Retrotransposon-based Plant DNA Barcoding

Plant DNA Barcoding and Phylogenetics, Pages xx-xx Eds. M. Ajmal Ali, Gábor Gyulai and Fahad Al-Hemaid © 2015 Lambert Academic Publishing, Germany 1 Retrotransposonbased Plant DNA Barcoding A.M. Alzohairy, G. Gyulai, M.M. Mustafa, S. Edris, J.S.M. Sabir, R.K. Jansen and A. Bahieldin Introduction Retrotransposons (RTs) are major components of most eukaryotic genomes; they are ubiquitous, dispersed throughout the genome, and their abundance correlates with the host genome sizes. Copy-and-paste life style of the RTs consists of three molecular steps, which involve transcription of an RNA copy from the genomic RT, followed by reverse transcription to cDNA, and finally a reintegration event into a new locus of the genome. This process leads to new genomic insertions without excision of the original RT. The target sites of insertions are relatively random and independent for different plant taxa; however, some elements cluster together in ‘repeat seas’ or have a tendency to cluster around the chromosome centromers and telomeres. The structure and copy number of retrotransposon families are strongly influenced by the evolutionary history of the host genome. Molecular barcoding of RTs play an essential role in all fields of genetics and genomics, and represent a powerful tool for molecular barcoding. To detect RT polymorphisms, marker systems generally rely on the amplification of sequences between the ends of the retrotransposon, such as long terminal repeats (LTR) of LTR-retrotransposons (LTR-RT) and the flanking genomic DNA. In this Chapter, we review the utility of some commonly used PCR-based molecular barcoding methods of LTR-RTs, including RBIP 1 (Retrotransposon-Based Insertion Polymorphism), REMAP (Retrotransposon-Microsatellite Amplified Polymorphism), SSAP (Sequence-Specific Amplified Polymorphism), IRAP (Inter Retrotransposon Amplified Polymorphism) and IPBS (Inter Primer Binding Sequence). Interspersed repetitive DNA sequences comprise a large fraction of the eukaryotic genomes. They predominantly consist of transposable elements (TEs) with two main families, Retrotransposons (Class I) and DNA transposons (Class II) (McClintock, 1984). Retrotransposons (RTs) are the most abundant class of TEs (IHGSC, 2001; Feschotte et al., 2002; Sabot and Schulman, 2006; Kalendar and Schulman, 2006). There are two major groups of RTs based on the presence vs. absence of long terminal repeats (LTRs), LTR-retrotransposons (LTRRTs) and non-LTR-retrotransposons. LTR-RTs comprise two main subgroups, copia (with high copy number) and gypsy (with high transposing activity) (Fig. 1). Both, copia and gypsy LTR-RTs, carry regulatory sequences of gene promoters such as CAAT box (e.g., CCATT), TATA box (e.g., TGGCTATAAATAG), transcription start (e.g., CCCATGG), polyadenylation signal (e.g., AATAAG), and polyadenylation start (e.g., TAGT) (Ramallo et al., 2008). All these domains are required for replication and integration of RTs (Sabot and Schulman, 2006; Mansour, 2008). The large internal domain of the LTRRTs encodes the structural proteins of the virus-like particle, which encapsulate the RNA copy of the RT, and the enzymes Reverse Transcriptase and Integrase (Fig. 1). The process is called transposition. There are three further non-autonomous, short derivative, recombinant LTR-RTs, LARD (Large Retrotransposon Derivatives), TRIM (Terminal Repeat Retrotransposon in Miniature) and solo-LTR (sequence carrying 5’ and 3’ LTRs only) (Xiong and Eickbush, 1990; Havecker et al., 2004; Jurka et al., 2007). The size of LTR-RTs varies from long (e.g., Bare1 copia LTR-RT at 13,271 bp, NCBI Z17327) to short (e.g., Bare1 copia solo-LTR-RT at 3,130 bp, NCBI AB014756; and the truncated RLC_Lara copia RT; at 735 bp, NCBI EF067844; TREP2298). In plants, LTR-RTs are more plentiful and active than non-LTRRTs (AGI, Arabidopsis Genome Initiative, 2000; Rice Chromosome 10 Sequencing Consortium, 2003; Alzohairy et al., 2012; 2013; 2014a,b). Due to the induction of chromosome recombinational processes during the meiotic prophase, active retrotransposons tend to lose their activity due to sequence breakage (Mansour, 2007; 2008; 2009; Alzohairy et al., 2012; 2013; 2014a,b). 