- Research article
- Open Access
Genomic characterization of putative allergen genes in peach/almond and their synteny with apple
© Chen et al; licensee BioMed Central Ltd. 2008
- Received: 15 July 2008
- Accepted: 17 November 2008
- Published: 17 November 2008
Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica).
Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01–3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2.
A total of 18 putative peach/almond allergen genes have been mapped on five linkage groups. Their positions confirm the high macro-synteny between peach/almond and apple. The insight gained will help to identify key genes causing differences in allergenicity among different cultivars of peach and other Prunus species.
- Linkage Group
- Peach Fruit
- Intron Size
- Apple Genome
- Peach Cultivar
In general, eating fruit is strongly recommended to improve health, but this can cause an allergic reaction in certain individuals. Most fruit crops in temperate and some subtropical zones belong to the Rosaceae family, mainly the subfamilies Maloideae and Prunoideae. Fruits from the Maloideae, like apple [1, 2] and pear , and Prunoideae such as peach [4, 5], sweet cherry [6, 7], plum , apricot  and almond  have been reported to cause allergic reactions in Europe. The annual peach production in China has been around 6 million tons in recent years, and has lead to both an increase in peach consumption and more prevalent fruit allergy.
It is considered that general avoidance of fruit has a negative effect on health in allergic patients and also affects their quality of life. Therefore, the low allergic property of fruit is worth considering in new peach breeding programs. As cultivars of the same species differ in allergenicity , the selection and breeding of new, low-allergenic cultivars is becoming feasible by a multidisciplinary approach, such as in the EU-SAFE project , where doctors and plant geneticists identified the apple cultivar 'Santana' as low allergenic in the majority of Dutch patients with a history of allergy to apple . A similar approach was initiated in peach in recent years through collaboration between China and Europe.
A variety of allergens from different fruits have been identified by means of experimental immunology and molecular biology, in particular by protein and gene identification and sequencing. From the structural and biochemical point of view, fruit allergens identified belong to a limited number of protein families . In the case of Rosaceae, the majority of allergens present in fresh fruit belong to four families: pathogenesis-related protein 10 (PR-10 protein, birch allergen Bet v 1 homologues), thaumatin-like proteins (TLP, PR-5 proteins), non-specific lipid transfer proteins (nsLTPs, PR-14 proteins) and profilins (PRF) . Whole, unprocessed fruit containing PR-10 protein elicit allergy, while TLP, LTP and profilins are s`proteins because of intra-molecular disulfide bonds. These four allergen families in Prunus fruits are already on the international allergen list http://www.allergen.org. In peach they are denoted as Pru p 1 (PR10), Pru p 2 (TLP), Pru p 3 (LTP), and Pru p 4 (profilin), and in almond as Pru du 1, 2, 3, and 4, respectively. Due to the common structure and properties of each protein family over a wide range of plant species, genera and even families, allergy cross-reactivity has been frequently observed, for example between Pru p 1 and Mal d 1 proteins from peach and apple respectively with Bet v 1 proteins in birch pollen . Peach allergy occurs more often in southern Europe, and two different peach allergy profiles have been observed in Spain. One of these is the oral allergy syndrome (OAS) caused by Pru p1 and Pru p 4, the other is the systemic gastrointestinal symptom caused by Pru p 3 . In China both profiles have also been reported after eating fresh peach . In addition, some contact with peach fruit hair or even with pollen during the blooming season caused adverse reactions (from some patients' claims).
Genomic characterization of the sequences and linkage map positions of the four apple allergen gene families were reported in 2005 [17–19], and subsequent associations between the genetic constitution and allergenicity of different cultivars gave the first indications on the isoallergens (genes) and protein variants (alleles) involved in low and high allergenic responses . Comparison between apple (Malus domestica) and Prunus linkage maps suggests a high level of synteny of the linkage groups between these two genera . For Prunus G1, the upper part contains several RFLP markers that aligned with apple LG13 and LG16 on which the majority of Mal d 1 genes are located, and the lower part aligned with LG8, where Mal d 4.03A is located. Prunus G3 aligns with apple LG9 where apple Mal d 2.01A/B and Mal d 4.01 were mapped. Therefore, some of the homologous allergen genes of Prunus fruit may be located at syntenic positions as compared with the apple genome.
