Hindawi
International Journal of Genomics
Volume 2021, Article ID 5532885, 8 pages
https://doi.org/10.1155/2021/5532885
Research Article
Characterizing Repeats in Two Whole-Genome Amplification
Methods in the Reniform Nematode Genome
S. T. Nyaku ,1 V. R. Sripathi,2 K. Lawrence,3 and G. Sharma2
1Department of Crop Science, College of Basic and Applied Sciences, University of Ghana, Legon, P.O. Box LG44, Ghana
2Department of Biological and Environmental Sciences, Alabama A & M University, Normal AL 35762, USA
3Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA
Correspondence should be addressed to S. T. Nyaku; seloame.nyaku@gmail.com
Received 14 January 2021; Accepted 26 February 2021; Published 8 March 2021
Academic Editor: Kacper Zukowski
Copyright © 2021 S. T. Nyaku et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
One of the major problems in the U.S. and global cotton production is the damage caused by the reniform nematode, Rotylenchulus
reniformis. Amplification of DNA from single nematodes for further molecular analysis can be challenging sometimes. In this
research, two whole-genome amplification (WGA) methods were evaluated for their efficiencies in DNA amplification from a
single reniform nematode. The WGA was carried out using both REPLI-g Mini and Midi kits, and the GenomePlex single cell
whole-genome amplification kit. Sequence analysis produced 4Mb and 12Mb of genomic sequences for the reniform nematode
using REPLI-g and SIGMA libraries. These sequences were assembled into 28,784 and 24,508 contigs, respectively, for REPLI-g
and SIGMA libraries. The highest repeats in both libraries were of low complexity, and the lowest for the REPLI-g library were
for satellites and for the SIGMA library, RTE/BOV-B. The same kind of repeats were observed for both libraries; however, the
SIGMA library had four other repeat elements (Penelope (long interspersed nucleotide element (LINE)), RTE/BOV-B (LINE),
PiggyBac, and Mirage/P-element/Transib), which were not seen in the REPLI-g library. DNA transposons were also found in
both libraries. Both reniform nematode 18S rRNA variants (RN_VAR1 and RN_VAR2) could easily be identified in both
libraries. This research has therefore demonstrated the ability of using both WGA methods, in amplification of gDNA isolated
from single reniform nematodes.
1. Introduction detection, and genotyping [3] and especially in sequencing
genomes of single nematodes. Two of the most used WGA
The reniform nematode is most commonly found within techniques include multiple-displacement amplification
the southern parts of U.S. and is also in many tropical (MDA) and Omniplex methods. The MDA method utilizes
and subtropical regions of the world [1]. Areas within the φ29 DNA polymerase, which is highly processive, and the
U.S. where this nematode has established include Alabama, activities of random exonuclease-resistant primers, in the
Florida, Arkansas, Hawaii, Louisiana, Georgia, Mississippi, amplification reaction [4]. In the Omniplex technique, the
South Carolina, North Carolina, and Texas. This nematode genomic DNA is fragmented into smaller fragments (e.g.,
has surpassed the root-knot nematode (Meloidogyne incog- 200-2000 bases) for the generation of a library, which is
nita) in Alabama, Louisiana, and Mississippi as the leading then amplified. A high-fidelity polymerase is involved in
pest of upland cotton [2]. the reaction which enables the library to be amplified sev-
Development of whole-genome amplification (WGA) eral thousand folds. Whole-genome sequencing has been
techniques for amplification of DNA from nanogram quanti- successfully used in sequencing of two human parasitic
ties has aided the success of most genetic research which once nematodes, including Wuchereria bancrofti [5] and Stron-
needed microgram quantities. These methods prove very gyloides stercoralis [6]. The usefulness of whole-genome
useful in applications such as forensic science, embryonic amplification methods has been applied in genome
disease diagnosis, microbial diversity, bioterrorism genome sequencing projects for the reniform nematode [7, 8].