2 (a) Gag-Pol Coding Region 5’ LTR PBS GAG AP INT RT RH PPT 3’LTR LTR3’ 5’ LTR PBS GAG AP RT RH INT PPT 3’ LTR Internal Domain (b) Figure 1. Schematic of structural differences between Copia (a) and Gypsy (b) LTR-RT families. 5`LTR - 5`-end of long terminal repeat; PBS - primer binding site; GAG - group-specific antigen (syn.: capsid protein gene); AP - aspartic protease gene; INT - integrase gene; RT - reverse transcriptase gene; RH - ribonuclease-H gene; 3`LTR - 3`-end of the long terminal repeat; PPT polypurine tract (Alzohairy et al., 2014b). Utilization of retrotransposons as molecular markers Molecular barcoding methods (Schulman, 2007) based on RTs rely on PCR, and detect large portions of the genome (Kalendar et al., 1999; Kalendar and Schulman, 2006; Venturi et al., 2006; Branco et al., 2007; Sanz et al., 2007; Mansour, 2008; Mansour et al., 2010; Poczai et al., 2013). Barcode and marker systems based on different RTs show different levels of resolution and can be chosen to fit the identification of a given genome (Leigh et al., 2003; Queen et al., 2004; Ashalatha et al., 2005; Chadha and Gopalakrishna, 2005; Tam et al., 2005; Teo et al., 2005; Brik et al., 2006; Kalendar and Schulman, 2006). Retrotransposonbased markers follow Mendelian inheritance with high levels of genetic variability (Manninen et al., 2000; Huo et al., 2009). Three different orientations of RTs are possible (i.e., head-tohead, tail-to-tail, and head-to-tail), either at a single locus, or inserted next to or within each other (nested RTs). This feature increases the variation available for revealing polymorphism within and among species. If the RT sequence and the adjacent genomic sequences are known, then all types of PCR-based molecular techniques can detect RT polymorphisms. As the new cDNA copy of RT integrate into a new locus of the genome the old copy persist in the genome across generations, and the variation between ancestral and derived RT loci can be revealed (Mansour, 2008). The presence of a given retrotransposon suggests its orthologue integration, while the absence indicates the plesiomorphic condition prior to integration (Kalendar, 2011). The presence vs. absence of RTs can be utilized to construct phylogenetic trees of species due to the distribution of retrotransposons across organisms. This is the reason that RTs have been suggested to provide powerful phylogenetic markers with little if any homoplasy (Shedlock and Okada, 2000, Schulman, 2007). 3 Primer design for detection LTR-RTs The LTR sequences are chosen to minimize the size of the target to be amplified. A primer facing outward from the 5’ LTR will necessarily face inward to a 3’ LTR of a neighboring LTR-RT, because the LTRs are direct repeats. The long sequences of LTR may also interfere with the production of amplicons within the size range of standard PCR. The conservative regions of LTR sequences are also used for designing inverted primers for Long-PCR, which can be used for cloning entire RTs and also for IRAP, REMAP and SSAP techniques. IRAP primers are designed for using single or double primers In REMAP, one primer is designed from the LTR and another from a nearby simple sequence repeats (microsatellites, syn.: SSRs). RBIP can detect both the presence and absence of the RT insertion using three primers to generate single-locus codominant markers. In SSAP, two primers are designed to produce amplification between RTs and adaptors ligated to a restriction site (usually MseI or PstI). In IPBS, primers are designed to match and amplify the conserved regions of the primer binding sequences (PBS). One or two primers can be used depending on the desired output of the experiments. Retrotransposon-Based Insertion Polymorphism (RBIP) RBIP (Flavell et al., 1998) detects retrotransposon insertions using a primer flanking the insertion site of the genome and another primer binding to the retrotransposon (Fig. 2). (a) U3 Flanking region TG R 3’LTR U5 CA PBS 5’LTR Internal Domain U3 TG R U5 CA Flanking region LTR Retrotransposon RBIP products RBIP products with RE insertion with RE insertion (b) Flanking region Flanking region RBIP products with no RE insertion Figure 2. RBIP (Retrotransposon-Based Insertion Polymorphism; Flavell et al., 1998) detects the presence (a) or absence (b) of retrotransposons in the host genome. Amplification takes place between retrotransposons (3` or 5` LTRs) and proximal flanking region of the genome. The alternative reaction takes place between the primers from the left and right flanks, which is inhibited in the full (RT-occupied) site by the length of retrotransposon, and able to amplify the shorter, empty (RT-unoccupied) site. (Primers are indicated as color arrows) (Alzohairy et al., 2014b). 