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18S and ITS1 Genomic sequence variations in Rotylenchulus reniformis isolates
in Alabama
Article  in  Journal of Cotton Science · January 2013
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The Journal of Cotton Science 17:184–194 (2013)  184
http://journal.cotton.org, © The Cotton Foundation 2013
MOLECULAR BIOLOGY AND PHYSIOLOGY
18S and ITS1 Genomic Sequence Variations in Rotylenchulus reniformis  
Isolates from Alabama
Seloame T. Nyaku*, Ramesh V. Kantety, Yonathan Tilahun, Kathy S. Lawrence,  
Khairy M. Soliman, Ernst Cebert, and Govind C. Sharma
ABSTRACT with clones grouping together irrespective of sex 
and isolate. Sequencing of one-third of the 18S 
Upland cotton, Gossypium hirsutum L., is and ITS1 rDNA regions provided clear evidence 
highly susceptible to infection by reniform nema- of intra-and inter-nematode variability, in addi-
tode (Rotylenchulus reniformis), which can cause tion to gene conversion events in the 18S rDNA of 
over 10% reduction of cotton yields in Alabama. individual male and female RN clones.
Detection of reniform nematode (RN) and analysis 
for molecular variation within its population is he reniform nematode (Rotylenchulus reniformis) 
important for understanding its interactions with Tis widely distributed in many tropical and 
cotton and other host plant species. Restriction subtropical regions of the world, where it parasitizes 
analysis of PCR products of ITS1 regions was about 300 plant species (Heald and Inserra, 1988; 
achieved using four restriction enzymes, HaeIII, Inserra and Dunn, 1992; Davis et al., 2003) in 
HhaI, MspI, and RsaI. These showed similar band- more than 38 countries in Africa, the Americas, the 
ing patterns for both male and female populations. Middle East, East Asia, and Australia (Luc et al., 
However, MspI digestion of ITS1 amplification 2005; Siddiqi, 1972). Within the United States, the 
products showed variants within the combined sex reniform nematode (RN) is established in at least ten 
and location effects primarily attributed to a 500 southern states, from North Carolina to New Mexico 
bp fragment that was absent in other restriction (Weaver et al., 2007). The wide distribution of this 
digestions. Intra-nematodal variations in 18S and nematode is a result of its diverse host range, and 
ITS1 rDNA were studied in detail by sequencing its ability to endure prolonged periods in dehydrated 
a minimum of ten clones in each individual male conditions while being dispersed to distant locations 
and female RN isolates in both directions. Multiple through dust storms (Gaur, 1988). Monoculture 
sequence alignment of the 18S rDNA sequences and the lack of genetic resistance in cotton further 
showed two major types of sequences within this enhance population increases of the RN (Gazaway 
gene for both male and female RN clones, which and McLean, 2003).
could be distinguished at 27 specific sites. Two In most populations reproduction is amphi-
distinct ITS1 fragments of lengths (550 bp and mictic with 40-60% males; however, there are also 
720 bp) were observed; referred to as ITS1S and parthenogenetic populations with few to no males 
ITS1L respectively. Neighbor-joining analysis was (Nakasono, 1977, 1983; Sivakumar and Seshadri, 
used in revealing the relationships and grouping 1971). Only the female RN parasitizes plant roots. 
characteristics between male and female RN clones, An immature female imbeds its head into root tissue 
while the tail end remains in the soil interspace. As it 
feeds and grows, the portion of the body inside the 
root enlarges. Young infective adult females mate 
S.T. Nyaku*, Department of Biological and Environmental 
Sciences, Alabama &M University, Normal, AL 35762 after establishing a feeding site and eggs are laid in 
[Current Address: Department of Crop Science, College of a gelatinous matrix (Birchfield, 1962; Linford and 
Agriculture and Consumer Sciences, University of Ghana, Oliviera, 1940; Nakasono, 1977; Peacock, 1956; 
P.O. Box LG44, Legon, Ghana]; R.V. Kantety, Y. Tilahun, Sivakumar and Seshadri, 1971).
