, 1994). Pinus species have a applications that required a broader genome coverage large genome (Ohri & Khoshoo, 1986; Wakamiya et (e.g. quantitative trait dissection studies). Two- a!., 1993) characterized by a high proportion of repeti- dimensional electrophoresis (2D-PAGE) of mega- tive DNA (Miksche & Hotta, 1973; Rake et a!., 1980; gametophyte proteins identified a larger number of Kriebel, 1985). Therefore, RAPDs and markers that loci. Bahrman & Damerval (1989) reported linkage are based on coding sequences could provide different analysis for 119 loci and Gerber et a!. (1993) reported coverage of the genome in pine. a 65 loci linkage map covering 530 cM of the maritime Each type of marker has advantages and limitations pine genome. Both isozymes and proteins correspond and many factors can influence choice of marker to coding DNA. Devey et a!. (1994) presented linkage systems for a given purpose. Marker based techno- groups in loblolly pine for 80 RFLPs detected using logies are being used for linkage map construction, cDNA probes. RFLPs typically sample genetic quantitative traits dissection experiments, germplasm variation in coding regions or directly adjacent to evaluation, genetic fingerprinting and manipulation of coding regions of the genome, and use low copy genes. Genomic maps can also be used to locate and clone genes of interest. In addition, they may provide *Correspondence: INRA, Laboratoire de Génétique et Améliora- information for understanding genome structure and tion des arbres forestiers, BP45, F-3360 Cestas, France. evolution (Neale & Williams, 1991). RFLP methods

1995 The Genetical Society of Great Britain. 661 662 C. PLOMION ETAL. are well suited for species maps because the same hybridization probes can be used in comparisons DNA extraction and RAPD procedure among species. Ahuja et al. (1994) showed that DNAsamples were prepared from needles of the mapped DNA probes from loblolly pine can be used to Corsican and Landes grandparents (accessions Cl 0 construct RFLP maps for other members of Pinaceae, and L146, respectively), and the F1 hybrid parent thus presenting an opportunity to compare the genome (accession H12), as well as from the megagame- in related pine species. Forest trees exhibit generally tophytes from Hi 2. DNA was extracted using the high levels of genetic diversity and are highly out- CTAB procedure described by Bousquet et al. (1990). crossed. As a result, linkage disequilibrium should be The DNA extracted from needle samples was purified low. Thus, alleles at quantitative trait loci (QTLs) and by centrifugation in a CsCI-ethidium bromide density alleles at marker loci should be randomly associated in gradient. The pine DNA was diluted to a working different genotypes (Strauss et al., 1992). Therefore the concentration of approximately 1 ng/pL, by ability to create maps for individual trees, and to assess comparison with the fluorescence of lambda DNA marker genotypes on hundreds of progenies, is essen- concentration standards on ethidium bromide-stained tial for breeding experiments that aim to use marker- agarose gel. RAPD reactions were performed using assisted selection. The RAPD technology appears to the method of Williams et a!. (1990) with 5—10 ng be well suited for developing single-tree maps. The template; 20 ng of ten-base primers from Operon major advantage of this technique is the rapidity of Technologies (Alameda, CA), kits A—Z;8ug/uL screening for polymorphisms, the identification of a non-acetylated bovine serum albumin and 1 unit of large number of markers and its potential automation Taq DNA polymerase. The mixture was covered with (Sobral & Honeycutt, 1993). However, synteny of 50 pL of mineral oil and amplifications were carried linkage groups with other species could be difficult to out in 96-well microtitre plates using an MJ Research established with RAPD markers, as noticed by Torres PT-100 thermal cycler (MJ Research, Watertown, eta!. (1993). MA). Amplification products were separated by We located 2-D protein polymorphisms that repre- electrophoresis on 2 per cent agarose gels, detected by sent coding DNA on a RAPD-based framework map staining with ethidium bromide, and the gels were of maritime pine (Pinus pinaster Ait., 2x =2n=24). photographed. RAPD fragments were sampled from Protein markers were used because (i) they only reveal the agarose gel by stabbing the fluorescing band with a gene products, (ii) they have been well studied in that pipette tip and rinsing the tip into 100 uL of 20 per species, and (iii) they provide interesting tools, as cent TE buffer (10 mM Tris-HCI, 0.2 m EDTA), and candidate genes, for genetic dissection of vigour and were stored at —20°Cuntil required for reamplifica- adaptative traits in the frame of the maritime pine tion. breeding programme. The objectives of this contribu- tion are threefold: (i) genetic map construction based on markers of coding and noncoding chromosomal Characterizationof genomic sequence complexity of RAPD markers regions, (ii) estimation of genome size of maritime pine, and (iii) characterization of RAPD fragments' internal Tocharacterize the copy number of RAPDs internal sequences for copy number in the genome. The map sequences, 53 RAPD fragments were labelled and and markers will be used for QTL mapping of height used as non-radioactive probes on dot blots (strip blots growth and adaptative traits in an F2 progeny of the with four dots containing 20 g, 2 ig and 0.2 ug of mapped tree. pine DNA and 20 ug of herring sperm DNA as a negative control), as described by Grattapaglia & Sederoff (1994). Materials and methods Proteinextraction and electrophoresis P/ant material Asample of 34 megagametophytes was individually The material used was 124 megagametophytes from crushed in 6uL/mg UKSbuffer(9.5 M urea, 5 m seeds of one inter-racial hybrid (parent F1: K2C03, 1.25 per cent SDS, 0.5 per cent dithiothreitol, Corsican x Landes) of maritime pine. Individual mega- 2 per cent pharmalyte pH 3—10 and 6 per cent Triton gametophytes were harvested after 2 weeks of germi- X- 100). Thirty-five microlitres of the supernatant was nation. Each megagametophyte was cut in two parts, submitted to electrofocusing in the first dimension, with one-quarter freeze-dried for RAPD assay, and followed by a second dimension electrophoresis three-quarters stored at —80°Cand devoted to protein (Bahrman & Thiellement, 1987). The gels were silver- analysis. stained according to Damerval et al. (1987) in the

