Polymorphism and Methylation of Four Genes Expressed in Salivary

MOLECULAR ENTOMOLOGY

Polymorphism and Methylation of Four Genes Expressed in SalivaryGlands of Russian Wheat Aphid (Homoptera: Aphididae)

LU GONG,1,2 FENG CUI,1,3 CHANGZHONG SHENG,4 ZHE LIN,3 GERALD REECK,4

JICHEN XU,2,5 AND LE KANG3,5

J. Econ. Entomol. 105(1): 232Ð241 (2012); DOI: http://dx.doi.org/10.1603/EC11289

ABSTRACT DNA methylation is a general epigenetic mechanism for plants, animals, and fungi toadapt to environmental variation. Two biotypes of the Russian wheat aphid (Diuraphis noxia), Biotype1 and Biotype 2, have different virulence to host plants. In this study, in addition to a high polymor-phism, DNA methylation at cytosines were observed in genomic fragments of four genes in Biotype1 and Biotype 2, after the genomic DNA was treated with sodium bisulÞte. These genes presumablyencode proteins and enzymes in salivary glands of aphids. The two Biotype 1 showed differentmethylation levels, that is, Biotype 1 showed a higher methylation on the four genes. Two thirds ofmethyl cytosines were in a sequence context of CHH (H � A, C, or T). Some polymorphism andmethylation sites were located at important positions in terms of enzyme function, such as close tocatalytic residues or inhibitor binding residues. These Þndings may provide clues to explore theevolutionary mode between Russian wheat aphid virulence and resistance genes of host plants.

KEY WORDS DNA methylation, salivary gland, virulence, biotype, sodium bisulÞte

Epigenetic systems play key roles in adaptation toenvironmental variation and regulation of phenotypicplasticity (Moczek and Snell-Rood 2008). Methylationof DNA at cytosine residues is one of the main epi-geneticmechanisms that regulategenomic imprinting,X-chromosome inactivation, silencing of transposons,and expression of endogenous genes in eukaryotes(Regev et al. 1998). DNA methylation is conserved inmany plants, animals, and fungi, and is thought tooccur “globally” in vertebrates, whereas invertebrates,plants, and fungi have “mosaic” methylation (Hender-son and Jacobsen 2007, Goll and Bestor 2005, Suzukiand Bird 2008). For many years it was thought thatvery little, if any, methylation occurred in insect ge-nomes. Recent evidence from several insect specieshas suggested a more complicated role for DNA meth-ylation in insects. For example, DNA methylation hasa key role in regulating queen determination in honeybees, Apis mellifera L. (Kucharski et al. 2008), andorganophosphate-resistant green peach aphids,Myzuspersicae (Sulzer), are methylated within the gene re-sponsible for the resistance phenotype E4 esterasegene (Field et al. 1996).

The Russian wheat aphid, Diuraphis noxia (Mord-vilko) (Homoptera: Aphididae), is one of the mostnotorious phloem-feeding pests, and mainly damageswheat, Triticum aestivumL., and barley,Hordeum vul-gareL. (Stoetzel 1987). It has a worldwide distribution(apart from Australia) across major small grain pro-duction areas, and causes signiÞcant yield and qualitylosses in several countries (Puterka et al. 1993, Zhanget al. 1999).

Based on the chlorotic damage responses to Russianwheat aphid feeding in barley and wheat cultivarscontaining resistance genesDn1 toDn9, Þve biotypeshave been designated (Burd et al. 2006): Biotype 1 isthe most avirulent biotype, which only can colonizecultivars carrying Dn1, Dn8, or Dn9; Biotype 2 is vir-ulent to cultivars containing any of theDn genes otherthanDn7;Biotype3 is themostvirulentbiotype,whichcan colonize cultivars carrying any of the Dn genes;and Biotype 4 and Biotype 5 are virulent to seven andÞve resistance cultivars, respectively. Although manydifferences in virulence were observed among thesebiotypes, their genetic differences do not seem asprominent, at least for Biotype 1 and Biotype 2, asdisclosed by the mitochondrial gene encoding cyto-chrome oxidase I and several simple-sequence repeatloci (Shufran and Payton 2009). In contrast, in a recentstudy, we observed large amounts of polymorphismsites in several Russian wheat aphid salivary-glandtranscripts, which encode proteins and enzymes ofsalivary glands, both within Biotype 1 and Biotype 2and between these two biotypes (Cui et al. 2011).

In this study, we aim to describe differences, if any,in DNA methylation levels at cytosine residues be-

1 These authors contributed equally to this work.2 National Engineering Laboratory for Tree Breeding, Beijing For-

estry University, Beijing, China.3 State Key Laboratory of Integrated Management of Pest Insects

& Rodents, Institute of Zoology, Chinese Academy of Sciences, Bei-jing, China.

4 Department of Biochemistry, Kansas State University, Manhattan,KS.

5 Corresponding authors, e-mails: [email protected]; [email protected].

0022-0493/12/0232Ð0241$04.00/0 � 2012 Entomological Society of America

tween Biotype 1 and Biotype 2 in the Russian wheataphid. We treated genomic DNA of parthenogenesisaphids with sodium bisulÞte, and compared the meth-ylation levels of four putatively salivary-gland tran-scribed genes that encode proteins and enzymes ofsalivary glands, between the two biotypes. The im-portance of methylation sites toward encoded en-zymes was predicted based on the conservation ofprimary sequences and the positions in tertiary struc-tures.