2 International Journal of Genomics
Existence of low-complexity regions (LCRs) [9] and their In order to verify the absence of bacterial contamination,
repeats (2.2%) within the genome of reniform nematode DNA isolated from the nematodes was amplified using the
serves as an indication of higher levels of variation [8]. universal bacterial primers to amplify the 16S rDNA region.
The objectives of this study were to characterize repeats Extracted DNA (2μl; ~1ng/μl) was transferred into PCR
in the reniform nematode genome through massively parallel tubes containing 2.5μl 10x buffer (Promega, Madison, WI,
genome sequencing, when two different whole-genome USA.), 2.0μl MgCl2 (25mM) (Promega, Madison, WI,
amplification (WGA) methods were employed. USA.), 0.5μl dNTPs (10mM), 0.5μl of primer (10μm) (syn-
thesized byMWG-Biotech AG, USA), and 0.2μl of Taq DNA
polymerase (Promega, Madison, WI, USA.), and double dis-
2. Materials and Methods tilled water added to a final volume of 25μl. Primers
PRBA338F (5′ AC TCC TAC GGG AGG CAG CAG 3′)
Eggs of the reniform nematode were extracted from the roots
of MicroTom tomato plants and were sterilized by immer- (Lane, 1991) and PRUN518R (5′ ATT ACC GCG GCT
sion in 10% Clorox solution and then shaken continually GCT GG 3′) [10] were used in the amplifications. The
for 4 minutes in a beaker. The solution was then poured PRBA338F and PRUN518R primers amplify the 338 to 518
through a 325-mesh sieve, nested on a 500-mesh sieve. The rDNA region and contain one variable loop of rRNA. A pos-
eggs were rinsed thrice with distilled water into a 10ml bea- itive control (Pseudomonas DNA) and a negative control
ker, to remove the residues of Clorox solution. (DNase-free water) were included in the amplifications.
A 325-mesh sieve and a 500-mesh sieve were sterilized by PCR reactions were performed in a Peltier Thermal Cycler
autoclaving using the dry cycle (120°C for 1 hr). Extracted (PTC) tetrad 2 DNA engine (Bio-Rad, Hercules, CA, USA).
PCR conditions were as follows: 94°reniform eggs were then poured onto the 325-mesh sieve C for 9min, then touched°
nested on the 500-mesh sieve. Clorox solutions of concentra- down using 9 cycles from 62 C for 30 sec to 57
°C for 30 sec
and 29 cycles of the following: 94°C for 30 sec, 57°tions 0.5%, 5%, and 10% were used for sterilizing the eggs. C for°
The eggs on the 500-mesh sieve were rinsed immediately 30 sec, and 72 C for 30 sec. The final extension phase was°
with about 300ml of sterilized distilled water to wash o 72 C for 7min. The quality of PCR products was checkedff
the Clorox solution for about 5 minutes and then transferred by electrophoresis of 6μl of PCR reaction in 1% agarose gel
into sterilized beakers with about 10ml of sterilized distilled with ethidium bromide staining. The gel was visualized and
water. One ml of the solution containing the sterilized eggs photographed under ultraviolet light, and the size of each
was placed onto the agar plates. These plates were sealed with PCR product was determined by comparing it with a
parafilm along its edges, covered with aluminum foil, and 100 bp DNA marker.
