DNA probe, was denatured for 6 min at 96˚C. The slides and hybridization mixture were incubated at 80˚C for 10 min and then maintained at 37˚C for 16 h. The slides were washed with 10% (v/v) formamide for 10 s, 2× SSC at 37˚C for 5 min (five times), and 0.2% (v/v) Tween-20 at room temperature for 5 min. Digoxigenin was detected using FITC-conjugated anti-digoxigenin antibody (Roche), and chromosomes were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Slides were viewed using a Leica DM2500 fluorescence microscope (Leica, Germany). Images were captured using the CV-M4+CL progressive scan charge-coupled device camera (DM2500, Leica) and analyzed using CytoVision software (version 7.3.1).
3.1. Identification of rt Fragments
The sequences of the expected 580 bp amplicons  were compared with sequences available in the NCBI and GIRI databases using BLAST  . In total, two independent rounds of PCR and cloning yielded 43 clones with homology to known retroelements in the NCBI and GIRI (http://www.girinst.org/) database. All these elements were selected for further analysis. All the clones are deposited in GenBank under accession numbers: KT900650?KT900692 (Supplementary material 2).
3.2. Characterization of LINE rt Sequences
Nucleotide sequences derived from the isolated rt fragments were aligned and used to construct a phylogenetic tree using the maximum-likelihood method in MEGA6. The identified rt sequences were classified into eight groups (Figure 1). Group I contained the most rt clones (41.8%, 18/43), followed by Group VI (30.2%, 13/43). These two groups accounted for 72.1% of the 43 clones and were further classified into several subfamilies. Additionally, sequences of clones from the same group were of almost the same length, and were highly similar (> 97%, except for Group II) (Table 1 and Supplementary material 2). The length of isolated LINE rt fragments ranged from 557 bp (MnoL_11, MnoL_13, and MnoL_15) to 595 bp (MnoL_31), with an average of 579 bp. The AT/GC ratio
Figure 1. Phylogenetic analysis of reverse transcriptases from mulberry LINE retrotransposons. All cloned nucleotide sequences of reverse transcriptase fragments from mulberry were aligned by muscle (version 3.8.31) under default parameters. The best substitution model (Tamura 3-parameter + G), which was tested by MEGA6, was used to construct a phylogenetic tree based on a maximum-likelihood method with the pairwise deletion in MEGA6. Only more than 50% of the frequency of replicate (1000 replicates) trees were shown. The sequences were classified into eight groups: Group I to Group VIII.
ranged from 1.26 (MnoL_20 and MnoL_37) to 1.98 (MnoL_42), with an average of 1.37, which indicated the rt sequences are rich in AT (Table 1 and Supplementary material 2). Pairwise comparisons revealed that similarity among 43 rt nucleotide sequences ranged from 31.8% (MnoL_20 and MnoL_31, MnoL_31 and MnoL_36) to 99.4% (MnoL_17 and MnoL_29) (Supplementary material 3). These results suggest that the rt sequences isolated were highly heterogeneous.
The alignment of amino acid sequences of multiple isolated mulberry LINE rt rfragments evealed that all sequences contained several premature stop codons, with the exception of the MnoL_10 sequence (Figure 2). The MnoL_42 sequence had 16 premature stop codons, which is the most of any clone. Additionally, the premature stop codons were in similar locations in the sequences from the same group (Figure 2). Furthermore, all sequences carry frameshift mutation, except for the MnoL_10, MnoL_23, and MnoL_31 sequences (Supplementary material 4).
Figure 2. Premature stop codon positions in reverse transcriptase sequences from mulberry LINE retrotransposons. The locations of premature stop codons in rt sequences were demonstrated using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/). Each straight red line represents one of the eight groups of reverse transcriptase sequences (from top to bottom: Group I to Group VIII). The blue line means reverse transcriptase fragments. Red block appeared in the blue line means premature stop codons.