4 The basic RBIP was developed for high-throughput applications by replacing gel electrophoresis with hybridization to a filter, and was developed by studying the PDR1 retrotransposon in Pisum sativum (Flavell et al., 1998). One of the disadvantages of this method is that it is more expensive and technically demanding compared to other methods. The method also allows the dot blot approach to be scaled down to microarrays with the attendant advantages in throughput using sensitive oligo-based hybridization to spotted PCR products (Flavell et al., 1998). RBIP requires information on the sequences of the 5’ and 3’ flanking regions of the retrotransposon insertions. One limitation of RBIP is due to size range of standard PCR (about 3-5 Kbp). By using three primers, RBIP can detect both the presence and absence of the TE insertion and generates single-locus codominant markers. RBIP can also generate a dominant marker when only two flanking primers are used (Ribaut and Hoisington, 1998). When RBIP detects the occupied and unoccupied RT sites together, the products blotted onto membrane are probed with a locus-specific probe. Empty sites are usually scored by amplification between the left and right flanks of the presumptive integration site with primers specific to both flanking regions. This method can detect genomic polymorphisms by using standard agarose gel electrophoresis, or by hybridization, which is more useful for automated and high throughput analysis. RBIP was successfully used to generate molecular barcodes to examine the evolutionary history among Pisum species (Vershinin et al., 2003; Jing et al., 2005). Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) REMAP (Kalendar et al., 1999) combines primers (Fig. 3) to RTs and locus-specific simple sequence repeats (SSRs) of the genome (Kalendar and Schulman, 2006; Mansour, 2008; Kalendar, 2011). This technique is applicable when SSR locates near the retrotransposons (Tsumura et al., 1996; Mansour, 2008; Kalendar, 2011). Amplification between retrotransposon and a nearby SSR requires neither digestion with restriction enzymes nor adaptor ligation to generate the marker bands. This protocol can be completed in 1-2 days (Kalendar and Schulman, 2006; Mansour, 2008, Kalendar, 2011) and has been used to measure diversity, similarity and cladistic relationships in many genotypes of Oryza sativa (Branco et al., 2007), rice pathogens (Magnaporthe grisea) (Chadha and Gopalakrishna, 2005), Spartina sp. (Baumel et al., 2002) and Avena sativa (Tanhuanpää et al., 2007). Sequence-Specific Amplified Polymorphism (SSAP) SSAP (Waugh et al., 1997) analysis (Fig. 4) was one of the first retrotransposon-based barcoding methods relying on AFLP (amplified fragment length polymorphism) (Vos et al., 1995). SSAP utilized the BARE-1 LTR-RT for molecular barcoding (Waugh et al., 1997) using one primer matching the end of an RT (e.g., 3’ LTR) and the other matching an AFLP-like restriction site (usually MseI or PstI) adaptor. Primer pairs contains two or three selective nucleotides of MseI or PstI (or any 5 restriction enzyme) adaptor primers and one nt selective nucleotide of 32 either P- or fluorescently-labeled retrotransposon-specific primers (Ellis et al., 1998). Microsatellite TG U3 R Microsatellite 3’LTR U5 CA PBS 5’LTR Internal Domain U3 TG R U5 CA LTR Retrotransposon REMAP products REMAP products Figure 3. REMAP (REtrotransposon-Microsatellite Amplified Polymorphism; Kalendar et al., 1999) amplifies genomic DNA stretches between LTRs of the LTR-RT and a nearby microsatellite (vertical bars). (Primers are indicated as color arrows) (Alzohairy et al., 2014b). Restriction site adaptor TGU3 R U5 CA Restriction site adaptor 3’LTR PBS 5’LTR Internal Domain TG U3 R U5 CA LTR Retrotransposon SSAP products SSAP products Figure 4. SSAP (Sequence-Specific