Materials and Methods

Insects. Populations ofD. noxiaBiotype 1, collectedfrom wheat Þelds near Hays, KS, and Biotype 2, col-lected from wheat Þelds near Briggsdale, CO (via theUSDA-ARS Plant Science Research Laboratory atStillwater, OK), were cultured in separate locations inthe greenhouse on susceptible ÔJaggerÕ wheat plants atKansas State University for use in the experiments.The identity of each biotype was veriÞed in diagnosticplant differential greenhouse assays in Stillwater, OK(Biotype 2), and Manhattan, KS (Biotype 1) (Qureshiet al. 2005).Genomic DNA Extraction and Sodium BisulfiteTreatment. Genomic DNA of Biotype 1 and Biotype2 was extracted from 10 adult aphids of parthenogen-esis using the CTAB method as described in Roger andBendich (1988). The DNA was divided in two aliquots:one was used directly as polymerase chain reaction(PCR) template and the other was treated by sodiumbisulÞte, which converts unmethylated cytosines touracils and leaves methyl cytosines unchanged in ge-nome, before used as PCR template. In total, 300 ng ofDNA was treated with EZ DNA Methylation-Gold Kit(Zymo Research, Irvine, CA), following the protocolof the manufacturer. After PCR ampliÞcation, the cy-tosines that turn to thymines were unmethylated sites

and the unchanged cytosines were methylation hap-pening sites in the genome.Gene Cloning, Sequencing, and Analysis. Genomic

fragments of four genes were cloned from genomicDNA of Biotype 1 and Biotype 2: dipeptidyl carboxy-peptidase, sucrase, trehalase, andC002.The primers foreach gene were designed based on our previous workon their cDNA sequences (Cui et al. 2011). The sizesof PCR products were estimated with reference totheir homologs in the pea aphid (Acyrthosiphon pisumHarris) genome (International Aphid Genomics Con-sortium 2010). Because of a long intron insertion be-tween two exons of sucrase in the pea aphid, this genewas divided into two fragments for PCR ampliÞcationin the Russian wheat aphid genome and named assucrase fragment 1 and sucrase fragment 2. The infor-mation of primer pairs and size of PCR product foreach gene fragment before and after the sodium bisul-Þte treatment were detailed in Table 1.

The 50 �l PCR mixture contained 20 ng of genomicDNA, 0.25 �M of each primer, 0.2 mM of each dNTP,and 2.5 U of LA Taq polymerase (Takara, Otsu, Shiga,Japan) in a 1 � reaction buffer. The LA Taq is athermostable DNA polymerase having 3� 3 5� exo-nuclease activity (proof reading activity). Under gen-eral PCR conditions, higher efÞciency and higher Þ-delity are obtained compared than conventionalTaqDNA polymerases. The PCR was run on a ther-mocycler (Eppendorf, Westbury, NY) with a dena-turing step at 94�C for 5 min, followed by 35 cycles of94�C for 30 s, 55�C or 52�C for 30 s, 72�C for 30 s or 1min, and a Þnal extension of 5 min at 72�C. Afterseparated and puriÞed from 1% agarose gel with Axy-Prep gel extraction kit (AXYGEN, Union City, CA),PCR products were connected with the pGEM-T easyvector (Promega, San Luis Obispo, CA) and trans-fected into DH5� cells. At least Þve independentclones for each gene before and after the sodium

Table 1. Information of primers and products of PCR amplification

Gene fragmenta Typeb Primer Product size (bp)

C002 control F: CAATACACGAGAGGTATGTT 261R: CTGTTTCATTGATTCTCCGTC

(JN092369) treatment F: TTATTGTTAGAGAGTTGAATATGA 220R: TTCATTAATTCTCCATCTTTAAAT

Dipeptidyl carboxypeptidas control F: ACATCGTTAAACTTGAGCGCA 198R: ATATTGGATGTGTCCCATCTC

(HQ709433) treatment F: TTTGGGATAAATTTATTTTGGAGA 162R: ATTAAATATATCCCATCTCATAAT

Sucrase fragment 1 control F: GTTGATTTCCAGTTAGGTGTATT 461R: TACTTAGTTTTCTTTTCCCTGG

(JN092368) treatment F: TTTGAATTATAGAAATTTATTTGTTA 213R: AATTCATCAACTAAATCTTCTCTT

Sucrase fragment 2 control F: GATGGTGGAATGTTACTCACC 516R: CGTTATACGAATTGTCCCAT

(JN092368) treatment F: TAGGTTTATGATATGATTAGATTAT 276R: ACATTTATATCAATCATACCTAATT

Trehalase control F: ATTGAAGCGGATTATTCTGTGA 644R: CCGTTTGTCCCGTTAGATAC

(JN092370) treatment F: ATTGATATTTAATATAAGTATATT 466R: TTTATCCCATTAAATACTATATC

a The registration no. in GenBank for each gene is in parenthesis.b PCR was performed using genomic DNA templates that were treated with sodium bisulÞte (treatment) or not (control).

February 2012 GONG ET AL.: DNA METHYLATION OF RUSSIAN WHEAT APHIDS 233

bisulÞte treatment were sent to BGI Company (Bei-jing, China) for sequencing.