The Genetical Society of Great Britain, Heredity, 74, 661—668. INTEGRATED RAPD—PROTEIN MAP OF MARITIME PINE 663 apparatus described by Granier & de Vienne (1986) allelic products of structural genes varying in position, and dried. 22 spots concerned presence/absence variations and three spots involved staining intensity variations (see Bahrman & Damerval, 1989; Gerber et 1993, for and nomenclature of RAPD and protein al., Scoring genetic analysis of each variation). They all segregated markers in a Mendelian fashion (a =0.01). From 520 Segregationof RAPD markers was recorded in four OPERON primers screened for polymorphisms, 142 sets of 31 different megagametophytes from HI 2. showed amplification and segregating RAPD markers DNA extraction, reaction mixture preparations, gel present in one grandparent as well as in the hybrid analysis and genotype scoring were performed inde- parent and absent in the other grandparent. Out of the pendently for each set. This replicated design provided 142 primers, 113 and 29 were used to produce RAPD a control that aimed to retain RAPD markers that markers on four and two replicates of 31 megagameto- amplified consistently in the studied population. phytes, respectively. Segregation of 470 RAPD RAPD fragments were named by the OPERON primer markers was scored in the whole experiment (Fig. 1). A code, followed by their molecular size in base pairs. total of 437 RAPDs were repeatable among the map- Because protein analysis requires elaborate laboratory ping replications. On average, one primer produced techniques, protein markers were recorded for only 34 three polymorphic RAPD fragments. Fragment sizes randomly chosen megagametophytes among the map- ranged from 194 to 2627 base pairs. They all con- ping sample of 124 megagametophytes. The dried gels formed to Mendelian segregation (a =0.01).The were visually scored on an illuminated box by super- nonreproducible RAPD bands were discarded from imposition. Three kinds of variations were scored in further analysis. They were typically very faint, and 2-D protein patterns: position (V), presence/absence often had a molecular weight > 2000 bp or <200 bp. (P) and staining intensity (I) variations. The name of each marker included the grandparental origin ('+' Constructionof the genomic map denoted markers inherited from the Corsican grandparent, '—'denotedmarkers inherited from the Landes Groupingand initial ordering of markers were carried grandparent). out at LOD 5.0 and Out of 471 markers (436 RAPDs and 35 proteins), 463 loci (436 RAPDs and 27 proteins) were assigned to 13 linkage groups. Linkageanalysis Eight of the protein markers were not linked to any Atotal of 463 genetic markers were tested for other locus when lowering the statistical stringency. departure from the1:1 Mendelian ratio of However, lowering the LOD score to 3.0 and keeping presence:absence of band. The linkage relationships of O to 0.30 would result in the merging of linkage group the markers were analysed with the Macintosh 1 and 13 as indicated by a faint line between markers MAPMAKER v2.O computer program (Lander eta!., R10_767/— and O18_1207/+ (Fig. 2). Few addi- 1987). Markers were considered to be linked when tional mapped markers would be needed to fill the gap their LOD score was 5.0andrecombination fraction between these two groups. Local mapping based tech- O 0.30. A subset of markers that could be ordered niques (Reiter et al., 1992) should facilitate this objec- with an interval support 4 (i.e. difference in log likeli- tive. The number of major groups corresponded to the hood between the best and alternative orders 4), 12 expected based on the known karyotype of mari- provided a framework map. Accessory markers that time pine (Saylor, 1964). could not meet this ordering criterion were located to A framework map was established using the RIPPLE the closest framework markers. Recombination command, to identify a subset of loci that could be distances of accessory markers to the nearest locally ordered with an interval support 4. Approxi- framework markers were incorporated in the marker mately 53 per cent of the markers were placed on the names. Recombination fractions were converted to framework map defining a total of 244 loci and 1860 map distances using Kosambi's mapping function. cM of map distance. The size of linkage groups ranged from 177.9 cM to 16.6 cM. The average distance between two framework markers was 8.3 cM, with only Results a few gaps exceeding 20 cM. Only one interval between two markers located in linkage group 5 was larger than Identification of polymorphic markers 30 cM. However, the LOD score for this interval was We scored 35 protein markers in 2-D protein patterns above the threshold 5. The majority of the intervals (72 obtained from germinated megagametophytes. Twenty per cent) were <10 cM. Most of the accessory markers spots belonging to 10 polypeptides corresponded to were placed within 5 cM of the nearest framework The Genetical Society of Great Britain, Heredüy, 74,661—668. 664 C. PLOMJON ETAL.