then kept at room temperature for 2 to 4 days for the eggs PCR products from seven individual female nematodes
to hatch. Some of the plates were also placed in the incubator were cloned into a plasmid vector using TOPO TA Cloning
set at 25°C for hatching of the eggs. Kit (Invitrogen-Life Technologies, Carlsbad, CA). The liga-
Extraction of DNA was initially from fourteen (14) single tion reaction was made up of 3μl of PCR product, 1μl of salt
sterilized female reniform nematodes, undertaken using the solution (1.2MNaCl and 0.06MMgCl2), 1μl of sterile water,
DNeasy blood and tissue kit (Qiagen, Maryland, USA) and 1μl of TOPO vector. Several clones were picked for ver-
according to the manufacturer’s recommended protocol. ification of inserts from PCR amplifications conducted, using
Two (2.0) μl of extracted DNA (~1ng/μl) was transferred each nematode clonal DNA with M13 forward and reverse
into PCR tubes containing 2.5μl 10x bu er (Promega, Mad- primers. PCR conditions were as follows: 94
°C for 5min,
ff
ison, WI, USA), 2.0μl MgCl (25mM) (Promega, Madison, then 40 cycles of the following: 94
°C for 30 sec, 55°C for
2
WI, USA), 0.5μl dNTPs (10mM), 0.5μl of primer (10μm) 1min, and 72
°C for 1min. The final extension phase was
°
(synthesized by MWG-Biotech AG, USA), and 0.2μl of Taq 72 C for 10min. Individual colonies were picked and placed
DNA polymerase (Promega, Madison, WI, USA), and the in separate 1.5ml centrifuge tubes with 1ml of liquid LB
required amount of double distilled water was added to make media containing 100μg/ml of ampicillin. These were shaken
′ at 37
°C for 24 hours at 300 rpm in an Innova 4300 rotary
up the final volume to 25μl. Primers 18SF (5 GCTTGTCTC
′ ′ incubator shaker (New Brunswick Scientific, Edison, NJ).AAAGATTAAGCC-3 ) and 18SR (5 -TGATCCWKCYG- Tubes containing the bacterial cells were centrifuged for 30
CAGGTTCAC-3′) which amplified the 5′ one-third of the seconds at 13,000 rpm in a Hermle MR-2 (National Labnet
18S rRNA gene used in the amplifications. Polymerase chain Company, Woodbridge, NJ) tabletop centrifuge to obtain a
reaction (PCR) was performed in a Peltier Thermal Cycler cell pellet. Plasmid DNA was isolated using a QIAprep Mini-
(PTC) tetrad 2 DNA engine (Bio-Rad, Hercules, CA, USA).
° prep kit (QIAGEN, Maryland, USA).Polymerase chain reaction conditions were as follows: 95 C
° Plasmid inserts from at least ten colonies originatingfor 5min, then 30 cycles of the following: 95 C for 30 sec, from each individual nematode were sequenced in both
57°C for 30 sec, and 72°C for 45 sec. The final extension phase directions with two vector primers, M13 F and M13R, using
was 72°C for 5min. The quality of PCR products was checked the Applied Biosystems (ABI) PRISM BigDye Terminator
by gel electrophoresis of 6μl of PCR reaction on 1% agarose cycle sequencing ready reaction kit (Applied Biosystems,
gel with ethidium bromide staining. The bands were visualized Foster City, CA) in an ABI 3100 nucleotide sequencer in
and photographed under ultraviolet light. The size of each the Center for Molecular Biology at Alabama A & M Uni-
PCR product was determined by comparing it to a 100bp versity. The sequences were then screened for homology,
DNA marker. to reniform nematode sequences using the standard
International Journal of Genomics 3
nucleotide-nucleotide BLAST (blastn) on the NCBI website M 1 2 3 4 5 6 7 8 9 10 M 1112 13 14
(http://www.ncbi.nlm.nih.gov). Sequences from nematodes
having high homology to reniform nematode sequences 600 bp
in the GenBank were identified, and the DNA from these
nematodes used for whole-genome amplification (WGA),
following the manufacturer’s protocol.
The DNA of four female reniform nematodes was used
for WGA after confirmation of the absence of bacterial Figure 1: Amplification of 14 female reniform nematodes using 18S
contamination. The WGA was carried out using both rRNA primers showing a 600 bp band. M: 100 bp molecular marker.