The obtained sequences were aligned using theonline ClustalW2 sever at EBI (www.ebi.ac.uk/Tools/clustalw2/index.html) to identify the polymorphismsites. The normalized CpG content (CpGO/E) for eachgene was calculated as

CpGO/E � PCpG/�PC*PG�

where PCpG, PC, and PG are the frequencies of CpG

dinucleotides, C nucleotides, and G nucleotides, re-

spectively, estimated from each gene.

Protein Structure Prediction.To determine the rel-

ative positions of the polymorphism and methylation

sites totheconservativeoractiveregionsofenzymes, the

deduced amino acid sequences of dipeptidyl carboxy-

peptidase, sucrase, and trehalase from Russian wheat

Fig. 1. Nucleotide and deduced amino acid sequences of cloned fragments ofC002 (A), dipeptidyl carboxypeptidase (B),sucrase (C and D), and trehalase (E) from Russian wheat aphid. Short dashes indicate introns.

234 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 1

aphids were sent to I-TASSER online sever (http://zhanglab.ccmb.med.umich.edu/I-TASSER) for tertiarystructure modeling. Three parameters were provided

along the predicted models: 1) C-score, a conÞdencescore for estimating the quality of predicted models,typically in the range of [-5, 2] (C-score of highervalue signiÞes a model with a high conÞdence); 2)TM-score; and 3) RMSD; both TM-score and RMSDare known standards for measuring the accuracy ofstructure modeling. A TM-score �0.5 indicates amodel of correct topology (Wu et al. 2007, Zhang2008). The primary amino acid sequences of these

three enzymes from Russian wheat aphids were

Table 2. Polymorphisms of C002 and dipeptidyl carboxypep-tidase from Biotype 1 and Biotype 2

Gene Biotype

PolymorphismClone

frequencyTotal sites(percenta)Nucleotide

amino acid

C002 Biotype 1 A56G N19S 1/5 4 (1.5%)A71G N24S 1/10T180C No 1/10A217C M73L 1/10

Biotype 2 C115A P39T 1/13 5 (1.9%)A136G R46G 1/13A144G No 1/13A186G No 1/13A201G No 1/6

Dipeptidylcarboxypeptidase

Biotype 1 A59G K20R 1/5 3 (1.5%)A141T No 1/5A148G N50D 1/10

Biotype 2 A30T Q10H 1/12 3 (1.5%)A81G No 1/12T168C No 1/12

The polymorphism sites in bold were observed in PCR productsafter methylation treatment. No, synonymous mutation.a The total no. polymorphism sites divided by the length of cloned

fragments.

Table 3. Polymorphisms of sucrase from Biotype 1 and Bio-type 2

Biotype

PolymorphismClone


amino acid

Biotype 1b T145C Ñ 1/10 14 (1.4%)C255T No 1/5A258G No 5/10A264G No 1/5C372T No 1/5A493G T136A 1/5T501C Y139H 1/5G583A A166T 1/5A668G Ñ 1/10C699A Ñ 1/10G751A No 1/10T773C No 1/10A791G M212V 1/10A946G No 2/5

Biotype 2 A26G N5S 2/7 21 (2.2%)A45G No 1/7T167C Ñ 1/11A258G No 2/7�7/11A262G R58G 1/11C374T T95I 1/7G483A No 1/5T489C No 1/5T613C W176R 1/5A668G Ñ 1/12T682C Ñ 1/12A694G Ñ 1/12A697G Ñ 1/12A727G No 1/12G747T R197I 1/12A780G D208G 1/12A815G T220A 1/5A824G T223A 1/12A877G No 1/5T907C No 1/5A946G No 2/5

The polymorphism sites in bold were observed in PCR productsafter methylation treatment. No, synonymous mutation;—, polymor-phism sites located in introns.a The total no. polymorphism sites divided by the length of cloned

fragments.b Sucrase fragment 1 and sucrase fragment 2were counted together

in this table.

Table 4. Polymorphisms of trehalase from Biotype 1 and Bio-type 2

Biotype

PolymorphismClone


amino acid

BWA1 G52C Ñ 1/5 24 (3.7%)A72T Ñ 1/5C95A Ñ 1/5T169G Ñ 1/5A210G Ñ 1/11A228G Ñ 1/5T265C W23R 1/11A296T Q33L 1/11C321T No 1/5A344G Q49R 1/11C379A R61S 1/11A407G K70R 1/11A438G Ñ 1/11T446C Ñ 1/11A458G Ñ 1/5A472G Ñ 1/11A473G Ñ 1/11T504C Ñ 2/5A509G Ñ 1/5A512G Y77C 1/11A533T E84V 1/11C536T T85I 1/5G538A G86S 1/11G541A G87R 1/5

BWA2 C52G Ñ 1/5 25 (3.9%)T73C Ñ 1/5A95C Ñ 1/5T96C Ñ 1/5T97C Ñ 1/5T148A Ñ 1/5G169T Ñ 1/5A248G Y17C 1/11T288C No 1/11A296G Q33R 1/11A311G Q38R 1/11A329G Q44R 1/5A344G Q49R 1/11A462G Ñ 1/11A480G Ñ 1/11C504T Ñ 1/5T506C Ñ 1/5T519C No 1/5A556G T92A 1/11A563G Q94R 1/11T572A F97Y 1/11T595C F105L 1/11T601C F107L 1/11A608G N109S 1/11A612G No 1/11

The polymorphism sites in bold were observed in PCR productsafter methylation treatment. No, synonymous mutation.—, polymor-phism sites located in introns.a The total no. polymorphism sites divided by the length of cloned

fragments.


aligned with the homologous enzymes from otherorganisms using the online ClustalW2 server at EBI.