4 4

Fig. 1 Segregation of RAPD markers. The first lane is a molecular weight size standard (stars from the top correspond to 1636, 1018, 510 and 396 base pairs). Other lanes show the separation of five RAPD fragments amplified from 31 genomic DNA samples, using primer J16. Segregating RAFD markers are indicated by arrows (arrows from the top correspond to RAPD markers J16_1118/+,J16....1056/+,J16965/—,J16_643/+,J16_479/—).

marker. The limited number of megagametophytes did struction procedure, should provide a high confidence not allow a precise estimate of the recombination for map length and loci order. distances and ordering of tightly linked markers. In a The 27 mapped protein loci were well distributed sample of 62 or 124 megagametophytes, approxi- throughout the genomic map (Fig. 2). A total of four, mately 95percent of the gametes will show no recom- three, two and one protein loci were mapped in two, bination in a 5 cM interval and provide no information two, five and three linkage groups, respectively. The on order. Assuming the same error rate in genotyping, three types of proteins (see Materials and methods) we also observed that the number of accessory RAPD were represented on the map. Protein loci were geno- markers placed at distances 6 cM from the nearest typed on only 34 megagametophytes, which did not framework marker was not much higher for 62 (two allow a precise estimation of two-point recombination sets of 31 megagametophytes) than for 124 mega- fractions. This could explain the high proportion of gametophytes (four sets): 22 per cent and 18 per cent unlinked protein markers (23 per cent) and the fact amplified from samples of 62 and 124 megagameto- that almost all proteins could not meet the local order phytes, respectively. Thus, there was little increase in criterion used for framework map construction (inter- precision on the relative position of accessory markers val support 4). Therefore, most protein loci were when genotyping 124 individuals instead of 62. placed as accessory markers. Assuming that the order of framework markers was correct, we used the SHOW n.w command to identify double recombinants that involved flanking loci. True Copynumber of RAPD fragments double recombination events should be rare, and an Outof 53 RAPD fragments, 11 per cent did not show excess of double recombination could indicate poten- detectable hybridization or gave a faint signal in the 20 tial scoring errors. Double crossovers were systema- pg dilution. They were classified as amplifying from ticaly re-examined on gel photos. When dubious data low-copy to moderately repetitive chromosomal points were found they were treated as missing data. regions (Fig. 3a,b). Twenty per cent gave a signal in the Then the ordering analysis was performed again. 20 1ug and 2 pg dilutions and were classified as ampli- Errors in genotyping were mostly from weak amplifica- fying from highly repeated regions (Fig. 3c); 69 per tion of a specific band, smearing problems or artefacts cent gave a signal in all dilutions and were classified as from the loading of wells in the gel. This data quality amplifying from very highly repeated regions (Fig. control, in combination with the framework map con- 3d,e). So, 89 per cent of the RAPD fragments were