K.M. Soliman, E.Cebert, and G.C. Sharma, Department Significant damage to cotton yields by RN oc-
of Biological and Environmental Sciences, Alabama 
&M University, Normal, AL 35762; and K.S. Lawrence, curs in Alabama (Gazaway et al., 2001), Louisiana 
Department of Biological and Environmental Sciences, (Overstreet, 1999), and Mississippi (Lawrence and 
Alabama &M University, Normal, AL 35762 McLean, 1999). In 2012, 105,402 bales of cot-
*Corresponding author: seloame.nyaku@gmail.com ton were estimated to have been lost in Alabama, 
NYAKA ET AL.: 18S AND ITS1 SEQUENCE VARIATION IN ROTYLENCHULUS 185
Louisiana, and Mississippi through RN infestation of rDNA (type I and II) and 8% of nucleotides 
(Blasingame and Patel, 2013). Monoculture and distinguished the variants (Carranza et al., 1996). 
the lack of genetic resistance in cotton, aid in the Within the near full 18S rDNA of an individual RN, 
maintenance of high population densities of the RN sequence variants (RN_VAR1 and RN_VAR2) exist, 
(Gazaway and McLean, 2003). Over the past decade and these can be distinguished using 96 specific base 
an average of 7% loss in yield was experienced in sites (Nyaku et al., 2013). The 18S rDNA is a slow 
Alabama, resulting in an annual loss of $126 million evolving region in living organisms, and thus forms 
(Blasingame et al., 2009). the basis for inference of phylogenetic history across 
Molecular characterization for nematode spe- taxa (Fitch et al., 1995; Nadler and Hudspeth, 1998).
cies and populations have been achieved using The aim of this study was to identify variation 
mitochondrial DNA (mtDNA), nuclear high copy among and within five populations from Alabama 
DNA, nuclear low copy DNA (Blouin, 2002; Car- using restriction digestion of ITS1, and to determine 
penter et al., 1992), simple sequence repeat (SSR) within nematode variation for the nuclear 18S and 
markers (Arias et al., 2009) and ribosomal DNA. ITS1 rDNA regions in single male and female RN 
The first ribosomal transcribed spacer (ITS1) is isolates from Alabama.
part of the eukaryotic cistron of ribosomal DNA 
located between the genes coding for 18S and MATERIALS AND METHODS
5.8S rRNA. Due to its non-coding structure, ITS1 
shows a high evolutionary rate and has been used Soil Sample Collection and Establishment of 
for phylogenetic studies of closely related species Reniform Nematode Populations. Samples of RN 
of animals, plants, and fungi at the population and infested soils were obtained from five cotton farms 
species level (Cherry et al., 1997; Chrisanfova et al., located in three counties in Alabama (Table 1). Each 
2008). Evidence exists suggesting that populations soil sample was thoroughly mixed and kept at 4°C; 
of nematodes can be analyzed through PCR-RFLP a 150 cm3 subsample was used for extraction of the 
digestion of the ITS1 region (Powers et al., 1997). nematodes. These populations were further main-
Variations in the ITS1 region in R. reniformis popu- tained on cotton cultivar ‘Delta and Pineland 425 
lations from Alabama show the appearance of two BG/RR’ (DPL 425) together with a mixed-Alabama 
main clusters with individual branches, confirming population in the greenhouse.
the presence of variability in the ITS1 rDNA region Reniform Nematode Extraction from Soil 
(Tilahun et al., 2008). Samples. The nematodes were extracted from the 
The 18S rDNA is approximately 1700 base pairs soil samples as described by Deng et al. (2008). The 
(bp) in length and 50% of the nucleotide variability suspension containing the nematodes was transferred 
is observed in the third of the sequence closest to into a Petri dish using a pipette, and placed on an 
the 5’ region. According to Hillis and Dixon (1991), Olympus SZH-ILLD dissection microscope (Olym-
intra-specific variation in this region is influenced pus optical Co. Ltd. Japan) at 10X magnification. 
by the availability of many copies of 18S rDNA per Individual nematodes were picked using a sterilized 
genome, and its homogenization through evolution. hook, and then placed in a drop of water on a glass 
Intra-individual variation occurrence within the 18S slide for morphological identification, under an 
rDNA of animal genomes is rare. However, 18S Olympus IMT-2 compound microscope (Olympus 
rDNA variation within a metazoan was first noted optical Co. Ltd. Japan). DNA was then extracted 
in Dugesia (Schmidtea) mediterranea, a free-living from individual male and female RN isolates using 
platyhelminth. This gene is made of two types two methods of isolation.
Table 1. GIS information for five Alabama (AL) cotton farm sites infested with RN sampled for this study.