3.3. Phylogenetic Analysis of LINE rt Clones
Phylogenetic analyses indicated that the mulberry LINE rt sequences are homologous to rt sequences in other species (Figure 3). One interesting feature of the result is that PTLRT19 grouped with other mulberry rt sequences (Group V, VI, VII), instead of other PTLRT (PTLRT12, PTLRT4, and PTLRT14) from Paeonia suffruticosa. In fact, P. suffruticosa and M. notabilis are distantly related taxa. The phylogenetic between the LINE-rt sequences and the host species trees were incongruous (APG, The Angiosperm Phylogeny Group, http://www.theplantlist.org).
Figure 3. Phylogenetic analysis of reverse transcriptase fragments from mulberry and other thirteen plant species. All nucleotide sequences of reverse transcriptase fragments from mulberry and the representative members of other thirteen plant species were aligned by muscle (version 3.8.31) with default parameters. Firstly, MEGA6 was used to find the best-fit substitution models for those datasets with default parameters. The best substitution model (Tamura 3-parameter + G) was used to construct a phylogenetic tree, using the maximum-likelihood method with the pairwise deletion in MEGA6. Frequency (>50%) of replicate trees in which the associated taxa clustered together in the bootstraps test (1000 replicates) were shown. Detailed information of other thirteen plant species used in the present research was shown in supplementary material 1.
3.4. Distribution of LINEs in the Mulberry Genome
Fluorescence in situ hybridization (FISH) was performed to study the distribution of these sequences along mulberry chromosomes. Chromosomal localization of the LINEs elements was performed using a heterogeneous probe cocktail containing all isolated clones. FISH with such a cocktail revealed that hybridization signals were mainly concentrated in subtelomeric and pericentromeric regions (Figure 4).
4.1. Characterization of LINEs
All cloned rt fragments could be classified into eight groups (Figure 1). Group I and VI consisted of the most rt clones (72.1%, 31/43). Only one clone (MnoL_42) contained in the Group VIII (Figure 1). The range of nucleotide sequence similarities between MnoL_42 and the other clones was only 32.8 to 37.2% (Supplementary material 3). Meanwhile, the number of premature stop codons in MnoL_42 was 16, which is much higher than in the other clones (Supplementary material 2). These results indicated that mutations accumulated progressively over evolutionary time. Combining these data with the phylogenetic analysis results (Figure 1) allows us to come to the conclusion that the MnoL_42 is an ancient LINE in mulberry.
The rt fragments amplified from the mulberry genome were highly heterogeneous. Almost all the 43 rt sequences described here contained frameshifts and premature stop codons (Figure 2 and Supplementary material 5), which were the main causes of the observed heterogeneity. These results are consistent with those observed in other plants, including Hordeum species  and Vicia species  . Furthermore, the rt fragments from clones within the same group exhibited very few differences, suggesting that the heterogeneity among rt sequences is also the result of base substitutions, deletions, and insertions. As shown in Figure 2, similar stop codon distribution patterns among sequences of the rt fragments from the same group suggested that they went through a similar evolutionary process.
Figure 4. Chromosomal distribution of mulberry LINE retrotransposons by fluorescence in situ hybridization experiment. Images were captured using the CV-M4+CL progressive scan charge-coupled device camera (DM2500, Leica) and CytoVision software (version 7.3.1). (a) Cytological detection of mulberry chromosome; (b) blue ﬂuorescence signals correspond to 4'-6-diamidino-2-phenylindole-stained DNA; (c) green ﬂuorescence signals correspond to hybridization sites of reverse transcriptase probes; (d) the overlaid images of (b) and (c). Scale bar: 5 μm.
As indicated in Table 1 and Supplementary material 2, the AT/GC ratio ranged from 1.26 to 1.98, with an average of 1.37. The results suggested that the rt sequences are rich in AT bases, which is important for the LINE copy and paste replication mechanism   . An intact LINE element contains two open reading frames, ORF1 and ORF2. The ORF2 contains reverse transcriptase, which is a critical enzyme responsible for the replication process of LINE   . One of the critical steps in the life cycle of LINE is that the ORF2 protein cleaves the first one DNA strand at the target. Due to the fact that the target sequence in this site is always rich, AT bases and the target site are usually similar to consensus TTAAAA   ; the AT bases content is high in the rt sequences to ensure that the target sites can be identified efficiently in the replication process of LINE.
