anestofltrretrotransposonsadjacentthedisease resistance ... · sequence in beta procumbenswas...

Hindawi Publishing CorporationInternational Journal of Plant GenomicsVolume 2009, Article ID 576742, 8 pagesdoi:10.1155/2009/576742

Research Article

A Nest of LTR Retrotransposons Adjacent the DiseaseResistance-Priming Gene NPR1 in Beta vulgaris L. U.S.Hybrid H20

David Kuykendall, Jonathan Shao, and Kenneth Trimmer

Molecular Plant Pathology Laboratory, ARS, USDA, Beltsville, MD 20705, USA

Correspondence should be addressed to David Kuykendall, [email protected]

Received 26 September 2008; Accepted 25 January 2009

Recommended by Cheng-Cang Wu

A nest of long terminal repeat (LTR) retrotransposons (RTRs), discovered by LTR STRUC analysis, is near core genes encoding theNPR1 disease resistance-activating factor and a heat-shock-factor-(HSF-) like protein in sugarbeet hybrid US H20. SCHULTE, a10 833 bp LTR retrotransposon, with 1372 bp LTRs that are 0.7% divergent, has two ORFs with unexpected introns but encoding areverse transcriptase with rve and Rvt2 domains similar to Ty1/copia-type retrotransposons and a hypothetical protein. SCHULTEproduced significant nucleotide BLAST alignments with repeat DNA elements from all four families of plants represented in theTIGR plant repeat database (PRD); the best nucleotide sequence alignment was to ToRTL1 in Lycopersicon esculentum. A secondsugarbeet LTR retrotransposon, SCHMIDT, 11 565 bp in length, has 2561 bp LTRs that share 100% identity with each other andshare 98-99% nucleotide sequence identity over 10% of their length with DRVs, a family of highly repetitive, relatively small DNAsequences that are widely dispersed over the sugarbeet genome. SCHMIDT encodes a complete gypsy-like polyprotein in a singleORF. Analysis using LTR STRUC of an in silico deletion of both of the above two LTR retrotransposons found that SCHULTE andSCHMIDT had inserted within an older LTR retrotransposon, resulting in a nest that is only about 10 Kb upstream of NPR1 insugarbeet hybrid US H20.

Copyright © 2009 David Kuykendall et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

1. Introduction

Retrotransposons are now recognized as movers and shapersof plant genome evolution (see reviews [1, 2]). Thatretrotransposon elements account for much of the sugarbeet(Beta vulgaris L.) genome was shown by the identification[3] of repetitive DNA sequences in Beta vulgaris similarto long interspersed nuclear elements (LINEs), a typeof retrotransposon without long terminal repeats (LTRs),and other repetitive DNA sequences that resembled LTRretrotransposons of the Ty1-copia class. A repeated DNAsequence in Beta procumbens was described as “Athila-like”[4] since it was deduced to be part of a long terminalrepeat with similarity to the Athila retrotransposon fromArabidopsis.

Prior to the present study, pDRV sequences [5] wereknown simply as a family of short highly amplified DNArepeats shown by fluorescent in situ hybridization (FISH)

technique to be widely dispersed over all 18 chromosomesof sugarbeet.

Vulmar1, a mariner-class DNA transposon in Beta vul-garis [6], is 3909 bp, has 32 bp terminal inverted repeats, andencodes, in a single ORF, a transposase with a characteristic“DDE” signature motif. Polymerase chain reaction (PCR)and fluorescent in situ hybridization (FISH) were used [6]to identify and to establish an abundance of En/Spm-liketransposons in sugarbeet.

Coe1, a DNA transposon within apparent LTRs and otherretrotransposon-like features, was discovered on a sugarbeetgenomic BAC carrying the NPR1 disease resistance-priminggene [7–9]. This recent discovery in Beta vulgaris of a unique16.3 Kb CACTA En/Spm-like transposon named Coe1 [7]was followed by the finding of conserved microsynteny ofNPR1 with another core plant gene whose predicted producthas high similarity to a DNA-binding HSF protein [8].About 70 Kb of repetitive DNA separates the HSF gene and

2 International Journal of Plant Genomics

NPR1 from another small core gene cluster with a CaMPgene specifying a signal peptide calmodulin-binding proteinand a gene encoding a CK1-class protein kinase gene [8],greatly extending and disambiguating the results of the initialsequencing and partial in silico analysis of an NPR1 gene-carrying sugarbeet BAC [9]. In summary, our laboratoryhas identified, sequenced, and annotated a bacterial artificialchromosome (BAC) carrying the NPR1 disease resistancepriming gene of sugarbeet, Beta vulgaris L. [7–9].