REPLI-g Mini and Midi kits (Qiagen, Maryland, USA), as
well as the GenomePlex single cell whole-genome amplifica-
tion (WGA4) kit (Sigma-Aldrich, MO, USA). Procedures for
amplifications were followed according to the manufacturer’s Repeat masker (http://www.repeatmasker.org/cgi-bin/
protocol. Concentrations of amplicons were determined WEBRepeatMasker) was used in characterization of the reni-
using a TKO 100 fluorometer (Hoefer Scientific Instruments, form nematode genome from each of the libraries and to
San Francisco). The PCR products obtained from bothWGA study its organization (Tables 2 and 3), for REPLI-g and
methods were further amplified using 18S and bacterial SIGMA libraries, respectively. The highest and lowest num-
primers. Real-time PCR was then used to validate results bers of repeats within the genome of RN from the REPLI-g
obtained from WGA amplifications using 16S rRNA gene- library were low-complexity repeats (87%) occupying a
specific primers in the Roche LightCycler 480 instrument region of 130,763 bp and satellites (<1%), occupying a region
(Indianapolis, IN, USA) to check for any bacterial contami- of 320 bp. Simple repeats were the second highest (6%)
nation. Whole-genomic-amplified DNA was purified using within the reniform nematode genome occupying a
the GenElute PCR clean-up kit (Sigma-Aldrich, MO, USA) 10,584 bp region. However, the reniform nematode SIGMA
and then quantified using a TKO 100 fluorometer (Hoefer genomic library had retrotransposable (RTE)/Bovine-B
Scientific Instruments, San Francisco). (Bov-B) repeats being the lowest in the genome (<1%) and
Purified gDNA libraries (10μg/μl) from pooled DNA of occupying a region of 66 bp. The highest repeats from this
selected four single female reniform nematodes were used library were the same as those from the REPLI-g library;
in 454 high-throughput sequencing at the Advanced Centre these were low-complexity repeats (86%), occupying a region
for Genome Technology (ACGT), University of Oklahoma of 304,237 bp, and the next highest repeats were simple
(Norman, OK). repeats (7.2%); these occupied a 32,111 bp region.
Gene Ontology (GO) distributions for the reniform nem- A comparison of the genome characteristics using the
atode genomic sequences were determined using Blast2GO repeat masker showed that the SIGMA library had four other
(http://www.blast2go.com) a functional annotation and visu- repeat elements (Penelope (long interspersed nucleotide ele-
alization tool. ments (LINE)), RTE/BOV-B PiggyBac, and Mirage/P-ele-
ment/Transib); these were absent in the library generated
using REPLI-g. The reniform nematode 18S rRNA variants
3. Results RN_VAR1 and RN_VAR2 [11] were used in blastn analysis
against the assembled reniform nematode 454 genome
Amplification using 18S rRNA primers produced a 600 bp sequences (Table 4). A combined total of 19 hits were noted
band for all the 14 female reniform nematodes (Figure 1). in both variants to the 454 genome sequences, and 5 contigs
The positive control (Pseudomonas DNA) amplified a from the genomic sequences fully overlapped for the cover-
200 bp band; however, all the reniform nematode samples age of the full 18S rRNA gene.