Abbreviation Location County/State Latitude Longitude Infested since
B Belle Mina Limestone, AL 86.89 W 34.66 N Early 1990s
S Shaw Limestone, AL 86.94 W 34.64 N Early 1990s
R Murphy Limestone, AL 86.75 W 34.59 N Late 1980s
T Thornton Lawrence, AL 87.37 W 34.73 N Early 1980s
W Whitehead Fayette, AL 87.73 W 33.84 N Late 1980s
JOURNAL OF COTTON SCIENCE, Volume 17, Issue 2, 2013 186
DNA Extraction (Method 1). This set of ex- at MWG-Biotech AG, USA), 0.3 µl of taq DNA 
tractions were performed solely on the Belle Mina polymerase (Promega, Madison, WI, USA) and 
isolates. DNA was extracted separately from ten indi- sterile PCR-grade water added to a final volume of 
vidual male and female RN isolates using a DNeasy 25 µl. The Nem_ITS1_F and Nem_ITS1_R primers 
Blood and Tissue Kit (Qiagen, Inc., Valencia, CA) were used to amplify the ITS1 region (Powers et al., 
according to the manufacturer’s protocol. 1997). To amplify the 5’ one-third of the 18S rRNA 
Polymerase chain reaction (Method 1).Two gene, the 18S_F (5’-GCTTGTCTCAAAGATTA-
micro-liters of extracted DNA (approximately 1.0 ng/ AGCC-3’) and18S_R (5’-TGATCCWKCYGCAG-
µl) from a single male and female RN was transferred GTTCAC-3’) primers were used. PCR reactions 
to PCR tubes containing 2.5 µl 10x high fidelity PCR were performed in a Peltier Thermal Cycler (PTC) 
buffer, 1.0 µl MgCl2 (50 mM), 0.5 µl dNTPs (10 tetrad 2 DNA engine (Bio-Rad, Hercules, CA, USA). 
mM), 0.5 µl of forward and reverse primers each (10 PCR conditions were: 95°C for 5 min, then 30 cycles 
µm) (synthesized by MWG-Biotech AG, USA), 0.2 of the following: 95°C for 30 s, 57°C for 30 s, and 
µl of high fidelity platinum taq (Invitrogen, Carlsbad, 72°C for 45 s. The final extension phase was 72°C for 
CA, USA.) and sterile DNase-free water added to 5 min. The quality of PCR products were checked by 
a final volume of 25 µl. Primer pairs Nem_18S_F electrophoresis of 6 µl of PCR reaction in 1% aga-
(5’-GGCGATCAGATACCGCCCTAGTT-3’) and rose gel with ethidium bromide staining. The bands 
Nem_18S_R (5’-TACAAAGGGCAGGGACG- were visualized and photographed under ultraviolet 
TATT-3’) were used to amplify a 600 bp region of light. The size of each PCR product was determined 
the 18S rRNA gene of the RN. The second pair of by comparison with a 100 bp DNA marker.
primers (Powers et al., 1997) for amplifying the first PCR-RFLP Analysis. Four restriction enzymes 
transcribed spacer region (ITS1) of the nematodes were used in the digestion of ITS1 amplicons of 
were Nem_ITS1_F (5’-TTGATTACGTCCCT- the ribosomal DNA from five individual male and 
GCCCTTT-3’) and Nem_ITS1_R (5’-ACGAGC- female RN isolates from the five Alabama popula-
CGAGTGATCCACCG-3’). Polymerase Chain tions. The restriction enzymes used were four-base 
Reaction (PCR) was performed in a Peltier Thermal cutters, HaeIII, HhaI, MspI, and RsaI. Amplicons 
Cycler (PTC) tetrad 2 DNA engine (Bio-Rad, Hercu- digested with these enzymes were separated on a 6% 
les, CA, USA). The PCR conditions were as follows: polyacrylamide gel using the CDASG-400-50: Dual 
94°C for 2 min, then 30 cycles of: 94°C for 30 s, Adjustable Mega Gel Kit System (CBS Scientific, 
60°C for 30 s, and 68°C for 1 min. A final extension CA) at 250V for 2 h and 40 min.