Results

Sequence Analysis of the Cloned Genes. The frag-ments of four genesC002, dipeptidyl carboxypeptidase,sucrase, and trehalase, presumably expressed in sali-vary glands of Russian wheat aphids, were cloned fromthe genomes of Biotype 1 and Biotype 2. The fragmentlengths of C002 and dipeptidyl carboxypeptidase were261 bp and 198 bp, respectively, and only covered exonregions (Table 1; Fig. 1A, B). For sucrase and trehalasethere were introns in the cloned fragments (Fig. 1C,

D, and E) and they presumably encoded 270 (twosucrase fragments together) and 120 amino acid resi-dues, respectively. These sequences were deposited inGenBank for reference. Based on the sequencing re-sults and suitability of primers, shorter fragments ofeach gene within the original sequencing regions wereampliÞed by PCR on sodium bisulÞte treated DNAtemplates for sequencing (Table 1).Sequence Polymorphism. To identify sequence

polymorphism of the cloned genes, at least Þve clonesfor each gene before and after the sodium bisulÞtetreatment were sequenced and compared with eachother in a mixture of 10 adult aphids of parthenogen-esis. More polymorphism sites were observed in su-crase (35) and trehalase (49) than in C002 (9) anddipeptidyl carboxypeptidase (6) (Tables 2 through Ta-ble 4). Trehalase had the highest variation rate amongthe four genes, that is, 3.7 and 3.9% of the clonedfragment in Biotype 1 and Biotype 2, respectively.Most of the variations were detected only once withinbiotypes, but some variations were found more thanonce, such as sites 26, 258, and 946 in sucraseof Biotype2 (Table 3). For the four genes, there were 45 poly-morphism sites in Biotype 1 and 54 in Biotype 2. Thisdifference was mainly because of sucrase, where Bio-type 1 and Biotype 2 had 14 and 21 polymorphism sites,respectively. The two biotypes only shared nine poly-morphism sites, that is, 258, 668, and 946 in sucrase(Table 3), and 52, 95, 169, 296, 344, and 504 in trehalase(Table 4).GeneMethylation.The methyl cytosines in the four

genes of the two biotypes were identiÞed by treatingthe genomic DNA of 10 parthenogenic adult aphidsfrom each biotype with sodium bisulÞte. Among thecloned 1,337 bp of genomic fragment (the total lengthof the four genes), there were 18 methylation sites inBiotype 1 and 12 in Biotype 2 (Table 5). The twobiotypes shared one methylation site, that is, the nu-cleotide 401 in trehalase. The methylation ratios, inBiotype 1 and Biotype 2, respectively, were 9.52 and6.35% when compared with the number of cytosines(189), and 1.35 and 0.90% when compared with thewhole length (1,337 bp). Eight methyl cytosines,

Table 5. Methylation sites of cloned genome fragments fromBiotype 1 and Biotype 2

Gene BiotypeSequence context of

methyl cytosineaAmino acid

changeClone

frequency

C002 Biotype 1 177 CCG No 1/10180 CAT No 1/10193 CTG No 1/10217 CTGb No 1/10

Biotype 2 151 CGG R51W 1/13Sucrase Biotype 1 145 CTA Ñ 1/10

755 CCA S200P 1/10773 CTA No 1/10802 CCT No 1/10814 CAC No 1/10

Biotype 2 152 CAT Ñ 1/12167 CAA Ñ 1/12632 CTG Ñ 1/12819 CAG S221L 1/12

Trehalase Biotype 1 265 CGG W23R 1/11289 CCT P31F 1/11290 CTT P31F 1/11299 CTT A34V 1/11401 CAG A68V 1/11439 CAT Ñ 1/11446 CTA Ñ 1/11560 CGC P93L 2/11581 CAA T100I 1/11

Biotype 2 259 CAA Q21stop 1/11281 CAT A28V 1/11288 CCC No 1/11401 CAG A68V 1/11410 CAA S71L 1/11595 CTC F105L 1/11601 CTC F107L 1/11

Shaded methylation sites are thymines in genome before sodiumbisulÞte treatment; nonshaded methylation sites are cytosines in ge-nome before sodium bisulÞte treatment. No, synonymous mutationwhen methyl cytosines convert to uracils in genome.—,methylationsites located in introns.aMethyl cytosine is the Þrst cytosine in the three nucleotides.b The methylation site is adenine in genome before sodium bisulÞte

treatment.

Table 6. Summary of methylation types in the two biotypes ofRussian wheat aphid according to the sequence context of methylcytosines

Biotype CG CHGa CHHa

Biotype 1 2 4 12Biotype 2 1 3 8Total 3 7 20

aH � A, C, or T.