In peach, differences in major allergen Pru p 1 (PR-10) and Pru p 3 (LTP) content among cultivars have been reported in recent years [11, 22], which encouraged us to conduct research similar to that done in apple [17–19]. Advances in three aspects make the genomic research of Prunus allergen genes possible. First, a set of candidate genes is now available based on previous medical and molecular biology research [23–25]. Second, a large number of peach fruit ESTs are available in databases (mainly from researchers in Chile, Italy and USA), allowing us to find different members of the four putative allergen gene families. Third, a highly saturated genetic Prunus reference map T × E (based on the F2 progeny of 'Texas' almond and 'Earlygold' peach) and a simple bin-mapping strategy  provides an efficient and reliable way to map new genes.
Basic genomic features of four putative allergen genes identified from the MB1-73 plant (F1 hybrid between 'Texas' almond and 'Earlygold' peach)
Pru p/du 1.01
Pru p/du 1.02
Pru p/du 1.03
Pru p/du 1.04
Pru p/du 1.05
Pru p/du 1.06A
Pru p/du 1.06B
Pru p 1.06C
Pru p/du 2.01A
Pru p/du 2.01B
Pru p/du 2.02
Pru p/du 2.03
Pru p/du 2.04
Pru p/du 3.01
Pru p/du 3.02
Pru p/du 3.03
Pru p/du 4.01
Pru p/du 4.02
Markers developed and cloning and sequencing primers for mapping
Allele length (nt)4
Pru du 1.01
Pru p 1.01
Pru p/du 1.02
Pru p/du 1.03
Pru p/du 1.04
Pru p/du 1.05
Pru p/du 1.06A
Pru du 1.06B
Pru p 1.06B
Pru p/du 1.06C
Pru du 2.01A
Pru p 2.01A
Pru du 2.01B
Pru p 2.01B
Pru p/du 2.02
Pru p/du 2.03
Pru p/du 2.04
Pru p/du 3.01
Pru p/du 3.02
Pru p/du 3.03
Pru p/du 4.01
Pru du 4.02
Pru p 4.02
Pru p/du 1 (PR-10) genes
Pru p/du 2 (TLP)
Based on the percentage amino acid identity among five Pru p 2 isoallergens, Pru p 2.01A, 01B and 2.02 were clustered with four apple Mal d 2 (01A, 01B, 02 and 03) (Figure 2B) proteins. Pru p 2.01A and 2.01B had 96.7% identity in their aa sequence, their closest homologous members being apple Mal d 2.01A and B. Pru p 2.03 and 2.04 are quite distant from 2.01 and 2.02 (Figure 2B) and no close counterparts were found in apple. The overall sequence identity level in amino acids among the four Pru p 2 proteins was around 40% (Figure 2B), but a long sequence region (DFYDVSLVDG [F/Y]NLPM) around position 130, and the typical 16 cysteine (C) to form 8 disulfide bonds, were conserved in all Pru p/du 2 members. Mature Pru p 2.01A, 02 and 04 proteins were acidic (pI around 4–5), whereas Pru p 2.01B and 3 were basic (pI 7.9–8.17). Mature Pru p 2.01 and 02 had a molecular weight of about 23 kDa. Mature Pru p 2.03 differed in length and weight with mature Pru du 2.03, with the molecular weight being 25.23 kDa and 27.46 kDa respectively. Mature Pru 2.04 had the highest number of aa (307) with a calculated molecular weight of 30.7 kDa. Unlike the Pru p 1 genes that were placed in a single bin, these five Pru p/du 2 members mapped on four linkage groups: Pru p/du 2.01 A and B on G3, -2.02 on G7, -2.03 on G8, and -2.04 on G1 (Figure 3A, C, D, E).
