| Literature DB >> 23593343 |
Characterization of the two intra-individual sequence variants in the 18S rRNA gene in the plant parasitic nematode, Rotylenchulus reniformis.
Seloame T Nyaku1, Venkateswara R Sripathi, Ramesh V Kantety, Yong Q Gu, Kathy Lawrence, Govind C Sharma.
Abstract
The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.Entities:
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Year: 2013 PMID： 23593343 PMCID： PMC3623918 DOI： 10.1371/journal.pone.0060891
Source DB: PubMed Journal: PLoS One ISSN： 1932-6203 Impact factor: 3.240
Introduction
Results
Table 1
Oligonucleotide primers for sequencing of the RN 18S rRNA gene.
| Oligo name | Primer sequence (5′-3′) | Direction |
| SSUF05 | GAAACTGCGAA(CT)GGCTCATT | Forward |
| SSUR81 | TGATCC(AT) (GT)C(CT)GCAGGTTCAC | Reverse |
| SSUF22 |
| Forward |
| SSUR13 |
| Reverse |
| T3 |
| Reverse |
| T7 |
| Forward |
Table 2
Variable sites identified among reniform nematode (RN) 18S rRNA variants.
| Variations at specific base locations in 18S clones (15) of rRNA variant 1 (RN_VAR1) | |||||||||||||||||||||||||
| 96 | 105 | 106 | 110 | 119 | 123 | 152 | 154 | 155 | 156 | 165 | 253 | 265 | 294 | 313 | 316 | 331 | 334 | 379 | 408 | 409 | 410 | 417 | 418 | 419 | |
| SSU1B8 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | G | C | T | G | A | T |
| SSU12A4 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU12A9 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU12A10 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU12A12 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU25A1 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU25A4 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU25A5 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU25A6 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | G | C | T | G | A | T |
| SSU25A8 | A | G | C | T | A | T | C | C | A | T | T | G | C | C | G | C | G | C | T | A | G | T | G | A | T |
| SSU25A10 | A | G | C | T | A | T | C | C | A | - | T | G | C | C | G | C | G | C | T | A | C | T | G | A | T |
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| Variations at specific base locations in 18S clones (34) of rRNA variant 2 (RN_VAR2) | |||||||||||||||||||||||||
| SSU1B1 | C | A | A | A | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU1B4 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | G | C | C | A | T | C |
| SSU1B5 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU1B6 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A1 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A2 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A3 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A5 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A6 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A7 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A8 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU12A13 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU25A11 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU25A12 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B1 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B2 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B3 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B4 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B5 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B6 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B7 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B9 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B10 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B11 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B12 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B13 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B14 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B15 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B16 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B17 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B18 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B19 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
| SSU13B20 | C | A | A | C | G | C | T | A | – | – | A | A | T | A | A | G | C | T | G | A | C | C | A | T | C |
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Table 3
Variable sites identified among reniform nematode (RN) 18S rRNA variants.
| Variations at specific base locations in 18S clones (15) of rRNA variant 1 (RN_VAR1) | ||||||||||||||||||||||
| 671 | 672 | 673 | 769 | 981 | 983 | 984 | 987 | 992 | 996 | 1284 | 1286 | 1300 | 1330 | 1331 | 1335 | 1411 | 1412 | 1415 | 1421 | 1428 | 1439 | |
| SSU1B8 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | - | C | G | A | C | G | A |
| SSU12A4 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU12A9 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU12A10 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU12A12 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU25A1 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU25A4 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU25A5 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU25A6 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU25A8 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | C | G | A |
| SSU25A10 | A | G | C | T | A | T | C | C | – | T | A | G | C | T | T | A | C | G | A | T | G | A |
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| SSU1B1 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU1B4 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU1B5 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU1B6 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU12A1 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU12A2 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | C | C | G |
| SSU12A3 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | C | C | G |
| SSU12A5 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU12A6 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU12A7 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU12A8 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU12A13 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU25A11 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU25A12 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B1 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B2 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B3 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B4 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B5 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B6 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B7 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B9 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B10 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B11 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B12 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B13 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B14 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B15 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B16 | G | A | G | - | G | C | G | T | T | C | G | G | T | – | – | G | A | T | G | T | C | G |
| SSU13B17 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B18 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B19 | G | A | G | - | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
| SSU13B20 | G | A | G | G | G | C | G | T | T | C | G | A | T | – | – | G | A | T | G | T | C | G |
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Figure 1Twenty-six conserved regions within the 18S rRNA gene of four juvenile female RNs.