Table 7. The normalized CpG contents (CpGO/E) and GC con-tents of the cloned four gene fragments of Russian wheat aphid

GeneC

contentG

contentCpG

contentCpGO/E

GCcontent

C002 0.20 0.26 0.064 1.23 0.46Dipeptidylcarboxypeptidas

0.17 0.20 0.019 0.56 0.37

Sucrase 0.17 0.21 0.033 0.92 0.38Trehalase 0.16 0.19 0.024 0.79 0.35

Table 8. Parameters of the predicted tertiary models by I-TASSER for the three enzymes from Russian wheat aphid

C-score TM-score RMSD

Dipeptidyl carboxypeptidase 1.50 0.92 0.06 0.5 0.5 ÅSucrase 1.12 0.87 0.07 3.7 2.5 ÅTrehalase 1.55 0.93 0.06 1.4 1.3 Å


when converted to thymines during transcription,could lead nonsynonymous mutations for each bio-type, Biotype 1 and Biotype 2. Most of the methyla-tions were detected once in 10Ð13 clones, and only themethylation at the site 560 in trehalaseof Biotype 1 wasobserved twice in 11 clones. Three types of methyl-ation were observed according to the sequence con-text of the cytosines, namely CG, CHG, and CHH(H � A, C, or T). Two thirds of DNA methylationhappened at CHH in the four genes in each biotype(Table 6).

The GC contents of the cloned fragments of thefour genes ranged between 0.35Ð0.46, and the nor-malized CpG contents (CpGO/E) ranged between0.56 and 1.23 (Table 7). Of the four genes, trehalasehad the most methylation sites, totally 16 in the twobiotypes. C002 had the most differentiated rates ofmethylation between the two biotypes, 1.8% in Bio-type 1 versus 0.45% in Biotype 2 in the 220 bp ofrange. There was no methylation site in the clonedfragment of dipeptidyl carboxypeptidase.Bioinformatic Analysis on the Teritary Structure ofThree Enzymes. The tertiary structures of deduceddipeptidyl carboxypeptidase, sucrase, and trehalase

from Russian wheat aphid were modeled by the I-TASSER online server. The accuracies of the esti-mated models for the three enzymes were high, asshown with large C-scores and TM-scores, and smallRMSD scores (Table 8). The tertiary structure ofdeduced C002 protein from Russian wheat aphid wasnot predicted because of lack of the homologous tem-plates from other organisms. The conserved polymor-phism and methylation sites causing nonsynonymousmutations of the three enzymes were labeled in theirpredicted structures.

According to the crystal structure of Drosophilaangiotensin I-converting enzyme (Kim et al. 2003),there were Þve catalytic residues in the cloned Rus-sian wheat aphid fragment, including H29, A30, H59,E60, andH63.The threepolymorphismsites,Q10,K20,and N50 that cause nonsynonymous mutations werelocated far away from these catalytic residues (Fig.2A).

For the sucrase of Russian wheat aphid there were12 polymorphism sites and two methylation sites thatcause nonsynonymous mutations (Table 3). The poly-morphism sites T95, W176, R197, T220, and T223, andthe methylation site S221 were conserved across dif-

Fig. 2. Predicted tertiary structures ofdipeptidyl carboxypeptidase (A), sucrase (B), and trehalase (C) from Russian wheataphid. The structure of sucrase incorporates the two fragments (sucrase fragment one and fragment 2). Yellow, catalyticresidues. Purple, polymorphism sites. Red, methylation site. Green, inhibitor binding sites. “m” in red, methylated polymor-phism sites.


ferent organisms (Fig. 3). According to the crystalstructure of Bacillus cereus, oligo-1, 6-glucosidase(Watanabe et al. 1997), D60, E128, and D194 werecatalytic residues. The polymorphism sites T95, R197,and T223 were close to these catalytic residues in thethree-dimensional structure of the enzyme (Fig. 2B).

For the trehalase of Russian wheat aphid there were21 polymorphism sites and 13 methylation sites that

cause nonsynonymous mutations (Table 4). The poly-morphism sites W23, Y77, G86, G87, Q94, and F97, andthe methylation sites W23, P31, and T100 were con-served through alignment of homologous trehalasesfrom various organisms (Fig. 4). Based on the knownthree-dimensional structure of a trehalase from Esch-erichia coli (Gibson et al. 2007), the cloned fragmentof Russian wheat aphid trehalase contained catalytic

A.pisum L G V F NS GS AWEWNE E RQQ Y Y L HQ F Q VK QP D L N YRNP S VRE E I K N T L L YWL 50T.adhaerens I S V F S - GS MWN YN T K RS Q F Y L HQ F L K E QP D L N Y T NP E V VQ AS YD V I S FWL 49S.purpuratus I G V F G - GS VWEWVE E RQQ F YMH A F L K E QP D L N Y I DG I VRDE MK D V VR FWM 49M.musculus L S V YG - N S SWH F DE VRK QC Y F HQ F L K E QP D L N F RNP A VQE E I K E I I T FWL 49H.sapiens L S V YG - N S SWH F DE VRNQC Y L HQ FMK E QP D L N F RNP D VQE E I K E I L R FWL 49D.rerio VS V F G - N S TWE YDE I RQQC Y F HQ F L K E QP D L N YRNP L V L QE MT D I I H FWL 49B.cereus WGA A F S GS AWQ YDE MT DE Y Y L H L F S K K QP D L NWDNE K VRQD V YE MMK FWL 50D.noxia L G V F NS GS AWEWNE E RQQ Y Y L HQ F Q VK QP D L N YRNP S VRE E I K N T L L YWL 50