phase of 72°C for 7 min concluded the amplifications. Cloning of PCR Products. The 18S and ITS1 
DNA Extraction (Method 2). DNA was extract- amplicons from ten individual male and ten female 
ed from five individual male and female RN isolates RN isolates, from the Belle Mina population, to-
from five Alabama populations, and from four indi- gether with four female RN isolates from the mixed-
vidual female RN isolates from the mixed-Alabama Alabama populations, were purified before cloning 
population soil samples, according to Floyd et al. using a QIAquick PCR Purification Kit (Qiagen, Inc., 
(2002), and maintained separately. The individual Valencia, CA, USA) according to the manufacture’s 
nematodes were picked and placed directly into 20 protocol. The purified fragments were then cloned 
µl of 0.25M NaOH in 0.2 ml tubes, and then kept at into a plasmid vector using TOPO TA Cloning Kit 
room temperature for 3-16 h (Stanton et al., 1998). (Invitrogen, Carlsbad, CA). The ligation reaction 
The lysate was then heated for 3 min at 95°C. Four was made up of 4.0 μl of PCR product, 1.0 μl of salt 
µl of 1M HCl and 10 µl of 0.5 M Tris-HCl buffer solution (1.2 M NaCl and 0.06 M MgCl2), and 1.0 
at pH 8.0 were added to neutralize the base. Five µl μl of TOPO vector. Several clones were picked for 
of 2% Triton X-100 was added, and the lysate was verification of inserts from PCR amplifications for 
heated again for 3 min at 95°C and stored at -20°C. each selected clone through colony PCR. This was 
Polymerase Chain Reaction (Method 2). performed by amplification of the clonal DNA using 
Extracted DNA (1 µl) of approximately 1.0 ng/ M13 forward (5’-TGTAAAACGACGGCCAGT-3’) 
µl was transferred to PCR tubes containing 2.5 and reverse (5’-AGCGGATAACAATTTCA-
µl 10x buffer (Promega, Madison, WI), 2 µl of 25 CAC-3’) primers. PCR conditions were as follows: 
mM MgCl2 (Promega, Madison, WI), 0.5 µl of 10 94°C for 5 min, then 40 cycles of the following: 
mM dNTPs, 0.5 µl of 10 µM primer (synthesized 94°C for 30s, 55°C for 1 min, and 72°C for 1 min. 
NYAKA ET AL.: 18S AND ITS1 SEQUENCE VARIATION IN ROTYLENCHULUS 187
The final extension phase was 72°C for 10 min. In- and ITS1 rDNA clones was examined. The analysis 
dividual bacterial colonies with inserts were picked was carried out using Ballerophon (http://comp-bio.
and placed into 96 well blocks with 1.3 mL of liquid anu.edu.au/bellerophon/bellerophon.pl) with default 
Luria- Bertani (LB) media containing 100 μg/mL settings (300 bp window). The Huber-Hugenholtz 
ampicillin and shaken at 37○C for 24 h at 300 rpm correction was used with Clustalw alignment (Hu-
in an Innova 4300 rotary incubator shaker (New ber et al., 2004). No chimeras were observed in the 
Brunswick Scientific, Edison, NJ, USA). The 96 sequences utilized in this study.
well blocks containing the bacterial cells were then Evolutionary relationships of taxa. The evo-
centrifuged for 12 min at 2,000 x g in a an Eppen- lutionary history was inferred using the Neighbor-
dorf 5804R centrifuge (Brinkmann Instruments Inc., Joining method (Saitou and Nei, 1987). The optimal 
Westbury, NY, USA) to obtain cell pellets. Plasmid tree with the sum of branch length was 0.52. The 
DNA from the bacterial cells was isolated using a percentages of replicate trees in which the associated 
QIAprep Mini-prep kit (Qiagen, Inc., Valencia, CA, taxa clustered together in the bootstrap test (1,000 
USA) according to the manufacturer’s protocol. replicates) were shown next to the branches. The 
Sequencing. Plasmid inserts from at least ten evolutionary distances were computed using the 
colonies originating from each individual nematode Maximum Composite Likelihood method (Tamura 
for the 18S and ITS1 rDNA regions were sequenced and Kumar, 2004) and are in the units of the number 
in both directions with the vector primers M13 of base substitutions per site. The analysis involved 
F and M13R, and T7 (5’-TAATACGACTCAC- 217 nucleotide sequences. All positions containing 
TATAGGG-3’) and T3 (5’-ATTAACCCTCACTA- gaps and missing data were eliminated. There were a 
AAGGGA-3’) primers for the mixed-Alabama and total of 496 positions in the final dataset. Evolution-
Belle Mina populations, respectively. Sequencing ary analyses were conducted in MEGA5 (Tamura 
was performed using the ABI PRISM Big Dye et al. 2011).
Terminator cycle sequencing ready reaction kit (Ap-
plied Biosystems, Foster City, CA) in an ABI 3100 RESULTS AND DISCUSSION
nucleotide sequencer.