Figure 2Neighbor-Joining (NJ) analyses for 44 RN 18S rRNA clones and nematodes in the super-families, Hoplolaimidae, Heteroderidae, Rotylenchulidae, and Meloidogynidae.
Table 4
Nematode species belonging to Tylenchoidea, GenBank accession numbers, and length of 18S rRNA sequence.
| Super-family | Nematodes | GenBank No. | Sequence length (bp) |
| Heteroderidae |
| FJ040403 | 1,725 |
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| FJ040404 | 1,725 | |
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| EU669915 | 1,725 | |
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| FJ040405 | 1,725 | |
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| EU669916 | 1,725 | |
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| AY284617 | 1,744 | |
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| EU306355 | 1,772 | |
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| FJ040402 | 1,724 | |
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| EU306357 | 1,788 | |
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| FJ040399 | 1,726 | |
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| FJ040400 | 1,726 | |
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| AY284618 | 1,746 | |
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| AY593880 | 1,728 | |
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| FJ040401 | 1,726 | |
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| EU682391 | 1,727 | |
| Hoplolaimidae |
| AY284609 | 1,634 |
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| AJ966503 | 1,796 | |
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| EU306356 | 1,796 | |
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| AY271723 | 1,811 | |
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| AJ966504 | 1,803 | |
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| AY284605 | 1,635 | |
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| AJ966486 | 1,790 | |
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| AY284606 | 1,755 | |
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| EU306354 | 1,802 | |
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| AY284607 | 1,751 | |
| Rotylenchulidae |
| EU306342 | 1,873 |
| Meloidogynidae |
| AY284621 | 1,730 |
Figure 3Neighbor-Joining (NJ) analysis for RN variants (RN_VAR1 and RN_VAR2) and nematodes in the super-families, Hoplolaimidae, Heteroderidae, Rotylenchulidae, and Meloidogynidae.
Table 5
Blast hits among RN 18S rRNA variants, and REPLIg 454 genomic library.
| Query and Subject | Total number of unique hits | Average % ID | Average alignment length | Average mis-matches | Average number of gaps | Average bit score |
| RN_VAR1 vs REPLIg genomic sequences | 83.00 | 98.97 | 400.43 | 2.23 | 1.55 | 749.64 |
| RN_VAR2 vs REPLIg genomic sequences | 236.00 | 99.25 | 441.40 | 1.07 | 2.08 | 832.79 |
Figure 4Multiple sequence alignment of the primary structure of the 18S rRNA gene in RN showing hairpin 17 corresponding to positions 391 - 455 in RN_VAR1 and RN_VAR2.
Figure 5The secondary structure of hairpin 17 of the 18S rDNA for RN_VAR1, RN_VAR2 of RN and 9 other nematode species.
The pairs of nucleotides peculiar to certain taxa are boxed in red at positions 17a, 17b, and 17c. Base pair for Daptonema, CA at position 17a is boxed in violet color.
The secondary structure of hairpin 17 of the 18S rDNA for RN_VAR1, RN_VAR2 of RN and 9 other nematode species.
Discussion
Materials and Methods
Materials Statement
Nematode Extraction from the Soil and DNA Extractions
Polymerase Chain Reaction (PCR)
Cloning of PCR Products
Sequencing
Alignment and Phylogenetic Analysis
Sequence Examination for Chimera Presence
Secondary Structure Analysis
Comparison of RN 18S rRNA variants to RN Genomic and EST Sequences
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