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(a) (b) (c) (d) (e) The RAPD primers used for mapping consisted of random sequences that should not discriminate coding and noncoding chromosomal regions. Therefore, and at least in conifer species, most RAPD loci are likely to fall within noncoding DNA. The characterization of the internal sequence of 53 RAPD fragments for copy number in the maritime pine genome showed that, although RAPD fragments mapped to unique genomic Fig. 3 Dot blot analysis of five RAPD fragments using a sites, most of them contained highly repeated chemiluminescence detection assay. Arrows from the top sequences. Conversely, protein markers sample regions correspond to 20 g herring sperm DNA as a negative of coding DNA. Our results showed that mapped control, and a serial dilution of 0.2 tg, 2 ug and 20 ug of protein markers were well distributed throughout the pine genomic DNA, immobilized on nylon membrane. genome of maritime pine. RAPD fragments containing low-copy to moderately repetitive sequences (a,b), highly repetitive sequences (c) and very highly repetitive sequences (d,e) are shown. Single-treemap vs. species map Aspecies consensus map of markers and traits could be difficult to use for breeding applications in alto- presumed to contain at least some high-copy gamous species with a wide genetic base, such as forest sequences, and were likely to amplify from highly trees (Grattapaglia & Sederoff, 1994). Marker:trait repetitive chromosomal regions. Thus, RAPD frag- associations are likely to be in linkage equilibrium in ments should be poor probes for hybridization experi- early generations of the breeding population and will ments (e.g. RFLP assay) in maritime pine. probably have to be established for each cross inde- pendently. Mapping of individual trees using markers Discussion specific to only one cross provides a powerful approach to genetic analysis of quantitative and Pine genome organization complex qualitative traits within families. However, genomic maps of individuals using RAPD markers can Gynmosperm species are characterized by: (i) their not readily be combined to make a concensus species antiquity (conifers appeared 140 millions years before map because the migration distance of a RAPD frag- the first angiosperm) and their longevity, (ii) the ment is not sufficient information to identify uniquely a absence of ploidy level and chromosome number specific locus across a species. Similar problems exist evolution (reviewed by Neale & Williams, 1991), and for RFLP probes that recognize several bands (e.g. (iii) the very high and consistent amount of DNA per Tanksley et at., 1988; Song et a!., 1991; Devey et a!., nucleus (Ohri & Khoshoo, 1986; Wakamiya et a!., 1994), a problem addressed by using probes that yield 1993). The technique of DNA reassociation kinetics only one band (e.g. Beavis & Grant, 1991). Further- applied to Pinus species (Miksche & Hotta, 1973; more, many individuals could be homozygous and the Rake et at., 1980; Kriebel, 1985) showed that 25 per marker would not be available for mapping in many cent of total DNA is low- to single-copy, 75 per cent crosses, depending on gene frequency. The criteria for being middle to highly repetitive. Thus, the vast establishing synteny using RAPD markers, or multiple majority of the DNA in the pine genomes is arranged in band RFLP markers must be more stringent, perhaps repeated sequence families. Most of this repeated DNA requiring parallel linkage groups having several does not encode proteins (Thompson & Murray, markers in the same order in different individuals. The 1981). If 60 000 genes are expressed during the life identity of some allozyme or protein markers (Gerber cycle of a plant (Kamalay & Goldberg, 1980), given an et a!., 1993) should be useful for establishing the average size of a gene of 2000 bp (exons only) and the correspondence of linkage groups in RAPD maps from size of maritime pine genome as 24 x 106 kbp, 0.5 per different trees. The distribution of the 27 mapped cent of the genome is likely to be coding DNA. This protein loci throughout the genomic map of maritime could be an underestimate because a significant pine is encouraging for that objective. A further advan- number of genes occur in multigene families (Kinlaw & tage to using proteins as genetic markers for the Gerttula, 1993; Ahuja eta!., 1994; Devey et a!., 1994). mapping of quantitative or qualitative traits is that the Thus, coding DNA may not represent more than a few polymorphism of a specific gene product could poten- per cent of the pine genome. This result agrees with the tially be responsible for the mapped quantitative effect estimated fraction of coding regions in plant species (Damerval et a!., 1994). Alternatively, a small number (Goldberg et a!., 1978; Thompson & Murray, 1981). of hypervariable microsatellite markers could be

The Genetical Society of Great Britain, Heredity, 74, 661—668. INTEGRATED RAPD-PROTEIN MAP OF MARITIME PINE 667 assayed in each cross to establish the correspondence DNA amount (Rees & Durrant, 1986; Tanksley eta!., of linkage groups. 1988).