Alignment and Phylogenetic Analysis. The Se- Restriction Digestion of the ITS1 rDNA Re-
qMan Pro program within the DNASTAR Lasergene gion of female and male reniform nematodes. The 
v8.0 software (DNASTAR Inc., Madison, WI) was undigested amplicons of the ITS1 region showed 
used in generating consensus sequences from both two fragment sizes of approximately 550 bp and 
forward and reverse sequences and any extraneous 750 bp, respectively. Restriction digestion of the 
sequences outside the respective amplification frag- ITS1 region for female and male RN isolates using 
ments trimmed-off. Multiple sequence alignment HaeIII produced fragments that ranged from 40 bp to 
and phylogenetic analysis were conducted using 500 bp. Fragments of digested PCR products ranged 
Molecular Evolutionary Genetics Analysis (MEGA) from 30 bp to 550 bp for HhaI, 230 bp to 550 bp 
software version 5.0 (Tamura et al., 2011). Identi- for MspI and 120 bp to 550 bp for RsaI. Banding 
cal sequences for both 18S and ITS1 regions were patterns for all the restriction enzymes were similar 
grouped into contigs and screened for homology to in both male and female RN isolates in the popula-
nematode sequences using the standard nucleotide- tions. The restriction enzyme, MspI produced a 500 
nucleotide BLAST [blastn] on the NCBI website bp band in the individual nematodes for female 
(http://www.ncbi.nlm.nih.gov/blast). Murphy, male WhiteHead and in both female and 
The mixed-Alabama and Belle Mina population male Belle Mina RN isolates (Fig. 1). Our study 
18S gene sequences have been deposited in Gen- involved the amplification of the ITS1 rRNA region 
Bank with accession numbers JN695066-JN695091, of five individual male and female RN isolates across 
and KF019952- KF020136 respectively. The ITS populations for a faster diagnosis for this pest, and to 
sequences for the mixed-Alabama and Belle Mina determine if sex-based differences existed. Banding 
populations have also been deposited in GenBank patterns analyzed showed that of the four restriction 
with accession numbers JN695092-JN695137, and enzymes used in digestion of the ITS1 region, MspI 
KF020137- KF020277 respectively. was the most-informative enzyme because a 500 bp 
Sequence examination for chimera presence. band was consistently produced in female, male; and 
Potential production of chimeric DNA in our 18S in both female and male nematodes from Murphy, 
JOURNAL OF COTTON SCIENCE, Volume 17, Issue 2, 2013 188
Whitehead and Belle Mina locations, respectively. Neighbor-Joining analysis showed two major 
The other restriction digestion enzymes produced groupings (A and B) (Figs. 2A and 2B, respectively) 
similar banding profiles and therefore did not differ- for the 18S rRNA gene in both the mixed-Alabama 
entiate nematode populations. Associations among and Belle Mina populations. Within each of these 
populations of three species of the stem nematode groups A and B, clone sequences from different 
Ditylenchus destructor, D. myceliophagus, and seven nematodes irrespective of the sex of nematode 
host races of D. dipsaci from different locations isolate clustered together. Twenty-seven parsimo-
have been verified using this method (Wendt et al., ny informative sites were used in distinguishing 
1993). They could differentiate among D. dipsaci the two major groups, with signs of inter-locus 
host races using southern blot hybridization with a gene conversions among the mixed-Alabama iso-
7.5 kb ribosomal cistron probe. This technique has lates (Table 2). Similarly, 27 sites could distinctly 
also been used in differentiating root-knot nematode separate the Belle Mina population sequences into 
species (Zijlstra et al., 1997). Xiphidorus species two types in both male and female RN isolates 
have also been deetermined using the restriction (Supplementary Tables 1 and 2). Within group 1, 
enzymes (TaqI, RsaI, and HinfI) after digestion of of the mixed-Alabama isolates, RN18S4A, RN-
the ITS1 region (Oliveira et al., 2004). 18S4B, RN18S4E, RN18S11B, and RN18S12G 
 Females Males clones had a total of six, six, ten, five, and seven 
 Whitehead              Shaw                    Murphy            Whitehead                 Shaw                  Murphy inter-locus gene conversion sites respectively, and 
1     2      3      4      5      6      7      8      9      10    11    12    13    14    15    16    17    18    19    20    21    22    23    24    25    26    27    28    29    30    M 
in group B, five, five, five, and eleven inter-locus 
500bp 
400bp gene conversion sites were observed for RN18S4F, 
RN18S5H, RN18S12H, and RN18S12D clones, 
respectively. The presence of indels at specific 
100bp loci were observed, these were at positions 68, 
Figure 1. Restriction Fragment Length Polymorphism from 406, and 407, respectively. Phylogenetic relation-
the digestion of ITS1 region of 5 female and five male ships among nematodes using DNA sequence 
reniform nematodes in three locations - WhiteHead (W), data from the 18S rDNA have been reported 
Shaw (S),and Murphy (R) digested using the restriction (Blaxter et al., 1998). Neighbor-joining analysis 
enzyme MspI and separated on 6% Polyacrylamide Gel on the 18S rDNA of the RN clones showed the 
(PAGE). Circled regions show presence of 500 bp bands 
among the amplicons. Lane M=100bp marker. presence of intra- and inter-nematode variation 