including the negative control (DNase-free water) showed Gene Ontology (GO) analysis performed on both geno-
no amplifications (Figure 2). mic libraries were grouped by molecular function, biological
Whole-genome amplification (WGA) performed on four process, and cellular component. By molecular function, ten
selected female nematodes, using the REPLI-g and SIGMA major GO distributions were assigned with a minimum of
kits, showed distinct amplifications (Figures 3 and 4). 200 sequences with an assigned gene function for the SIGMA
Real-time PCR analysis confirmed Pseudomonas DNA library. These were protein binding, ATP binding, trans-
(positive control) as of a bacterial origin with the highest con- porter activity, transcription factor activity, binding, DNA
centration, but no evidence of bacterial contamination was binding, protein binding, iron ion binding, oxidoreductase
detected from the reniform nematode DNA (data not activity, catalytic activity, and zinc ion binding with 875,
shown). 405, 400, 276, 274, 255, 255, 225, 212, and 210 sequences,
A total of 4Mb and 12Mb of sequence data generated respectively. The percentage contributions by each of these
from the REPLI-g and SIGMA genomic libraries were con- functions are shown (Figure 5). Similarly, ten major GO dis-
structed from the reniform nematodes (RN). In order to tributions were observed by molecular function for the
reduce data redundancy, the sequences were assembled for REPLI-g library. These were protein binding, DNA binding,
quality and length improvements, generating 28,784 and ATP binding, nucleic acid binding, GTP binding, catalytic
24,508 contigs for the REPLI-g and SIGMA libraries, respec- activity, GTPase activity, structural molecule activity, RNA-
tively, using the Lasergene software (Table 1). directed DNA polymerase activity, and RNA binding. These
4 International Journal of Genomics
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Table 1: Reniform nematode assembly characteristics for REPLI-g
and SIGMA genomic libraries.
200 bp Assembly characteristics REPLI-g SIGMA
Total number of contigs 28,784 24,508
Assembly combined length 4,418,499 bp 12,732,631 bp
Figure 2: Amplification of 14 female reniform nematodes using
Number of sequences < 200 bp 21,125 1903
bacterial primers. M: 100 bp marker; 1: Pseudomonas DNA (+ve
control); 2: sterilized DNAse-free water (-ve control); 3-14 are Number of sequences between 7656 20,305
reniform nematode amplifications. 200 bp & 1 kb
Number of sequences between
3 2243
1 kb & 5 kb
M 1 2 3 4 M1
Number of sequences between
0 57
5 kb & 10 kb
10 kb
Table 2: Summary of repeats within the reniform nematode
genome using the REPLI-g library.
1 kb
Type of element No. of elements Length occupied (bp)
Retroelements 50 5682
L2/CRI/Rex (LINEs) 8 608
BEL/Pao (LTR) 6 305
Gypsy/DIRS1 (LTR) 36 4769
DNA transposons 70 5188
Figure 3: Whole-genome amplification (WGA) of 4 female Hobo-activator 10 829
reniform nematodes using REPLI-g Mini and Midi kits. M: 1 kb Tc1-IS630-Pogo 47 3615
marker; M1: 00 bp marker; 1: 2.5 μl (26 ng/μl) of DNA using the MuDR-IS905 8 490
Mini kit; 2: 5.0 μl (26 ng/μl) of DNA using the Mini kit; 3: 2.5μl Unclassified 6 447
(560 ng/μl) of DNA using the Midi kit; 4: 5.0 μl (775 ng/μl) of
DNA using the Midi kit). Small RNA 39 6023
Satellites 2 320
Simple repeats 231 10,584
Low complexity 3311 130,763
M 1 2 3 4 5 Total 3824 169,623
By biological process, eight major GO distributions were
observed, each had a minimum of 100 sequences, contribut-
ing to that function or annotation for the SIGMA library.
These include regulation of transcription, transport, meta-
bolic process, electron transport, two-component signal
400 bp transduction system, proteolysis, peptidyl-histidine phos-
phorylation, and signal transduction with 637, 585, 525,
412, 262, 175, 125, and 110 sequences, respectively. The per-
centage contributions by each of these processes are shown
(Figure 7). However, by biological process, seven major GO
distributions were rather observed for the REPLI-g library,
each of which had a minimum of 9 sequences contributing
Figure 4: Whole-genome amplification (WGA) of 4 female to that function or annotation. These were nematode larval
reniform nematodes using SIGMA kit. M: 100 bp marker; 1: -ve development, reproduction, positive regulation of growth,
control (no DNA); 2: +ve control (human genomic DNA_D7192)- embryonic development, regulation of transcription, DNA
45 ng/μl; 3: RN DNA sample 1A (143 ng/μl), 4: RN DNA sample
12A (145 ng/ l); 5: RN DNA sample 13A (134 ng/ l). metabolic process, and locomotion with 15, 14, 13, 13, 11,μ μ
10, and 9 sequences, respectively. The percentage contribu-
tions by each of these processes are shown (Figure 8).