Interestingly, there were no frameshifts or premature stop codons in MnoL_ 10 (Figure 2 and Supplementary material 5). All rt fragments from mulberry, with the exception of clone MonL_10, represented potential pseudogenes (possessed stop codons or frameshifts). It would be worthwhile carrying out further research on MonL_10, which may be a potential active transposable element.
4.2. Phylogenetic Analysis of LINEs
Interestingly, we found a phenomenon that PTLRT19 grouped with other mulberry rt sequences (Group V, VI, and VII), instead of other PTLRT (PTLRT12, PTLRT4, and PTLRT14) from Paeonia suffruticosa (Figure 3). While the two species (P. suffruticosa and M. notabilis) are distantly related taxa according to the APG (The Angiosperm Phylogeny Group, http://www.theplantlist.org). Similarity phenomenon had been found in other studies. For example, twenty-six genomes harbor at least one case of horizontal TE transfer (HTT), which may be important in TE-driven genome evolution, and these HTTs involve species as distantly related as palm and grapevine, tomato and bean, poplar and peach, and so on  .
Table 1. Length, AT/GC ratio, and similarity [range (average)] of reverse transcriptase fragments from mulberry LINE retrotransposons.
It is hypothesized that HTT may be the reason for this phenomenon observed in our research. Further analysis was performed in this work. Accordingly, three criteria have been defined for the detection of HTTs: (i) patchy distribution of the TEs in phylogenies, (ii) high sequence similarity of the TE between distantly related taxa, and (iii) phylogenetic incongruence between the TE and host species trees    . In the present work, although there is phylogenetic incongruence between the TE and trees of the two species, the range of nucleotide sequence similarities between PTLRT19 and mulberry rt sequences (Group V, VI, and VII) is only 0.520 to 0.562 (Supplementary material 4). These results suggest that the conclusion for PTLRT19 is that it is uncertain whether it represents a horizontal transfer event.
4.3. Chromosomal Localization of LINE Retrotransposons
The LINE distribution patterns are associated with LINE functions. For example, FISH experiments in Cannabis sativa suggested that differential accumulation of LINE retrotransposon elements onto the Y chromosome leads to sex chromosome heteromorphism  . Although the chromosomal distribution of LINEs has been analyzed in only a few plant species, the FISH results here revealed that the distribution of LINEs in mulberry chromosomes was similar to that in sugar beet and peanut chromosomes   . Furthermore, the weak hybridization signals observed in this study indicated that LINEs were not abundant in the mulberry genome (Figure 4). Most of the hybridization signals were concentrated in subtelomeric and pericentromeric regions. The subtelomeric and pericentromeric regions are generally considered to correspond to the constitutively heterochromatic region. In fact, there is extensive DNA methylation in these regions  . Thus, we hypothesized that the tendency for LINEs to insert into these regions may be related to DNA methylation in mulberry.
Meanwhile, recent reports suggest that LINE insertion into promoters can influence promoter functionality and gene regulation, resulting in up or down regulation of reporter genes  . Insertion of a LINE into a gene can induce alternative splicing or change gene expression patterns, which can result in a change in the function of the gene  . Although we currently have no evidence that the mulberry LINEs described here are active and functional, previous studies have indicated that some LINEs are active and functional in other species  . So, our future studies will attempt to characterize the functions of mulberry LINEs more comprehensively, considering their localization in subtelomeric and pericentromeric regions.
The authors declare that they have no conflict of interest. The authors thank all the laboratory members who provided advice during this work. This project was funded by “Fundamental Research Funds for the Central Universities (XDJK2015C116)” and “China Postdoctoral Science Foundation (2016M592622)”.
Conflict of Interest
All authors declare that there is no conflict of interest.