A.pisum GRG VDG F R F D A VN Y L F E RE D L ADE P K S N - - K I G - - Y L D T D YDS L T H T S T L 96T.adhaerens NK G VDG F R I D A I K H V F E NP L L P DE VRNP N F K P G - - - - E P P YS S L I HNE T T 95S.purpuratus E RG ADG L R VD A V AQ L I E DH YMRDE P P DP A YK P T GK E T RP QWT S L L HN Y T F 99M.musculus S K G VDG F S F D A VK F L L E AK D L RNE I Q VN T S Q I P - - D T V T H YS E L YHD F T T 97H.sapiens T K G VDG F S L D A VK F L L E AK H L RDE I Q VNK T Q I P - - D T V T Q YS E L YHD F T T 97D.rerio K K G VDG F RMD A VK HML E A T H L RDE P Q VNP DQDP - - S T VD T E F E L YHD Y T Y 97B.cereus E K G I DG F RMD V I N F I S K E E G L P T VE T E E E G Y VS G - - - H K H F MNGP N I H K Y 97D.noxia GRG VDG F R F D A VN Y L YE RE D L ADE P K S N - - K I G - - Y L D T D YDS L T H T S T L 96

A.pisum DQP E T YS I VRQWR - QML DS YR T RE K K T K F MMVE C Y - - S P F DK T L L Y YGS N 143T.adhaerens D YP P L HQ L CK DWR - K V I D K F S T - S QK P R F A VGE A Y - - N P I R E I MK Y YG T N 141S.purpuratus NHP E HH T S I R SWRS D VMDR YS T - E P N YR FMMT E T Y - - D E P AN L L D Y YG T E 146M.musculus T Q VGMHD I VRD F R - Q T MNQ YS RE P GR YR FMG AE AS - AE S I E R T MMY YG L P 145H.sapiens T Q VGMHD I VRS F R - Q T MDQ YS T E P GR YR FMG T E A Y - AE S I DR T VMY YG L P 145D.rerio T Q AG L HE I L T DWR - I QMD T YS RE P GR YR FMVME S YD YE E I D K T MR Y YG T N 146B.cereus L HE MNE E V L S H YD I MT VGE MP G V T T E E AK L Y T GE E - R K E L QMV F Q F E HMD 146D.noxia DQP E T Y T I VRQWR - Q V L DS YR T RE K K T K F MMVE C Y - - S P F NK T MMH YGNN 143

A.pisum S E P G AH F P F N F L F I G T F DQQS D A AK VHNM I K SW I RGMP T GMWP NWV L GNH 193T.adhaerens - GDE F H F P F N F F L L T L G - - D F T G T G VNK T VE DWMS H AP RCGWP NWV VGNH 188S.purpuratus DG AE AD F P F N F Q L I S L N ADN L S GNK V F Q L VDDWMK V T L GDKWP NWV I GNH 196M.musculus F I QE AD F P F NK Y F T T I - - G T L S GH T V YE V I T SWME NMP E GKWP NWMTGGP 193H.sapiens F I QE AD F P F NN Y L S ML - - D T VS GNS V YE V I T SWME NMP E GKWP NWM I GGP 193D.rerio Y AK E S D F P F N F Y L L D L P - DN L S GN Y AK S I VE RWMS NMP K GKWP NWV VGNH 195B.cereus L DS GE GGKWD VK P CS L L - - - - - - - T L K E N L T KWQK A L E H T GWNS L YWNNH 189D.noxia S E P G AH F P F N F L F I G T F DQQS D A AQ VHDM I R SWMYGMP T GMWP NWV L GNH 193

A.pisum DN AR V AS RS NP - - - - - - ML VDG L HM I QH L L P R T S V T Y YGDE L G L I D T T VR 237T.adhaerens DNNR I S K RRGN - - - - - - A Y VR A VN A I N L L L P G T P T T Y YGE E I NMQH VD I D 232S.purpuratus DN F R AGHR L GK - - - - - - Q F AR A AN V L N L L L P G T P T T Y YGE E L GME H I S V T 240M.musculus E T P R L T S R VGS - - - - - - E Y VN AMHML L F T L P G T P I T Y YGE E I GMGD I S V T 237H.sapiens DS S R L T S R L GN - - - - - - Q Y VN VMNML L F T L P G T P I T Y YGE E I GMGN I V A A 237D.rerio DK P R I GS S AGK - - - - - - E Y VR A L NML L L T L P G T P T T Y YGE E I GMVD VN I S 239B.cereus DQP R V VS R F GNDGMYR I E S AK ML A T V L HMMK G T P Y I YQGE E I GMT N VR F E 239D.noxia DN AR V AS R T NP - - - - - - ML VDG L HM I QH L L P G T S V T Y YGDE L GM I D T N VR 237

A.pisum WDQ T VDP AG L N - - - - - - - - - - - - - - - VGP YR F L K F S RDP VR T P F PWDS S Y 272T.adhaerens L S E AK DP F A L Q - - - - - - - - - - - - - - - N - P D I YK T VGRDP E R T P MQWNRS A 266S.purpuratus F E E T QDP S GK N - - - - - - - - - - - - - - - N - P CCWE A YS RDP E RS P MQWN T E K 274M.musculus N F NE S - - - - - - - - - - - - - - - - - - - - - - - - - - - YD S T T L VS K S P MQWDNS S 260H.sapiens N L NE S - - - - - - - - - - - - - - - - - - - - - - - - - - - YD I N T L RS K S P MQWDNS S 260D.rerio V I QDP AG - - - - - - - - - - - - - - - - - - - - - - - - Q YDP S RDP QR T P MQWNNE L 265B.cereus S I D E YRD I E T L NMYK E K VME RGE D I E K VMQS I Y I K GRDN AR T P MQWDDQN 289D.noxia WDQ T VDP AG L N - - - - - - - - - - - - - - - VGP YR F L K F S RDP VR T P F PWDNS Y 272