within this gene from single male and female RN 
Sequencing Analysis for 18S rRNA gene. New isolates. This variation was confirmed through 
rDNA sequences (211) were generated from indi- grouping of clones from the same nematode with 
vidual male and female RN isolates. These sequences those from other nematodes irrespective of the 
comprised of 26, 95, and 90 sequences from four sex. Tilahun et al. (2008) also found variation 
female, ten male and ten female RN isolates from within the 18S rDNA region among seven popu-
the mixed-Alabama and Belle Mina populations, re- lations from Alabama. However, intra-individual 
spectively. The 18S data generated after performing nematode variation could not be determined 
MSA showed 689 characters made up of a number of from a particular sex because sequence data was 
sites. Sites within the aligned sequences were con- generated from a pooled population of eight RN 
served and parsimony-informative sites, represent- isolates. In our study, intra-individual variation 
ing 615 (89.3%), and 74 (10.7%) sites, respectively. was clearly determined because, single male and 
These regions showed single base change, no base female RN isolates were used for DNA isola-
change, two or more base changes and a combina- tion, amplification, cloning, and sequencing of 
tion of singletons and parsimony-informative sites, selected rDNA regions. The six clone sequences 
respectively. The 211 sequences exhibited average from Tilahun et al. (2008) included in our analy-
nucleotide frequencies of 24.2%, 24.5%, 23.3%, and sis also showed these sequences clustering with 
28.0% for thymine (T), cytosine (C), adenine (A) other RN clones in both groups confirming the 
and guanine (G), respectively. Estimate of the evolu- presence of variation within this gene. Eukaryotes 
tionary divergence among the 18S rDNA sequences exhibiting intra-individual variation in 18S rRNA 
showed an overall average pairwise distance of 0.02 gene include Plasmodium (Rogers et al., 1995), 
among the sequences. Acanthamoeba (Ledee et al., 1998), and Trypano-
NYAKA ET AL.: 18S AND ITS1 SEQUENCE VARIATION IN ROTYLENCHULUS 189
soma cruzi (Stothard et al., 2000). Powers et al., chromosomes (Eickbush and Eickbush , 2007). 
(2005) detected significant variation within the Another possible explanation of two main types 
18S gene of Meloidogyne spp, however, this gene of sequences within an individual male or female 
could not be used in distinguishing M. arenaria, RN 18S gene is the presence of two nucleolar 
M. incognita, and M. javanica. Explanations for organizing regions which undergo crossing-over 
the variation within the 18S rRNA gene have been occasionally producing small gene conversion 
through models relating to concerted evolution tracts in this gene. Studies in our laboratory have 
occurring within the rDNA locus due to unequal shown the presence of two variants (RN_VAR1 
crossovers and gene conversions. Unequal cross- and RN_VAR2) for the 18S RNA gene in the RN, 
over between sister chromatids occurs more often with a 5% difference distinguishing among these 
compared to exchanges between homologous variants (Nyaku et al., 2013).
Table 2. Parsimony-informative and inter-locus gene conversion sites within the 18S rRNA clones of female reniform nematode 
isolates from mixed-Alabama population.
Group 1 variations at specific base locations in 18S rRNA clones (14)
61 63 64 68 73 78 390 392 407 438 439 442 519 521 524 530 537 549 551 552 562 563 569 601 624 633 655
RN18S4A G C G T T C A G C T T A C G A C G A C G C C T C T G C
RN18S4B G C G T T C A G C T T A C G A C G A C G C C T C T G C
RN18S4E G C G T T C G A T - - G C G A C G A C G C C T C T G T
RN18S4G A T C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S5A A T C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S5B A T C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S5C A T C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S5D A T C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S5E A T C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S11B A T C C T T G A T - - G C G A C G A C G C C T C T G C
RN18S11C A C C C - T A G C T T A C G A C G A C G C C T C T G C
RN18S12B A T C C T T A G C T T A C G A C G A C G C C T C T G C
RN18S12F A T C C T T A G C T T G C G A C G A C G C C T C T G C
RN18S12G A T C C T T A G C T T A C G A C G A C G G A A T C A T
Group 2 variations at specific base locations in 18S rRNA clones (12)
RN18S12H A T C C T T G A T - - G A T G T C G T T G A A T C A T
RN18S4C G C G T T C G A T - - G A T G T C G T T G A A T C A T
RN18S4D G C G T T C G A C - - G A T G T C G T T G A A T C A T
RN18S12D A T C C T T G A T - - G A T G T C G T T G A A T C A T
RN18S12E A T C C T T A G C T T A A T G T C G T T G A A T C A C
RN18S5F G C G T - C G A T - - G A T G T C G T T G A A T C A T
RN18S5G A C G T T C G A T - - G A T G T C G T T G A A T C A T
RN18S11A G C G T - C G A T - - G A T G T C G T T G A A T C A T
RN18S12A G C G T - C G A A - - G A T G T C G T T G A A T C A T
RN18S12C G C G T - C G A T - - G A T G T C G T T G A A T C A T
RN18S5H A T C C T T G A T - - G A T G T C G T T G A A T C A T
RN18S4F A T C C T T G A T - - G A T G T C G T T G A A T C A T
Highlighted regions in yellow show gene conversion sites and positions with gaps are 73, 438, and 439 shown in green 
color.
