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Repetitive Regions

     

    The outputs of repeatmasker report that 65.70% of the 150,000bp sequence is covered by transposable elements (TEs) (Fig. 1) and mask these regions by Ns.  While performing downstream analyses using unmasked sequences gives us information about these regions, examining the content of masked sequence is also important, since repeatmasker sometimes produces spurious hits (either too short or too low sequence similarity) and some genes may harbor TE-derived parts (Rebollo et al. 2012).

    seq1_rm_maize.png

    Figure 1. Repeatmasker masked 65.70% of the sequence as transposable elements.

    Results

    Here we used BLASTx and BLASTn as complements to the output of repeatmasker.

    Full-length Sequence (150,000bp)

    g1_maizeTE.gif

    Full-length sequence blastn against maize TE database

    Sequences Splitted by Gene Models

    Based on ab initio results which produced 5 potential genes, the sequence is splitted into 6 regions to analyse separately.  Each sequence was blasted against Swiss-Prot and Maize TE database

    For blastx resuts, see NCBI BLAST BWGSGU9T01R, valid through 12.6.

    • Seq 1: [1, 44006]

             masked_01.png  

    BLASTx: seq 1 against Swiss-Prot.  Top hit: Ty3-I gag-pol polyprotein (E-value: 3e-44)

     

    masked01_maizeTE.gif

    BLASTn: seq 1 against Maize TE database

     

    • Seq 2: [47229, 61189]

      masked_02.png

    Seq 2 against Swiss-Prot.  

    Top hit: Retrovirus-related Pol polyprotein from transposon TNT 1-94 (E-value: 5e-103)

    masked02_maizeTE.gif

    Seq 2 against maize TE database

    • Seq 3: [62729, 82396]

      masked_03.png

      Seq 3 against Swiss-Prot. Top hit: Copia-protein (E-value: 1e-93)

      (left: 1-aminocyclopropane-1-carboxylate oxidase 1, E-value: 2e-59...)

      masked03_maizeTE.gif

      Seq 3 against maize TE database

     

    • Seq 4 [89907, 101948]

      masked_04.png

      Seq 4 against Swiss-Prot.  

      Top hit (right): Retrovirus-related Pol polyprotein from transposon TNT 1-94 (E-value: 2e-151)

      (Left: U-box domain-containing protein 34, e-value: 3e-102)

      masked04_maizeTE.gif

      Seq 4 against maize TE database

    • Seq 5 [113683, 138555]

      masked_05.png

      Seq 5 blastx against Swiss-Prot

      Top hit: 30S ribosomal protein S4.  E-value: 4e-110; 177/201 aa matched at 100% identity.

      masked05_maizeTE.gif

      Seq 5 blastn against maize TE database

      Further blastx against nr using ~1000bp around the suspicious region (figure below) suggested similar result, that a partial sequence from chloroplast is detected.  It includes a partial ribosomal S4/S9 N-terminal domain and a full-length S4/Hsp/tRNA synthetase RNA-binding domain.  Moreover, the ~550bp match is embedded within a retrotransposon insertion.

      S4.png

    • Seq 6 [143927, 150000]

      masked_06.png

       

      Seq 6 against Swiss-Prot.  Top hit (right):Retrovirus-related Pol polyprotein from transposon TNT 1-94 (E-value: 5e-54)

      left: Putative transcription factor bHLH041, E-value: 8e-05

      masked06_maizeTE.gif

      Sep 6 against maize TE database

    Conclusions

    Repeatmasker results were mostly supported by DNA-level evidence using a maize TE database.  The transposable elements that helped shape this genomic region is LTR elements.  No full-length transposable elements were detected.  Protein-level evidences were also examined using blastx against well-annotated protein databases.  In addition to TE-related proteins, however, there were also a few interesting cases where additional proteins were detected as appeared in Seq 3, Seq 4, Seq 5 and Seq 6.  

    The hit to transcription factor in Seq 6 have high E-values and low % identity, so it is not considered to be a real hit.  The hit to 1-aminocyclopropane-1-carboxylate oxidase 1 in Seq 3 seemed to be a part of the gene model (see Gene 2).  However, it is not supported by further EST/cDNA data (data not shown).  

    The most interesting case was in Seq 5, where there's a ~550bp match to chloroplast ribosomal proteins at 100% identity.  The match was embedded a retrotransposon which also had high % identity at 95% and 96% at each side.   It is possible that the chloroplast sequence was brought to this area by a retrotransposon "read-out" event (Kashkush et al. 2002), but the exact mechanism that caused the pattern is unknown.

    Overall, the repetitive region analysis in this 150,000bp genome sequence revealed the fragmented nature of repeats, and that transposable elements played a significant role in shaping the genome architecture in maize.  

    Reference

    Kashkush, Khalil, Moshe Feldman, and Avraham A. Levy. "Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat." Nature genetics 33.1 (2002): 102-106.

    Rebollo, Rita, Mark T. Romanish and Dixie L. Mager “Transposable elements: an abundant and natural source of regulatory sequences for host genes” Annual Review of Genetics 46 (2012): 21-42

    Wicker, Thomas, et al. "A unified classification system for eukaryotic transposable elements." Nature Reviews Genetics 8.12 (2007): 973-982.

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