Pru p/du 3 (nsLTP)
Four peach and almond nsLTP members were traced from the NCBI DNA database, three of them have complete coding sequences, the fourth (GenBank:BQ641139) is a partial almond cDNA sequence distant from the other three. Genomic cloning and sequencing was done with the first three members Pru p/du 3.01-03 [GenBank: EU424264-EU424269]. Pru p/du 3 gene sequences have two exons and one intron (Table 1). The size of the second exon was conserved and extremely short (10 nt), while the first was variable (344, 362 and 341 nt). Sequences from peach and almond LTPs are highly conserved in exon and intron size, with only one base pair length difference in introns of Pru p/du 3.02 and 3.03 (Table 1).
Pru p/du 4 (profilin, PRF)
This study provides basic genomic sequences and map positions of four allergen families in Prunus persica (peach) and Prunus dulcis (almond). This information is a prerequisite for unravelling the genetic basis of allergenic characteristics of Prunus fruits, and is likely to be similar for other Prunus species due to the high level of synteny of the species within this genus .
Gene families and relevance of individual genes in allergenicity
Members of four gene families related to allergenicity, pathogenesis related 10 proteins (PR-10), thaumatin-like proteins (TLP), lipid transfer proteins (LTP) and profilin (PRF) on the five Arabidopsis thaliana chromosomes traced from the GenBank database
A limited number of putative allergen genes may be expressed in consumed fruits, their expression profiles at mRNA and protein level are essential to allergenicity. Evidence of cDNA present in the ripe fruit is not sufficient. The need to qualify and quantify their individual or combined allergenicity is a major challenge for further investigation, where the application of new experimental and computational tools in genomics and proteomics will be crucial. Results in this area are starting to emerge and integrate, such as differential expression of LTP 1 (Pru p 3.01) and LTP2 (Pru p 3.02) , the expression studies of Pru p 3.01 (Pp-LTP1 in two peach genotypes , where major differences were detected in expression at mRNA and protein accumulation levels between them, and the research on IgE binding capacity , where specific IgE-binding protein regions of Pru p 3 were found for two types of allergy (OAS and systemic) by the mimotope mapping technique. Of four Pru p 2 isoallergen genes, a clone PpAz44 [GenBank: AF362988] matching to Pru p 2.01 was abundantly expressed in ripe peach fruit, while clone PpAz8 [GenBank:AF362987] (Pru p 2.02) was undetected by northern blot analysis . Pru p 2.03 and Pru 2.04 were identified from the fruit ESTs, the latter may be in large amount of transcript, since 29 ESTs can be assembled into one consensus gene. A very low level of Pru p 4 (profilin) was has been identified as pollen-associated allergen, and two isoallergen genes were isolated from fruit tissue . Different Pru p 1 genes expression was not reported previously, our preliminary expression study was performed by means of real-time PCR for six Pru p 1 isoallergen genes on full ripe fruits of two cultivars. The result showed that the most abundantly expressed member is Pru p 1.01, followed by Pru p 1.06, then very low expression level for Pru p 1.04 and -1.05, while Pru p 1.02 and -03 were hardly detected (data not shown).
In apple, a very recent comprehensive gene expression of four classes of putative allergens in apple fruit has gained insight of the genetic and environmental factors affecting the allergenic potential of apples . They showed that some putative members are statistically different in transcript accumulation in ripe fruit, while others were not detectable. This was a very nice reference to think about comparative transcription and protein expression for all set of peach putative allergen genes among diverse peach cultivars.
We found that the putative genes encoding the major allergens reported in peach fruit, Pru p 1 and Pru p 3 , were located on two linkage groups, G1 and G6. Based on their bin map positions, we can use SSR markers tightly linked or flanking these two major Prunus allergen genes for a diversity survey of a large number of genotypes to search for potential candidates for further sequencing of the allergen genes. Clinical evaluation of allergenicity for different peach cultivars is needed for association analysis to find low allergenic alleles.