JOURNAL OF COTTON SCIENCE, Volume 17, Issue 2, 2013 190
Sequence Analysis of ITS1 ribosomal DNA of individuals of a population, for the presence of gaps 
region. New ITS1 sequences (185) generated from within the sequence. The identification and character-
individual male and female RN isolates consisted ization of diallelic insertion-deletion polymorphisms 
of 145 and 40 new ITS1 sequences made up of two for example in the human genome has been observed, 
types of fragments with average lengths, 549.3 and this constituted 2,303 indels within 330 candidate genes 
720.8 bp and hereafter, called ITS1S and ITS1L, (Tusher et al., 2004). Intra-nematodal variation has been 
respectively. These ITS1S sequences consisted of observed within the ITS-2 region of the swim bladder 
29, 39, and 77 sequences from four female, ten nematodes, Cystidicola spp. (Miscampbell et al., 2004). 
female, and ten male RN isolates from the mixed- Two ITS-2 variants differed at four nucleotide positions 
Alabama and Belle Mina populations respectively. which were all from different populations.
Similarly, the ITS1L sequences consisted of much Findings from this study and from eukaryotic 
smaller sequence numbers made up of 16, 12, and species for transcribed and non-transcribed regions of 
12 sequences for mixed-Alabama and Belle Mina 18S gene for RN are not exceptions to the concept of 
populations, respectively. The ITS1 fragments were concerted evolution. This is believed to homogenize 
made up of conserved and parsimony-informative multiple copies of rDNA within species and promote 
sites representing 406 (69.2%), and180 (30.8%), optimal translation efficiency, and reduce intra-specific 
respectively for ITS1S, and 516 (69.7%) and 224 variance. Estimate of evolutionary divergence among 
(30.3%), respectively for ITS1L. Within the ITS1S the sequences showed the more divergence in ITS1 
sequences, the average nucleotide frequencies were sequences, than 18S sequences, with ITS1L sequences 
27.1%, 24.7%, 24.4%, 23.9% for T, C, A, and G, having the greatest divergence. Sequence similarities 
respectively. The larger ITS fragment (ITS1L) had were more prevalent in ITS1S than in ITS1L, thus im-
average nucleotide frequencies of 25.7%, 25.9%, plying a more recent origin of ITS1S than more diverse 
22.2%, and 26.2%, for T, C, A, and G, respectively. ITS1L. A higher degree of size variation was noted in 
Neighbor Joining analysis on ITS1S and ITS1L ITS1L, and the rates of variation in ITS1 regions dif-
sequences produced consensus trees showing clone fered between clones compared to the 18S sequences. 
sequences from disparate nematodes clustering together Factors associated with extreme size variation include 
(Figs. 3 and 4). However, two clones EC07 and EC10 high substitution rates and duplication of repeat se-
did not cluster with any of our sequences. The overall quences. Reports on size variation between closely re-
average pairwise distance among the ITS1S and ITS1L lated taxa indicate lengths not exceeding 400 bp, except 
sequences was 0.03 and 0.05, respectively. Sequenc- for the Schistosoma japonica (Platyhelminthes) and the 
ing of the ITS1 region of rDNA showed considerable Anopheles gambiae (Insecta) species, with 900 bp and 
intra- and inter-nematodal variation within individual 3,000 bp sizes respectively (von der Schulenburg et al., 
male and female RN isolates. This variation might have 2001). Ladybird beetles (Coleoptera: Coccinellidae) 
resulted from differences in the rates of ITS1 evolu- have an ITS1 region ranging from 791 bp up to 2,572 
tion occurring within the nematodes. Variation within bp, which shows increased level of variability.
this region was also observed by Tilahun et al. (2008), 
although individual nematodal variation could not be CONCLUSIONS
accessed because amplifications were from pooled RN 
DNA. In another study, similar variations within the Our results show the presence of variants of 
ITS1 region were observed for the amphimictic popu- nematodes within Alabama populations based on the 
lations from cotton-growing regions in mid-land U.S., following: (1) the presence of the 500 bp band observed 
and those from Brazil, Colombia, Hawaii, and Hondu- in some of the PCR-RFLP amplifications in the ITS1 di-
ras (Agudelo et al., 2005). However, a parthenogenetic gests, and (2) the presence of 27 parsimony-informative 
population from Japan could be distinguished from the bases distinctly separating the 18S rDNA clones in 
other populations using the ITS1 region. Indels and these populations into two major types of sequences. 