A.pisum N 273T.adhaerens N 267S.purpuratus N 275M.musculus N 261H.sapiens N 261D.rerio N 266B.cereus H 290D.noxia N 273

Fig. 3. Alignment of the deduced amino acid sequence of sucrase from Russian wheat aphid with its homologs from otherorganisms. Accession numbers of these homologs in GenBank are NP 001119607 for Acyrthosiphon (A.) pisum,XP 002108399 forTrichoplax(T.)adhaerens, XP 001183808 forStrongylocentrotus (S.)purpuratus, NP 033231 forMus(M.)musculus, BAG52443 forHomo(H.) sapiens, XP 685969 forDanio (D.) rerio, and N1UOK A forBacillus (B.) cereus. Triangledesignates catalytic residue; hollow pentagram polymorphism site; solid circle methylation site. The complete consensus siteswere highlighted in black. The sites with 60% similarity were highlighted in gray.


residue E75 and inhibitor binding residues E90 andY91. The polymorphism sites, Y77, G86, G87, Q94, andF97, and the methylation sites A28, P31, P93, and T100were locatedclose toeither thecatalytic residueor theinhibitor binding residues (Fig. 2C).

Discussion

In this study, high polymorphism and DNA methy-lations at cytosines were observed in four genes, pre-sumably encoding proteins and enzymes in salivaryglands, of two biotypes of Russian wheat aphid. Bio-type 1 showed a lower polymorphism and a highermethylation level than Biotype 2 for the four genes.Some polymorphism sites and methylation sites werelocated at important positions in terms of enzymefunction. For example, the P31F of trehalase, in whichtwo cytosines in the code are methylated, was locatedin the catalytic region; the P93L of trehalase,where themethyl cytosine was detected twice, was locatedclosely to inhibitor binding sites. Whether these poly-morphism sites and methylation sites of the four pro-teins are related to the different virulence of the twobiotypes need be veriÞed in further work.

Based on the study of 1,337 bp of gene body area inRussian wheat aphid genome, we detected that 6.35 to9.52% of cytosines were methylated. This methylationlevel is much higher than in other insects, estimatedfrom the entire genome level, such as 0.17% in thehoney bee (Lyko et al. 2010), 0.11% in the silkworm

(BombyxmoriL.) (Xiang et al. 2010), and 0.69% in thepea aphid (A. pisum) (Walsh et al. 2010). This unusualhigh methylation level in Russian wheat aphid couldbe factual, considering that the measured genes wereputatively expressed in salivary glands of aphids andencode proteins in saliva; however, the limited lengthof sequence checked may not reßect the epigeneticpicture of the whole genome. Another unusual phe-nomenon in Russian wheat aphid is that two thirds ofmethyl cytosines were in a sequence context of CHH,whereas most methylations happened at CG in thehoney bee, silkworm, and pea aphid (Lyko et al. 2010,Walsh et al. 2010, Xiang et al. 2010). It is generallythought that CG methylation is maintained by Dnmt1DNA methyltransferases, and CHH methylation ismaintained by Dnmt3 methyltransferases (Goll andBestor 2005, Feng et al. 2010). Considering such a highratio of CHH methylation from these four genes, theDnmt3 methyltransferases may play a key role on themaintenance of DNA methylation in Russian wheataphid.

The GC contents of the cloned fragments of the fourgenes of Russian wheat aphid ranged between 0.35Ð0.46, similar to the pea aphid, whose genome has a GCcontent of 29.6% and whose transcripts show a higherGC content of 38.8% (International Aphid GenomicsConsortium 2010). The pea aphid, like the honey bee,exhibits a bimodal distribution of the frequency ofgenes with different ratios of CpGO/E, suggesting twobroad classes of genes with different methylation sta-

S.frugiperda L K QS GG L D F P GG VP T S L I R S GE QWD F P N VWP P E VS I V VN A I E N I G T P E AS 50P.hypochondriaca L T RNG I L D YMGG T P AS L F P T GE QWD L P N AWP P L QS I I VQ A L RNS NE E S AE 50T.molitor I T QN - MMD YHGG I P T S L S H T GE QWD YP N AWP P MQS I I VMG L DK S GS YR AK 49B.mori L DK VK MD I F E GG I P T T F E H T GE QWD YP N AWP P L Q Y I V I MG L AN T GHP E AM 50H.sapiens L E DNR I L T YQ YG I P T S L QK T GQQWD F P N AWAP L QD L V I RG L AK AP L RR AQ 50O.cuniculus L QDS Q I L NHRHG I P T S L QN T GQQWD F P N AWAP L QD L V I RG L AK S P S AR T Q 50A.franciscana L E K S N A L K F P GG VP T S L MQ T S QQWD F P NGWP P L QHML VMG L DK T GDP R AK 50E.coli - - - - - - L L QP GG L N T T S VK S GQQWD AP NGWAP L QWV A T E G L QN YGQ - - - K 41D.noxia I E AD YS V I F N - G T P T S L YNS S QQWD F P N AWP P L Q A F I I QS L DK T QQK L AQ 49