Comparative mapping between Prunus and Malus using the sequence and position of allergen genes
Dirlewanger et al.  compared the Prunus and apple genomes using a limited number of common markers. They found that three Prunus linkage groups (G1, G3 and G4) were syntenic to seven apple linkage groups, G1 to LG13 and LG16 at the top and LG8 at the bottom, G3 to LG9 and LG17, and G4 to LG5 and LG10. Information is also available on the correspondence between homoeologous linkage groups within the apple genome obtained initially with RFLPs by Maliepaard et al. , to which various allergen genes and SSRs were recently added by Gao et al. [17–19] and Celton et al. , respectively. With the availability of genomic sequences and the linkage map positions of the four allergen-families in apple (26 apple allergen genes on eight linkage groups) [17–19] and peach (18 genes on 5 linkage groups, this study), we have additional information useful for updating the synteny between these two species.
Three of the eight Prunus bins where we located putative allergen genes corresponded to known syntenic and colinear regions of the apple genome (Figure 3): the Pru p/du 1 on G1, corresponding to regions with clusters of homologous genes (Mal d 1) of apple on LG13 and LG16, Pru p/du 4.01 on the lower part of G1 and Mal d 4.03 on LG8, and Pru p/du 2.01A/B on G3 and Mal d 2.01A/B on LG9. Three more syntenic regions were detected in this study: Pru p/du 4.02 on G7 and the region of LG2 where Mal d 4.02 maps, Pru p/du 3.01-03 on G6 corresponding to Mal d 3.01 on LG12 and Mal d 3.02 on LG4; and Pru p/du 4.01 which, in addition to the region of LG8 already mentioned, is syntenic with the upper part of LG15 where Mal d 4.03B maps. No counterparts were found to the Pru p/du 2.02, -03, and -04 genes, which were mapped on G7, G8 and G1, respectively, on the apple genome. The positions of these three genes in the Prunus genome indicate syntenic regions of apple where additional Mal d 2 genes may be located. Because Mal d 1 on apple LG 13 and LG16 are clustered in a region of about 2 cM, so we can deduce our mapped Pru p 1 genes are also in a cluster.
There are two hypotheses on the origin of the two genomes that generated the apple amphydiploid genome (2n = 34). One proposes that these were from members of two Rosaceae subfamilies, one from the Amygdaloideae (thus closer to Prunus with x = 8) and another from the Spiraeoideae (x = 9) . The other hypothesis proposes that both ancestral genomes had a basic chromosome number of x = 9 at least one being Spiraeoideae (x = 9) . Combining map position and sequence comparison, the cluster of Pru p 1 genes mapped on Prunus G1 is more similar to that of the Mal d 1 genes of LG16 than to LG13: a) Mal d 1 isoallergen genes were classified into four subfamilies and members of sub-families were distributed asymmetrically to LG 13 and LG16 (Figures 2, 3), i.e. LG13 contains Mal d 1 members from subfamilies I and IV, but LG16 contains representative members of the four subfamilies; b) Pru p 1 genes fall into the four subfamilies as defined in apple, and had a higher deduced amino acid sequence similarity to the Mal d 1 members on LG16 (Figure 2 and data of percentage amino acid identity in the Results section). These results favor the two primitive genomes for apple and one is closer to Prunus than the other.
Bin mapping feasibility and effectiveness
The bin mapping approach provides a fast and cheap way to map new genes  that has worked very well in our hands. Once we cloned and sequenced the two putative alleles for each gene from MB1-73, we either created an allele-specific marker and genotyped the eight plants of the bin set, or cloned the fragments from the individuals of the bin set and sequenced them to determine their genotypes. One of the advantages of using the T × E population for bin mapping is that, due to its inter-specific nature, polymorphism is very high . The disadvantage of the bin mapping approach is that there is a loss of precision in marker location (8–25 cM), but this can be solved for markers of particular interest by running the whole population to establish a more precise position or narrow down a region where closely related genes or common RFLP markers in apple has been fine mapped. For example, we estimate Pru p/du 1 genes (in bin 1:14) are in a cluster and likely around 13 cM from the top based on location of Mal d 1 genes and anchor marker MC001 (Figure 3A). A very large number of ESTs from peach fruit are on public databases. This information is essential to find candidate genes to be mapped, and their positions compared to that of major genes or quantitative trait loci of interest mapped using phenotypic data. Bin mapping, as in this case, can facilitate the positioning of these candidate genes. Exchange of DNA and genotype data of the T × E bin set in the peach research community is helping candidate gene mapping, with genetic analysis and breeding applications following on.