substitutions were observed in our ITS1 sequences after Phylogenetic analysis of the multiple sequence align-
MSA was performed. Although mutation rates occur ments revealed the existence of two putative 18S 
at higher rates in loops and stems of rRNA (Dixon and rRNA gene sequence types and many ITS1 sequence 
Hillis, 1993), mechanisms involved may be different variants within individual male and female RN isolates. 
(Tusher et al., 2004). Identification of short diallelic Sequencing of 18S and ITS1 rDNA regions was criti-
indels is facilitated by examining aligned sequences cal in detecting differences between individual clones.
NYAKA ET AL.: 18S AND ITS1 SEQUENCE VARIATION IN ROTYLENCHULUS 191
ACKNOWLEDGEMENT Cherry, T., A.L. Szalanski, T.C. Todd, and T.O. Powers.1997. The 
internal transcribed spacer region of Belonilamus (Nemata: 
This work was supported by USDA-CSREES grant Belonoaimidae). J. Nematology. 29:23-29.
# 2004-38814-15160 to Ramesh V. Kantety, USDA- Chrisanfova, G.G., D. A. Charchevnikov, I. O. Popov, S. V. Zino-
CSREES grant # ALAX-011-706 to Ramesh V. Kantety, vieva, and S. K. Semyenova. 2008. Genetic variability and differentiation of three Russian populations of potato cyst 
ALAX-011-206 to Govind C. Sharma and NSF Plant nematode Globodera rostochiensis as revealed by nuclear 
Genome Research award # 0703470 to Ramesh V. Kan- markers. Russ. J. Genetics. 44: 533-538.
tety, whose untimely passing occurred in the process of Davis, R.F., S.R. Koenning, R.C. Kemerait, T.D. Cummings, and 
reviewing this manuscript. We would like to acknowl- W.D. Shurley. 2003. Rotylenchulus reniformis management 
edge the informatics support offered by Ms. Padmini in cotton with crop rotation. J. Nematol. 35:58-64.
Sripathi and other anonymous reviewers who assisted Deng, D., A. Zipf, Y. Tilahun, G.C. Sharma, J. Jenkins, and K. 
in improving the manuscript. This is journal article # Lawrence. 2008. An improved method for the extraction of 
631 of Alabama A&M Agricultural Experiment Station. nematodes using iodixanol (OptiPrepTM). Afri. J. Micro-biol. Res. 2:167-170.
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NYAKA ET AL.: 18S AND ITS1 SEQUENCE VARIATION IN ROTYLENCHULUS 193
Figure 2a Figure 2b
Figure 2. Phylogram generated from Neighbor-Joining analysis for 18S clone sequences of female and male reniform 
nematodes from mixed-Alabama and Belle Mina populations. The percentages of bootstrap replicates supporting the clades 
are indicated at the branch points. Bootstrap values greater than 50% are shown beside nodes. RN= Reniform nematode; 
18S =18S rRNA gene; 01-10 = reniform nematode isolate IDs; A, B, C, D, E, F, G, H/01-15= clone IDs, BM=Belle Mina, 
F=Female, M=Male.
JOURNAL OF COTTON SCIENCE, Volume 17, Issue 2, 2013 194
Figure 4. Phylogram generated from Neighbor-Joining 
analysis for ITS1-720 sequences for female and male 
reniform nematodes from mixed-Alabama and Belle 
Mina populations. The percentages of bootstrap replicates 
supporting the clades are indicated at the branch points. 
Bootstrap values greater than 50% are shown beside nodes. 
RN= Reniform nematode; ITS1L= Large ITS1 rDNA; 01-
10 = reniform nematode isolate IDs; A, B, C, D, E, F, G, 
H/01-18= clone IDs, BM=Belle Mina, F=Female, M=Male.
Figure 3. Phylogram generated from Neighbor-Joining 
analysis for ITS1-550 sequences for female and male 
reniform nematodes from mixed-Alabama and Belle 
Mina populations. The percentages of bootstrap replicates 
supporting the clades are indicated at the branch points. 
Bootstrap values greater than 50% are shown beside nodes. 
RN= Reniform nematode; ITS1S = Small ITS1 rDNA; 01-
10 = reniform nematode isolate IDs; A, B, C, D, E, F, G, 
H/01-20= clone IDs, BM=Belle Mina, F=Female, M=Male.
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