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 McClintock, B. (1950) The Origin and Behavior of Mutable Loci in Maize. Proceedings of the National Academy of Sciences of the United States of America, 36, 344-355.
 Bucher, E., Reinders, J. and Mirouze, M. (2012) Epigenetic Control of Transposon Transcription and Mobility in Arabidopsis. Current Opinion in Plant Biology, 15, 503-510.
 Du, J., Tian, Z., Hans, C.S., Laten, H.M., Cannon, S.B., Jackson, S.A., Shoemaker, R.C. and Ma, J. (2010) Evolutionary Conservation, Diversity and Specificity of LTR-Retrotransposons in Flowering Plants: Insights from Genome-Wide Analysis and Multi-Specific Comparison. The Plant Journal, 63, 584-598.
 Wicker, T., Sabot, F., Hua-Van, A., Bennetzen, J.L., Capy, P., Chalhoub, B., Flavell, A., Leroy, P., Morgante, M., Panaud, O., et al. (2007) A unified Classification System for Eukaryotic Transposable Elements. Nature Reviews Genetics, 8, 973-982.
 Noma, K., Ohtsubo, E. and Ohtsubo, H. (1999) Non-LTR Retrotransposons (LINEs) as Ubiquitous Components of Plant Genomes. Molecular and General Genetics, 261, 71-79.
 Heitkam, T. and Schmidt, T. (2009) BNR—A LINE Family from Beta vulgaris—Contains a RRM Domain in Open Reading Frame 1 and Defines a L1 Sub-Clade Present in Diverse Plant Genomes. The Plant Journal, 59, 872-882.
 Belancio, V.P., Hedges, D.J. and Deininger, P. (2008) Mammalian Non-LTR Retrotransposons: For Better or Worse, in Sickness and in Health. Genome Research, 18, 343-358.
 Schwarzsommer, Z., Leclercq, L., Gobel, E. and Saedler, H. (1987) Cin4, an Insert Altering the Structure of the A1 Gene in Zea mays, Exhibits Properties of Nonviral Retrotransposons. The EMBO Journal, 6, 3873-3880.
 Higashiyama, T., Noutoshi, Y., Fujie, M. and Yamada, T. (1997) Zepp, a LINE-Like Retrotransposon Accumulated in the Chlorella Telomeric Region. The EMBO Journal, 16, 3715-3723.
 Vershinin, A.V., Druka, A., Alkhimova, A.G., Kleinhofs, A. and Heslop-Harrison, J.S. (2002) LINEs and gypsy-Like Retrotransposons in Hordeum Species. Plant Molecular Biology, 49, 1-14.
 Komatsu, M., Shimamoto, K. and Kyozuka, J. (2003) Two-Step Regulation and Continuous Retrotransposition of the Rice LINE-Type Retrotransposon Karma. The Plant Cell, 15, 1934-1944.
 Yamashita, H. and Tahara, M. (2006) A LINE-Type Retrotransposon Active in Meristem Stem Cells Causes Heritable Transpositions in the Sweet Potato Genome. Plant Molecular Biology, 61, 79-84.
 Piskareva, O., Ernst, C., Higgins, N. and Schmatchenko, V. (2013) The Carboxy-Terminal Segment of the Human LINE-1 ORF2 Protein Is Involved in RNA Binding. FEBS Open Bio, 3, 433-437.
 Malik, H.S., Burke, W.D. and Eickbush, T.H. (1999) The Age and Evolution of Non-LTR Retrotransposable Elements. Molecular Biology and Evolution, 16, 793-805.
 Kubis, S.E., Castilho, A.M., Vershinin, A.V. and Heslop-Harrison, J.S. (2003) Retroelements, Transposons and Methylation Status in the Genome of Oil Palm (Elaeis guineensis) and the Relationship to Somaclonal Variation. Plant Molecular Biology, 52, 69-79.