S.frugiperda V L A F E T AQ T F VRS CHWG FQE - - YK QMF E K YD AE NP GK F GGGGE Y T VQ F G F 98P.hypochondriaca K L AK E L A I RWL R ANHK G YS Q - - S GQMF E K YD A L NP GK F GGGGE Y V VQE G F 98T.molitor Q L ARE L ARRWVK AN L I G F RQ - - T GE MF E K YN VE VP GQNGGGGE Y V VQS G F 97B.mori R Y A T E L A T KWVRS N F E VWK Q - - K A AML E K YD A T I L GG L GGGGE Y V VQ T G F 98H.sapiens E V A F Q L AQNW I R T N F D V YS Q - - K S AMYE K YD VS NGGQP GGGGE YE VQE G F 98O.cuniculus E V A F Q L AQNW I R T N F D V YS Q - - R S AMYE K YD I S N - AQP GGGGE YE VQE G F 97A.franciscana E L A F D L AQRWV F NN YE A F T QS L P N AMF E K YD V T V VG L P GGGGE YD VQ L G F 100E.coli E V AMD I SWH F L T N VQH T YDR - - E K K L VE K YD VS T T G T GGGGGE YP L QDG F 89D.noxia Q V A F K L AE VWL RS N YK S F AE - - K S MMF E K YD V L AS GE I GGGGE Y T P Q T G F 97

S.frugiperda GWS NG AG L E F MK K YGE G L T ADDS 121P.hypochondriaca GWT NG V V YE F L NS YP N - A T P DDN 120T.molitor GWT NG V V L E F I NQ F - - - - - - - - - 111B.mori GWS NG V VMS L L NR YGD V I S A ADN 121H.sapiens GWT NG V V L ML L DR YGDR L T S G AK 121O.cuniculus GWT NG V A L ML L DR YGDR L S S G T Q 120A.franciscana GWT NG V VMD F L VK YGE R L T S VRD 123E.coli GWT NG V T L K ML D L I C P K E QP CDN 112D.noxia GWT NG V V F E F L NRWGD T VS NG T N 120

Fig. 4. Alignment of the deduced amino acid sequence of trehalase from Russian wheat aphid with its homologs fromother organisms. Accession numbers of these homologs in GenBank are Q0ZIF5 for Spodoptera (S.) frugiperda, Q8MMG9for Pimpla (P.) hypochondriaca, P32359 for Tenebrio (T.) molitor, Q3MV18 for Bombyx (B.) mori, O43280 for Homo (H.)sapiens, P19813 for Oryctolagus (O.) cuniculus, QBLC for Artemia (A.) franciscana, and P13482 for Escherichia (E.) coli.Triangle designates catalytic residue; hollow pentagram polymorphism site; solid circle methylation site; square inhibitorbinding site; solid pentagram methylated polymorphism site. The complete consensus sites were highlighted in black. Thesites with 60% similarity were highlighted in gray.


tus, namely low and high CpGO/E classes (Interna-tional Aphid Genomics Consortium 2010). TheCpGO/Ecutoff values are 0.82 in the pea aphid and 0.72in the honey bee (Hunt et al. 2010). As a close relativeof the pea aphid, Russian wheat aphid may show asimilar bimodal distribution of the frequency of geneswith different ratios of CpGO/E. If so, the dipeptidylcarboxypeptidas (CpGO/E � 0.56) and trehalase(CpGO/E � 0.79) may belong to a low CpGO/E class,and C002 (CpGO/E � 1.23) and sucrase (CpGO/E �0.92) may belong to a high CpGO/E class in Russianwheat aphid. Both low and high CpGO/E classes ofgenes were detected to have DNA methylation in thisstudy, implying that the methylation level of Russianwheat aphid may not be low.

Before the sodium bisulÞte treatment, two situa-tions of methylation sites were observed in the threegenes of Russian wheat aphid: 1) cytosines detected inall clones and 2) thymines detected in all clones (Ta-ble 5). Minor clones showed methyl cytosines afterthe sodium bisulÞte treatment in both the situations ofmethylation sites. We speculate that in the secondsituation the mutations from methyl cytosines to thy-mines were Þxed in the adult stage under positiveselection. Another possibility is that most of thyminesat these sites were not from methyl cytosines and theywere factual polymorphism sites.

Our work revealed DNA methylation in severalgenes that are putatively expressed in salivary glandsof Russian wheat aphid, and documented differentmethylation levels between two Russian wheat aphidbiotypes with different virulence toward hosts. TheseÞndings may provide clues to explore the evolutionarymode between Russian wheat aphid virulence andresistance genes of host plants.

Acknowledgments

We thank Professor Michael Smith of Kansas State Uni-versity for endowing Russian wheat aphid biotypes 1 and 2and Montana Atwater for the manuscript edition. This workis supported by the fund of Chinese Academy of Sciences(KSCX2-EW-N-05) and the special fund for Agro-ScientiÞcResearch in the Public Interest (201103022).

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Received 28 August 2011; accepted 7 October 2011.


Polymorphism and Methylation of Four Genes Expressed in Salivary

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