Eighteen putative Prunus allergen genes were located on five of the eight linkage groups of this genus. Genomic characterization of Prunus allergens provided further evidence for synteny between Prunus and Malus. This information will be directed towards identifying the key genes or isoforms causing the differences in allergenicity among different cultivars by additional gene and protein expression and genetic association studies. These results are a first step towards the understanding of allergenicity caused by peach fruit and for the development of new cultivars with enhanced health properties and subject to specific recommendations on fruit consumption.
PCR cloning and sequencing for targeting allergen genes to identify different gene members and alleles was mainly on the 'Texas' (T) almond and 'Earlygold' (E) peach hybrid (MB1-73) used to generate the reference mapping population (T × E) and it is now used by the international Prunus community. Therefore, two putative alleles, one from 'Texas' (T) and another from 'Earlygold' (E), will be simultaneously amplified with a pair of primers based on a reference sequence. For T and E allele-specific marker tests, DNA from T and E were used as controls. Eight plants were used for the bin set (the set of plants used for bin mapping): the grandparent E, the parent MB1-73, and six plants from the F2 progeny (# 5, 12, 23, 30, 34, 83) . DNA samples were isolated and provided by IRTA using standard protocols .
PCR primers, cloning and sequencing
PCR primer pairs used for genomic cloning of the different peach/almond allergen genes and for (bin) mapping the genes
Primer sequence (5' – 3')
Pru p/du 1.01/05
Pru p/du 1.02
Pru p/du 1.03
Pru p/du 1.04
Pru p/du 1.05
Pru p/du 1.06A/C
Pru p/du 1.05
Pru p/du 1.06A/B
Pru p/du 1.06C
Pru p/du 1.06A
Pru p/du 2.01A/B
Pru p/du 2.02
Pru p/du 2.02
Pru p/du 2.03
Pru p/du 2.03
of 29 ESTs
Pru p/du 2.04
Pru p/du 3.01
Pru p/du 3.02
Pru p/du 3.03
Pru p/du 4.01
Pru p/du 4.02
Genome walking was applied to gDNA of MB1-73 using the Universal Genome Walker kit (Clontech, Palo Alto Calif.) for gene sequences that were not cover the ORF or to avoid the mistakes induced by primer sequence, or to explore polymorphisms in the flanking regions. Four libraries were constructed using Dra I, EcoR V, Pvu II and Stu I enzymes to digest 2.5 μg of gDNA from MB1-73. Adaptors were ligated to the digested DNA fragments. Two gene specific primers (GSP, Table 4) and two adaptors were used for nest PCR. The product from two of the four libraries was excised from the gel and subsequently purified, ligated, transformed and sequenced as described in the above section.
DNA sequences were analysed and single nucleotide polymorphisms (SNPs) were identified using the SEQMAN program (Lasergene 6, Madison, Wis.). Intron sizes were deduced by comparing the genome sequences with cDNA (ESTs) sequences or by putative conserved splicing patterns in plants. The phylogenetic tree was created and percentage sequence identity calculated using Clustal W by Megalign (Lasergene 6). The GenDoc program http://www.psc.edu/biomed/genedoc was used for multiple DNA and amino acid sequence alignment.
Allele-specific markers and bin mapping approach
Three types of molecular markers were used: 1) two dominant single nucleotide amplification polymorphism (SNAP) markers  representing T and E respectively, 2) co-dominant markers based on length differences due to Indels and variation in the number of simple sequence repeats (SSRs) and 3) SNP markers based on direct sequencing of the PCR products from 8 bin samples. Markers 1) and 2) were used for the mapping of Pru p 1, 2, and 4 genes and 3) for Pru p 3 genes.