Genomesize of maritime pine Acknowledgements RAPD assay was completed in the Forest Biotech- Theprotein data produced by Bahrman & Damerval The (1989) and Gerber et al. (1993) suggested a genome nology Group at North Carolina State University. We size of approximately 2000 cM for maritime pine thank Dario Grattapaglia for assistance with the (Gerber & Rodoiphe, 1994). However, framework characterization of genomic sequence complexity of map procedures had not been used to construct these RAPD markers and spending much time discussing two protein-based maps. This may lead to over- genetics. We thank Antoine Kremer for critical reading estimates for genetic distances and total map length. of the manuscript. This work was supported by a grant Maritime pine has 12 metacentric chromosomes of from the Institut National de la Recherche Agrono- mique (AlP Génome des plantes cultivées 1994) and approximately equal size (Saylor, 1964). Linkage from the USDA Forest Service (Coop Agreement No groups 1—12 had approximately the same length (about 155 cM) and therefore should provide almost com- 29-1009). plete coverage of the genome. In addition, the genome size of the presented framework map (1860 cM) References agreed with what has been found for other dense AHUJA,M. R., DEVEY, M. E., GROOVER, A. T., JERMSTAD, K. D. AND linkage maps of loblolly pine, constructed with NEALE, D. B. 1994. Mapped DNA probes from loblolly pine approximately 400 RAPD markers (H. Amerson & P. can be used for restriction fragment length polymorphism Wilcox, personal communication). The relationship mapping in other conifers. Theor. App!. Genet., 88, between recombination rates and genome size has 279—282. been a matter of speculation for many years. Grant ARUMUGANATHAN, K. AND EARLE, E. D. 1991. Nuclear DNA content of some important plant species. Plant. Mo!. Biol. (1958) predicted that plants with long generation Rep., 9,208—218. times, such as pine, will have genetic systems that BAJ-IRMAN, N. AND DAMERVAL, c. 1989. Linkage relationships of promote recombination. Short-lived annual plants, loci controlling protein amounts in maritime pine (Pinus such as Arabidopsis, should have genetic systems that pinaster Ait.). Heredity, 63, 267—274. restrict recombination. One mechanism to promote BAHRMAN, N. AND THIELLEMENT, H. 1987. Parental genome recombination could be an increased number of expression in synthetic wheats (Triticumturgidum sp. x T. chromosomes. Grant (1958) also speculated that long- tauschii sp.) revealed by two-dimensional electrophoresis lived organisms such as pine might have a higher of seedling proteins. Theor. App!. Gene!., 74, 218—223. chiasma frequency to promote recombination. Our BEAVIS, W. D. AND GRANT, D. 1991. A linkage map based on data for pine, however, do not support this idea. The information from four F2 populations of maize, Zea mays total map distance per chromosome was approximately L.. Theor. App!. Genet., 82,636—644. BINELLI, G. AND BUCCI, G. 1994. A genetic linkage map of Picea 1.55 Morgans for pine and 1.30 Morgans for abies Karst., based on RAPD markers, as a tool in popula- Arabidopsis (Reiter el a!., 1992). Maritime pine and tion genetics. Theor. App!. Genet., 88, 283—288. Arabidopsis have approximately 2 pg and 0.03 pg of BOUSQUET, 3.,SIMON,L. AND LALONDE, M. 1990. DNA amplifica- DNA per chromosome, respectively (Ohri & Khoshoo, tion from vegetative and sexual tissues of trees using poly- 1986; Arumuganathan & Earle, 1991). Thus, on a merase chain reaction. Can. J. Forest Res., 20, 254—257. chromosomal basis, maritime pine has approximately BROWN, J. AND SUNDARESAN, v. 1991. A recombination hotspot 57-fold more DNA per cM than Arabidopsis. How- in the maize Al intragenic region. Theor. App!. Gene!., 81, ever, the number of crossovers per chromosome was 185—188. almost equivalent and did not seem to be really CONKLE, M. T. 1981. Isozyme variation and linkage in six affected by the DNA content and the proportion of conifer species. Proceedings of the Symposium on coding DNA. Although large and small genomes could Isozymes of North American Forest Trees and Forest differ in the organization and structure of genomic Insects. USDA For. Serv. Gen. Tech. Rep., PSW, 48, 11—17. DNA (John & King, 1980; Flavell et a!., 1985; Brown DAMERVAL, C., LEGUILLOUX, M., BLAISONNEAU, J. AND de VIENNE, D. & Sundaresan, 1991), the mechanism of crossing-over 1987. Simplification of the Heukeshoven and Dernick's must be highly conserved on a chromosomal basis and silver staining of proteins. Electrophoresis, 8, 158—159. independent of physical map size and the fraction of DAMERVAL, C., MAURICE, A., JOSSE, J. M. AND de VIENNE, D. 1994. coding DNA. This observation is consistent with other Quantitative trait loci underlying gene product variation: a results showing that recombination per chromosome novel perspective for analysing regulation of genome was approximately constant despite large differences in expression. Genetics, 137, 289—301.

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