had 25, 25, 18, 14, 8, 6, 5, 5, 5, and 5 sequences contributing to By cellular component, seven major GO distributions
these functions. The percentage contributions by each of were observed for the SIGMA library, each of which had a
these functions are shown (Figure 6). minimum of 100 sequences contributing to that function or
International Journal of Genomics 5
Table 3: Summary of repeats within the reniform nematode Molecular function
genome using the SIGMA genomic library.
6%
Type of element No. of elements Length occupied (bp) 6% 26%
7%
Retroelements 79 12,340
Penelope (LINEs) 2 227 8%
L2/CRI/Rex (LINEs) 26 2956
8%
RTE/BOV-B (LINEs) 1 66 12%
BEL/Pao (LTR) 5 251 8%
Gypsy/DIRS1 (LTR) 45 8840 8% 12%
DNA transposons 79 6882
Hobo-activator 13 1045
Tc1-IS630-Pogo 47 4716
ATP binding Protein binding
MuDR-IS905 7 322
Transporter activity Iron ion binding 
PiggyBac 2 103
Transcription factor activity Oxidoreductase activity
Other (Mirage,
2 105 Binding Catalytic activity
P-element, Transib)
DNA binding Zinc ion binding
Unclassified 36 6549
Small RNA 121 24,200 Figure 5: Gene Ontology (GO) distributions for reniform
Satellites 8 1785 nematode genomic sequences as determined by Blast2GO and
Simple repeats 491 32,111 grouped by molecular function for the SIGMA library.
Low complexity 5890 304,237
Total 6854 406,735
4. Discussion
Mobile genetic elements (retrotransposons) have the ability
Table 4: Summary of blast hits among reniform nematode 18S of generating DNA through reverse transcription of RNA;
variants and 454 genomic sequences. this DNA is then inserted into the eukaryotic genome.
The horizontal gene transfer between bird and nematode
REPLI-g 454 SIGMA 454
RN 18S rRNA variant
sequences sequences genomes took place in two pantropical waves, 425–22 mil-
lion years ago (Myr ago) involving the Brugia/Wuchereria
Total number of hits (RN_VAR1) 54 244 lineage and 420–17 Myr ago involving the Loa lineage
Overlapping contigs
16 16 [12]. A number of these elements were observed within
(nonredundant) (RN_VAR1) both genomic libraries of the reniform nematode. Classes
Total number of hits (RN_VAR2) 56 244 of retrotransposons include long-terminal-repeat (LTR) ret-
Overlapping contigs
16 13 rotransposons; these elements have mechanisms possessed
(nonredundant) (RN_VAR2) by vertebrate retroviruses. Elements BEL/Pao and Gypsy/-
DIRS1 were present in both genomic libraries of the reniform
nematode. Depending on the type of reverse transcriptase
(RT) domain possessed by the LTR retrotransposons, these
annotation. These were integral to membrane, cytoplasm, were categorized into Ty1/copia, Bel, and Ty3/gypsy groups.
membrane, plasma membrane, intracellular membrane, cell In Saccharomyces cerevisiae, Ty1 and Ty3 are very well studied
outer membrane, and outer membrane-bound periplasmic and characterized; on the other hand, copia, Bel, and Gypsy
space with 1,000, 575, 540, 425, 250, 145, and 100 sequences, are found in Drosophila melanogaster.
respectively. The percentage contributions by each of these Another group is the tyrosine recombinase retrotran-
components are also presented for detailed examination sposons which have similar properties to those of LTR ret-
(Figure 9). The REPLI-g library however had ten major GO rotransposons; however, they possess integrase instead of
distributions for the cellular component, each of which had recombinase, as they integrate into the host chromosomes.