With SNAP markers, the annealing temperature is usually critical. It was optimised by gradient PCR using gDNAs of 'Texas' almond and 'Earlygold' peach (the two parents of MB1-73) as contrasting templates, and then applied to genomic DNA of 8 bin samples. Indels and SSR markers were revealed by 6% PAGE and silver staining. For direct sequencing, after PCR amplification (GoTaq, Promega), an aliquot of the PCR reaction was loaded on an agarose gel to check for specific amplification. If the PCR produced a single fragment, the remaining PCR reaction was purified using Roche High Pure PCR Product purification kit (Germany). An aliquot of 0.5 μl to 5 μl of purified PCR was used as template for sequencing with forward and/or reverse primers. SNPs and/or indels were detected using Sequencher software, version 4.7.
For mapping, we followed the strategy of "selective" or "bin" mapping [26, 37] where, based on the data of a detailed map already constructed (in this case the TxE map), a few plants are selected having a high number of recombination breakpoints and an approximately even distribution of these breakpoints throughout the genome. The joint genotype of these plants for a new marker defines a single map position, allowing its mapping with less time and cost, and with a moderate loss of precision compared with the use of the whole population. For Prunus, a set of only eight plants: one of the parents ('Earlygold') the F1 hybrid (MB1-37) and six plants (# 5, 12, 23, 30, 34 and 83) from the TxE progeny, was selected by Howad et al.  allowing to establish the position of any marker to 67 fragments of the genome ("bins") of an average size of 7.8 cM. The identification of each bin is unequivocal except for two pairs (2:45/3:04 and 5:41/8:30), which have an identical genotype and require the genotype of an additional F2 plant (#27) to separate them. By comparing the genotype appeared in H and E, we were able to distinguish T allele from that of E. Marker tests were performed on these eight individuals of the bin set that were genotyped as homozygous for the 'Texas' allele (noted as A), homozygous for the 'Earlygold' allele (B) or heterozygous as MB1-73 (H). The resulting segregation pattern was compared to the defined data for each bin, and the matching bin was identified .
Nomenclature of the putative allergen sequences
We followed the systematic allergen nomenclature  for the four different protein classes and encoding genes. Generally, allergens are designated by the first three letters of the name of the genus, followed by a space and the first one or two letters of the species name. These letters are usually followed by a space and an Arabic number according to the order in which the allergens were identified, whereby the same number is used to designate homologous allergens of related species. Additional numbers and letters can be added to distinguish different isoallergens and variants . Accordingly, PR-10, TLP, LTP and PRF proteins are denoted as Pru p 1 to 4 in peach and Pru du 1 to 4 in almond. Amino acid sequences with more than 5% dissimilarity usually belong to a different isoallergen and are distinguished by a two-digit code. If there are several genes within isoallergen group (on the DNA sequence identity, usually more than 95%), they are further distinguished by adding a capital letter after their isoallergen name, such as Pru p 2.01A and Pru p 2.01B. This procedure has been employed in apple  and birch . The plant material in this study is an interspecies hybrid between peach and almond; we assigned two alleles of the same gene locus by its original species source, from Prunus persica as Pru p and from Prunus dulci as Pru du. Therefore, Pru p/du refers to a gene in interspecies hybrid parent MB1-37.
This study has been carried out with financial support from the China Ministry of Education (107064 and 1108), Natural Science Foundation of China (30771496), Science and Technology Department of Zhejiang Province (2007C22068) and the 111 project (B06014). It was supported in part by the Commission of the European Communities for the project ISAFRUIT (Contract FP6-FOOD-CT-2006-016279). Prof. Xu Changjie and Dr. Zhang Bo are acknowledged for their advice and assistance on the lab work, and Dr. Luud Gilissen for his critical reading of this manuscript.
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