LTR retrotransposons from the Citrus x clementina genome: characterization and application

Tree Genetics & Genomes (2018) 14:43 https://doi.org/10.1007/s11295-018-1257-x ORIGINAL ARTICLE LTR retrotransposons from the Citrus x clementina genome: characterization and application Dongliang Du 1 & Xiaoyun Du 2 & Matthew R. Mattia 1 & Yanbo Wang 2 & Qibin Yu 1 & Ming Huang 1 & Yuan Yu 1 & Jude W. Grosser 1 & Fred G. Gmitter Jr 1 Received: 5 October 2017 / Revised: 15 May 2018 / Accepted: 17 May 2018 # The Author(s) 2018 Abstract Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire, and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel, and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis. Keywords Citrus . LTR retrotransposons . Gypsy . Copia . IRAP . SSAP Introduction Retrotransposons (RT) are a type of transposable element (TE) that moves through the genome via an RNA intermediate in a process that resembles Bcopy and paste^ (Wicker et al. 2007). Retrotransposons can be separated into two major subclasses, Dongliang Du and Xiaoyun Du contributed equally to this work. Communicated by W.-W. Guo Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11295-018-1257-x) contains supplementary material, which is available to authorized users. * Fred G. Gmitter, Jr 1 Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL 33850, USA 2 Yantai Academy of Agricultural Science, Yantai 265500, Shandong, China long terminal repeat retrotransposons (LTR-RTs) and nonLTR retrotransposons, based on their structure and transposition mechanism (Todorovska 2007). A typical LTR-RT contains two highly similar long terminal repeats (LTRs), a primer-binding site (PBS), a polypurine tract (PPT), and two genes necessary for their retrotransposition, gag and pol (Du et al. 2010). The majority of LTR-RTs can be divided further into Copia and Gypsy superfamilies according to the order of proteinase (PR), integrase (IN), reverse transcriptase (RT), and RNase H (RH) domains in Pol (Domingues et al. 2012). The domains of Gypsy elements are arranged as LTR-GAG-PRRT-RH-IN-LTR, whereas the Copia elements are organized as LTR-GAG-PR-IN-RT-RH-LTR (Wicker et al. 2007). Gypsy and Copia superfamilies can be further classified into lineages and families with phylogenetic analysis of protein domain sequences that are usually supported by differences in structure (such as size of LTRs and elements) (Wicker et al. 2007). In land plants, four Copia (Retrofit, Tork, Sire, and Oryco) and six Gypsy (Athila, Tat, Renia, CRM, Galadriel, and Del) lineages were reported (Llorens et al. 2011). 43 Page 2 of 14 LTR-RTs can be used as a molecular marker system because of their high copy number, widespread distribution, and high heterogeneity (Kumar and Hirochika 2001). Several types of LTR-RT molecular markers have been developed, such as inter-retrotransposon amplified polymorphism (IRAP), sequence-specific amplified polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism (REMAP), insertion site-based polymorphism (ISBP), and retrotransposon-based insertion polymorphism (RBIP) (Flavell et al. 1998; Kalendar et al. 1999; Paux et al. 2010; Waugh et al. 1997). Most notably, IRAP markers amplify the intervening region between two retrotransposons to show polymorphisms (Kalendar et al. 2011; Kalendar et al. 1999). IRAP is used frequently because of the easy development of one outward-facing LTR-derived primer and generation of marker bands without digestion and ligation. SSAP exploits LTR-RT polymorphisms by amplifying the region between a retrotransposon and adjacent restriction site in the genome creating additional polymorphisms that can be used to differentiate closely related accessions (Syed et al. 2005). SSAP is especially useful for clone identification (Bretó et al. 2001; Venturi et al. 2006; Zhao et al. 2010). LTR-RT markers have been used widely for pedigree analysis, population structure, fingerprinting, linkage, and genetic mapping in several plant species (Branco et al. 2007; Farouji et al. 2015; Huo et al. 2009; Jia et al. 2009; Kalendar et al. 2011; Mandoulakani et al. 2015; Queen et al. 2004; Smykal 2006; Sun et al. 2015). The development of next-generation sequencing technologies has allowed for huge numbers of retrotransposon sequences to be generated, providing new opportunities for molecular marker development (Barghini et al. 2014; Cossu et al. 2012; Du et al. 2010; Xu and Du 2013; Zhang et al. 2012). Many types of molecular markers have been used to characterize the phylogenetic relationships of Citrus accessions and relatives. Previously used markers include random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), and sequence-related amplified polymorphism (Barkley et al. 2006; Federici et al. 1998; Uzun et al. 2009; Yamamoto et al. 1993). These marker types could not fully reveal the origin and taxonomy of citrus. There are few studies that use LTR-RT based makers in citrus (Asins et al. 1999; Bernet and Asins 2003; Biswas et al. 2010a; Biswas et al. 2010b; De Felice et al. 2009; RicoCabanas and Martinez-Izquierdo 2007). However, small numbers of LTR-RT based primers were developed in these studies. In this study, we identified and characterized fulllength LTR-RTs in the C. x clementina genome for use as molecular markers. The LTR-RT markers were tested for transferability among and differentiation between Rutaceae species. The fluorescence-labeled SSAP system used in this study yields a high-throughput and highly efficient mutant identification syste (...truncated)