a minimum of 3 sequences contributing to that function or The second group is the non-LTR retrotransposons, which
annotation. Their organellar and membrane-bound distribu- do not possess either inverted or tandem terminal repeats;
tions were cytoplasm, integral to membrane, mitochondrion, these have poly(A) tails at their 3′ ends, and their 5′ ends
membrane, nucleus, endoplasmic reticulum, transcription have variable deletions (5′ truncations). These elements
factor complex, inclusion body, ribosome, and plasma mem- encode open reading frames (ORFs), which are prone to
brane with 17, 11, 11, 8, 5, 4, 3, 3, 3, and 3 sequences, respec- mutations [13]. Examples of these elements are LINEs
tively. The percentage contributions by each of these (long interspersed nucleotide elements), which are autono-
components are also presented (Figure 10). mous and about 5-10 kb in length; the other is SINEs (short
6 International Journal of Genomics
Molecular function
4%
4% 4%4% 22%
5%
7%
22%
12%
16%
Protein binding Catalytic activity
DNA binding GTPase activity
ATP binding Structural molecule activity
Nucleic acid binding RNA-directed dna polymerase
activity
GTP binding
Figure 6: Gene Ontology (GO) distributions for reniform nematode genomic sequences as determined by Blast2GO and grouped by
molecular function for the REPLI-g library.
Biological process
4% (GIY-YIG), and for reverse transcription, they make use of
4% the ends of chromosomes to serve as primers. Two
6% 22% Penelope-like retrotransposons were identified in only the
9% SIGMA genomic library of the reniform nematode. The third
group of transposable elements is DNA transposons; these
have short terminal inverted repeats (TIRs) of about 2-
15% 21% 10 bp at their terminals, with a long ORF, which codes for
protein domains possessing transposase (TR) and DNA
19% binding mechanisms. These elements were present in both
genomic libraries of the reniform nematode. Transposable
elements could either be autonomous (1000-4000 bp) or
nonautonomous (100-3000 bp); i.e., an example is Miniature
Regulation of Two component signal Inverted Repeat Transposable elements (MITEs). Transposi-
transcription transduction system tion of DNA transposons occurs in a variety of ways; how-
Transport Proteolysis ever, their duplications occur when DNA is being replicated
Metabolic process Peptidyl histidine [16]. The “cut-and-paste” mode of action is usually utilized
Electron transport phosphorylation during DNA transposition in majority of eukaryotes; here,
Signal transduction the element is cleaved and transported to another location
by the TR. However, the rolling circle (RC) mechanism is
Figure 7: Gene Ontology (GO) distributions for reniform used by other eukaryotic elements during DNA transposi-
nematode genomic sequences as determined by Blast2GO and tion; this is similar to that occurring in prokaryotes [17]. In
grouped by biological process for the SIGMA library. a recent study, DNA transposons or LTR retroelements
(20.6Mb and 11.3Mb, respectively) were identified in reni-
form nematode genomic assembly (RREN1.0, GCA_
interspersed nucleotide elements), which are nonautono- 001026735.1) of size 314Mb [8].
mous and about 100-400 bp in length [14]. The Penelope- The majority of repeats within both reniform nematode
like retrotransposons and LINEs found in Drosophila virilis genomic libraries were low-complexity repeats (>86%).
[15]. This element inserts into the host genome and their Low-complexity regions (LCRs) occur in many genomes
structures that are very diverse compared to other classes of and among protein families [9]. The diversity occurring
retrotransposons. These elements have the Uri domain among the amino acid sequences of these regions is low,
International Journal of Genomics 7
Biological process Cellular component
11% 4% 4%18% 4%
12% 4%
25%
16% 6%
13%
7%
15% 15% 16%
12%
16%
Nematode larval Regulation of
development transcription
Cytoplasm Endoplasmic reticulum
Reproduction DNA metabolic process Integral to membrane Transcription factor
Positive regulation of Locomotion
growth rate Mitochondrion complex
Inclusion body
Embryonic development Membrane
Ribosome
Nucleus
Figure 8: Gene Ontology (GO) distributions for reniform Plasma membrane
nematode genomic sequences as determined by Blast2GO and
grouped by biological process for the REPLI-g library. Figure 10: Gene Ontology (GO) distributions for reniform
nematode genomic sequences as determined by Blast2GO and
grouped by cellular component for the REPLI-g library.