Amplified Polymorphism; Waugh et al., 1997) amplifies sequence region between the retrotransposon and a restriction site anchored by an adaptor. Primers (color arrows) used for amplification match the adaptor (broken box) and the retrotransposon (in the LTR box, e.g., U3', R, and U5'). (Alzohairy et al., 2014b). SSAP primers are often designed to the LTR region, but could also match to an internal sequence of the RT, like the polypurine tract (PPT), which is found internal to the 3'-LTR of retrotransposons (Ellis et al., 1998). Non-selective primers could also be used when restriction enzymes have a long recognition site sequence, or when the copy number of the RTs is low. The number of selected bases may be increased in the case of high-copy-number families. The use of single or double enzyme digestions (or infrequent cutting enzymes) allows the survey of all insertion sites for a given RT, and can be considered as a variant of anchored PCR. The quality of SSAP pattern depends on the SSAP primers used. Primers that give highly polymorphic, clear, and reproducible SSAP banding patterns are candidate primers for subsequent work. Amplified fragments are commonly separated on 6% polyacrylamide sequencing gels and visualized by autoradiograph. SSAP usually displays a higher level of polymorphism as compared to AFLP (Ellis et al., 1998; Nagy et al., 2006; Syed et al., 2006; Venturi et al., 2006), and has been extensively used in Hordeum 6 vulgare (Leigh et al., 2003), Triticum spp. (Queen et al., 2004), Aegilops spp. (Nagy et al., 2006), Avena sativa (Yu and Wise, 2000), Malus domestica (Venturi et al., 2006), Cynara cardunculus (Lanteri et al., 2006), Lactuca sativa (Syed et al., 2006), Pisum sativum and other Fabaceae (Ellis et al., 1998; Jing et al., 2005), Capsicum annuum and Solanum lycopersicum (Tam et al., 2005) and Ipomoea batatas (Tahara et al., 2004). SSAP was also used for cladistic molecular barcodes to resolve evolutionary history in Nicotiana (Petit et al., 2007), Vicia (Sanz et al., 2007), Oryza (Gao et al., 2004), Triticum (Queen et al., 2004) and Zea (García-Martínez and Martínez-Izquierdo, 2003). Inter-Retrotransposons Amplified Polymorphisms (IRAP) There are many techniques that are based on inter-repeat amplification polymorphism such as REMAP (Kalendar et al., 1999 ; Kalendar and Schulman, 2006), inter-MITE amplification, and IRAP (Kalendar et al., 1999) (Fig. 5). IRAP is based on the fact that retrotransposons generally cluster together in ‘repeat seas’ surrounding ‘genome islands’, and may be nested within each other (Kalendar et al., 1999; Mansour, 2008). By this way, IRAP detects insertional polymorphisms of retrotransposons by amplifying the DNA sequences of two neighboring retroelements such as LTR-RTs and SINE-like sequences (Kalendar et al., 1999). ACU5 R 5’LTR U3 GT TG U3 LTR Retrotransposon R U5 CA PBS Internal Domain PBS 5’LTR Internal Domain LTR Retrotransposon IRAP products Figure 5. IRAP (Inter-Retrotransposons Amplified Polymorphisms; Kalendar et al., 1999) amplifies genomic DNA stretches between abundant dispersed repeats, such as the LTRs of two LTR-RTs. The primers (color arrows) point outwards from the LTRs of LTR-RTs to amplify region between two LTR-RTs (Alzohairy et al., 2014b). IRAP does not require restriction enzyme digestion or ligation (Kalendar and Schulman, 2006; Mansour, 2008; Kalendar, 2011). Different retrotransposon insertions increase the number of sites amplified and sizes of inter-RT fragments, which can be used as marker to detect genotype polymorphism. One or two PCR primers can be used for IRAP. The primers should be pointing outwards from the LTRs of RT to amplify the region between two RTs (Kalendar, 2011). The two primers could be designed to either the same or different RT families. IRAP can be also carried out with a single primer, which matches either the 5’ or 3’ end of the LTR but oriented away from the LTR itself. The copy number of RTs, size and insertion pattern can affect the complexity of the fingerprinting pattern (Mansour, 2008; Mansour et al., 2010). The pattern obtained with two 7 primers does not likely represent simply the sum of the products obtained with each primer individually. In the case of retrotransposons dispersed