 Hill, P., Burford, D., Martin, D.M. and Flavell, A.J. (2005) Retrotransposon Populations of Vicia Species with Varying Genome Size. Molecular Genetics and Genomics, 273, 371-381.
 Samoluk, S.S., Robledo, G., Podio, M., Chalup, L., Ortiz, J.P.A., Pessino, S.C. and Seijo, J.G. (2015) First Insight into Divergence, Representation and Chromosome Distribution of Reverse Transcriptase Fragments from L1 Retrotransposons in Peanut and Wild Relative Species. Genetica, 143, 113-125.
 Berg, C.C. (2005) Moraceae Diversity in a Global Perspective. Proceedings of a Symposium on Plant Diversity and Complexity Patterns—Local, Regional and Global Dimensions. Biologiske Skrifter, 55, 423-440.
 Darias-Martin, J., Lobo-Rodrigo, G., Hernandez-Cordero, J., Diaz-Diaz, E. and Diaz-Romero, C. (2003) Alcoholic Beverages Obtained from Black Mulberry. Food Technology and Biotechnology, 41, 173-176.
 Konno, K., Ono, H., Nakamura, M., Tateishi, K., Hirayama, C., Tamura, Y., Hattori, M., Koyama, A. and Kohno, K. (2006) Mulberry Latex Rich in Antidiabetic Sugar-Mimic Alkaloids Forces Dieting on Caterpillars. Proceedings of the National Academy of the United States of America, 103, 1337-1341.
 Daimon, T., Taguchi, T., Meng, Y., Katsuma, S., Mita, K. and Shimada, T. (2008) Beta-Fructofuranosidase Genes of the Silkworm, Bombyx mori: Insights into Enzymatic Adaptation of B. mori to Toxic Alkaloids in Mulberry Latex. Journal of Biological Chemistry, 283, 15271-15279.
 He, N., Zhang, C., Qi, X., Zhao, S., Tao, Y., Yang, G., Lee, T.H., Wang, X., Cai, Q., Li, D., et al. (2013) Draft Genome Sequence of the Mulberry Tree Morus notabilis. Nature Communications, 4, Article No. 2445.
 Doyle, J. (1991) DNA Protocols for Plants. In: Hewitt, G.M., Johnston, A.W.B. and Young, J.P.W., Eds., Molecular Techniques in Taxonomy, Springer, Berlin, Heidelberg, 283-293.
 Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 2725-2729.
 Dhellin, O., Maestre, J. and Heidmann, T. (1997) Functional Differences between the Human LINE Retrotransposon and Retroviral Reverse Transcriptases for in Vivo mRNA Reverse Transcription. The EMBO Journal, 16, 6590-6602.
 El Baidouri, M., Carpentier, M.C., Cooke, R., Gao, D., Lasserre, E., Llauro, C., Mirouze, M., Picault, N., Jackson, S.A. and Panaud, O. (2014) Widespread and Frequent Horizontal Transfers of Transposable Elements in Plants. Genome Research, 24, 831-838.
 Schaack, S., Gilbert, C. and Feschotte, C. (2010) Promiscuous DNA: Horizontal Transfer of Transposable Elements and Why It Matters for Eukaryotic Evolution. Trends in Ecology & Evolution, 25, 537-546.
 Sakamoto, K., Ohmido, N., Fukui, K., Kamada, H. and Satoh, S. (2000) Site-Specific Accumulation of a LINE-Like Retrotransposon in a Sex Chromosome of the Dioecious Plant Cannabis sativa. Plant Molecular Biology, 44, 723-732.
 Kubis, S.E., Heslop-Harrison, J.S., Desel, C. and Schmidt, T. (1998) The Genomic Organization of Non-LTR Retrotransposons (LINEs) from Three Beta Species and Five Other Angiosperms. Plant Molecular Biology, 36, 821-831.
 Dhadi, S.R., Xu, Z., Shaik, R., Driscoll, K. and Ramakrishna, W. (2015) Differential Regulation of Genes by Retrotransposons in Rice Promoters. Plant Molecular Biology, 87, 603-613.