Cellular component
5% 3% simple repeats could be either be microsatellites or minisatel-
lites. Microsatellites contribute to evolution within organ-
8%
33% isms [22] and are also subjected to changes in their lengths
(replication slippage). Replication slippage does not occur
14% frequently; its occurrence is once every 1,000 generations
[23]. The presence of simple repeats in the reniform nema-
tode genome may serve as markers for the identification of
18% resistance in plants attacked by this nematode. Another19% transposon identified in the SIGMA library is the PiggyBac
transposon. This has been used in transposon mutagenesis
in insects (e.g., Drosophila melanogaster). The Piggyback
transposase activity was observed in D. melanogaster when
Integral to membrane Intracellular the mutator elements in this organism present on the X chro-
Cytoplasm Cell outer membrane mosome in males were used in providing this activity
through a Hermes-based jump starter element; the α-1-tubu-
Membrane Outer membrane bound
periplasmic space lin promoter was used in this process [24]. In transposon
Plasma membrane mutagenesis, transposal elements are able to insert them-
selves into genomic loci resulting in gene disruption.
Figure 9: Gene Ontology (GO) distributions for reniform
nematode genomic sequences as determined by Blast2GO and
grouped by cellular component for the SIGMA library. 5. Conclusion
This research has revealed the ability of using both WGA
and any variation occurring may range from areas with one methods in amplification of gDNA from individual reniform
or many amino acids at specific positions [18]. Meiotic nematodes with both techniques useful in increasing DNA
recombination has been implicated in the fast evolutions of concentrations and identification of repeats, which can also
LCRs [19]. Repeats are also present in genomes of prokary- be applied in other species of nematodes. The highest repeats
otes, contributing to genetic variations in these organisms in both libraries were low complexity; however, the lowest
[20]. The highest source of variation in LCRs is from the tan- repeats for the REPLI-g and SIGMA libraries were satellites
dem repeats which could expand and contract [21]. The and RTE/BOV-B repeats, respectively.
prevalence of high low-complexity repeats within the reni-
form nematode genome suggests high levels of variation in Data Availability
its genome. These low-complexity repeats occupied about
2.2% of the reniform genome [8]. The raw reads for RN genomic sequences were submitted
Simple repeats were the next highest repeats within the to the NCBI Sequence Read Archive (accession Nos.
reniform nematode genome; these were about 6% and 7.2% SRX099033 and SRX099034) for REPLI-g and SIGMA
of total repeats for the REPLI-g and SIGMA libraries. These libraries, respectively.
8 International Journal of Genomics
Conflicts of Interest human pathogenic nematodes,” Nature Communications,
vol. 7, no. 1, 2016.
The authors declare that there are no conflicts of interest [13] T. H. Eickbush and V. K. Jamburuthugoda, “The diversity of
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This work was supported by USDA-CSREES grant # 2004- [15] M. B. Evgen'ev, H. Zelentsova, N. Shostak et al., “Penelope, a
38814-15160 and ALAX-011-706 to Ramesh V. Kantety, new family of transposable elements and its possible role in
ALAX-011-206 to Govind C. Sharma, and NSF Plant hybrid dysgenesis in Drosophila virilis,” Proceedings of the
Genome Research award # 0703470 to Ramesh V. Kantety. National Academy of Sciences, vol. 94, no. 1, pp. 196–201,
This study is dedicated to the late Dr. Ramesh V. Kantety. 1997.
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