within the genome, IRAP produces too many fragments to give good resolution on gels, or no products because target amplification sites are too far apart to generate amplicons. Yet, IRAP overcomes some of the drawbacks of other techniques. Unlike SSAP, IRAP does not require either radioactivity or fluorescent labeling of primers. The method was used widely for BARE-1 RT of the Hordeum vulgare genome to measure diversity of genotypes (Kalendar et al., 1999; Manninen et al., 2000, Manninen et al., 2006). IRAP was also used for barcoding of genotypes of Oryza sativa (Branco et al., 2007), Musa (Ashalatha et al., 2005; Teo et al., 2005), Brassica (Tatout et al., 1999), Spartina (Baumel et al., 2002), Triticum (Boyko et al., 2002) and Solanum (Mansour et al., 2010). Inter Primer Binding Sequence (IPBS) IPBS method (Kalendar et al., 2010) is frequently used for displaying retrotransposon polymorphisms (Fig..6). The need for sequence information to design IPBS primers is the case in all RT-based molecular barcoding techniques. IPBS tends to overcome this problem (Kalendar et al., 2010) as the primer binding sequence (PBS) is part of the internal domain of retrotransposons. IPBS utilizes the highly conserved regions of PBS site for tRNAs (Kalendar et al., 2010). While the process of reverse transcription is conserved among all retroviruses, the specific tRNA capture varies for different retroviruses and retrotransposons. Thus, the IPBS amplification method can be useful for all retroviruses Met Lys Pro that contain conservative PBS sites for tRNAi , tRNA , tRNA , Trp Asn Ser Arg Phe Leu Gln tRNA , tRNA , tRNA , tRNA , tRNA , tRNA or tRNA (Kalendar et al., 2010). 5’LTR Internal Domain PBS U5 AC 5’LTR R U3 U3 GT TG LTR Retrotransposon U5 CA PBS Internal Domain LTR Retrotransposon aggctctgataCCA-NNN-TG gggctctgataCCA-NNN-TG tggcaatggaaCCA-NNN-TG ccttgccgataCCA-NNN-TG agctcacgatgCCA-NNN-TG ggctcatgatgCCA-NNN-TG tggcaacggcgCCA-NNN-TG cagcggagtcgCCA-NNN-TG primers R CA-NNN-TGGtatcagagcct CA-NNN-TGGttccattgcca CA-NNN-TGGtatcggcaagg CA-NNN-TGGcatcgtgagct CA-NNN-TGGcatcatgagcc CA-NNN-TGGcgccgttgcca CA-NNN-TGGcgactccgctg CA-NNN-TGGtatcagagcat IPBS products primers Figure 6. IPBS (Inter Primer Binding Sequence; Kalendar et al., 2010) method utilizes the conserved sequence of PBS of LTR-RTs for screening retrotranspososns. Sequences shown are conserved regions of PBS used for primer (color arrows) design (Alzohairy et al., 2014b). As in plant species RTs are nested, mixed, inverted or truncated in the geneome, RTs can be easily amplified using conservative PBS primers. PCR amplification occurs between two nested PBSs of two neighboring LTR-RTs (Fig. 6). 8 PBS sequences can also be used for detecting other retrotransposons when the retrotransposon density is high within the genome (Kalendar, 2011). Retrotransposon movements and recombinations can also be monitored because new inserts or recombinations will be polymorphic, which will appear only in plant lines in which the insertions or recombinations have taken place. Conclusions Several retrotransposon-based molecular barcoding systems were developed based on PCR amplifications between sequences of RTs and the flanking DNA of the host genome (Kalendar and Schulman, 2006). These marker systems were found to be highly effective tools for tracking transpositions and diversities of RTs, and determining phylogenetic relationships of plant taxa (Hamdi et al., 1999; Shedlock and Okada, 2000). Many reports also suggest that the differences in genome size observed in the plant kingdom are related to variations of RTs activity and consequently their content, which suggests that RTs play important roles in the evolution of genome sizes (Vitte and Panaud, 2005; Alzohairy et al., 2012; 2013; 2014a,b). Other studies used LTR-RT barcoding detected the effects of environmental stresses on the reactivation of retrotransposons and hence their genetic diversity (reviewed in Alzohairy et al., 2014a). Many applications were also reported for study of phylogeny, genetic diversity and the functional analyses of genes using LTR-RT based barcoding (Waugh et al., 1997; Flavell et al., 1998; Kalendar and Schulman, 2006; Mansour, 2008; Roos et al., 2004). 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