Class I transposable elements which use reverse tran-scriptase to transpose via an RNA intermediate are termedretrotransposons. In order to identify possible LTR retro-transposons with LTRs, an intergenic region of repetitiveDNA was examined by LTR STRUC analysis, and this reportdetails the discovery of a nest of retrotransposons about10 Kb upstream from the NPR1 disease resistance gene insugarbeet H20. This nest appears to have formed when botha copia-type and a gypsy-type elements inserted within anolder LTR retrotransposon. Two full-length sugarbeet LTRretrotransposons are described herein for the first time.

2. Materials and Methods

Identification of a sugarbeet BAC carrying the NPR1 diseaseresistance control gene was described [9]. Genbank acces-sion DQ851167 represents a partial sequence; the 38.6 Kbsegment was the largest contig at that time. Subsequentlythe entire 130 Kb contiguous fragment was sequenced andannotated (Genbank accession EF101866). Basic methodsused for DNA sequence analysis were described [9], and con-struction of the BAC library was detailed [10]. In the presentstudy, LTR analyses of the NPR1 BAC were performed usingLTR STRUC [11], and LTR Finder [12]. Programs, einverted[13] (http://bioweb.pasteur.fr/seqanal/interfaces/einverted),and EMBOSS (http://emboss.sourceforge.net/) [13] wereused to identify inverted repeats, and repeats were also foundusing NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/).An EST database for sugarbeet (http://genomics.msu.edu/sugarbeet/blast.html) was employed for both nucleotideand protein BLAST to explore possible functional geneexpression [14]. Subsequent analysis of DNA sequencedata was performed using Lasergene version 6 (DNASTAR,Madison, Wis, USA). BLAST was used to identify the mostsimilar protein products of LTR retrotransposons in otherplant species. Multiple alignments were performed usingMegAlign from the DNASTAR suite. Neighbor joining tree,or cluster analysis, was performed using MEGA 4 software(http://www.megasoftware.net/).

3. Results and Discussion

A genomic NPR1 disease resistance priming gene-carryingBAC [7–9] was subjected to LTR STRUC and LTR FINDERanalyses, and two distinct full-length LTR retrotransposonswere identified (Figure 1). Depicted are RTR1 and RTR2,two LTR retrotransposons that we also term SCHULTE andSCHMIDT, respectively, as well as a previously describedelement, Coe1, a DNA transposase gene within apparent

LTRs and other retrotransposon-like features [7]. Theserepetitive DNA elements are intergenic, between two smallclusters of core genes: HSF and NPR1 genes separatedfrom CaMP and CK1PK genes encoding a signal peptidecalmodulin-binding protein and a “casein kinase 1-classprotein kinase,” respectively.

SCHULTE, a 10 833 bp long LTR retrotransposon, has1372 bp LTRs sharing only 99.3% nucleotide sequenceidentity. The 0.7% divergence in the LTRs of SCHULTEindicates about ten base substitutions occurred since inser-tion/transposition. This old, somewhat degraded retro-transposon has two ORFs encoding a Ty1/copia-like inte-grase/reverse transcriptase and a hypothetical protein(Figure 2). Unexpected introns, uncharacteristic of retro-transposon genes, may be the result of frameshifts and pointmutations. SCHULTE has 98% nucleotide sequence identityover ≥9 Kb with a 9.7 Kb DNA fragment (DQ374026)and 1.3 Kb of a 5.3 Kb DNA fragment (DQ374025), eachfragment of BAC62 [14]. BAC62 carries a Beta vulgaris L.genomic region adjacent a Beta procumbens translocationcarrying a nematode resistance gene [15], thus BAC62 hasa SCHULTE-like retrotransposon.

Named to honor an author of the first-described physicalmap of the afore-mentioned region, SCHULTE is the firstfull-length retrotransposon sequence from Beta vulgaris tobe reported. Since two out of the three B. vulgaris BACssequenced to date, BAC62 and the NPR1-carrying BAC, carrya SCHULTE-like element, there are likely a very large numberof SCHULTE-like LTR retrotransposons in the sugarbeetgenome. However, FLC, or the flowering control gene-carrying BAC [16], did not carry a SCHULTE-like element.

SMART analysis showed that the predicted product ofthe SCHULTE reverse transcriptase gene has rve and Rvt2protein domains. An alignment by MegAlign of the con-served rve and Rvt2 (Figure 3) domains of similar Ty1-copia-like plant retrotransposon-encoded proteins, identified byBLAST, were analyzed by neighbor joining in MEGA 4 toassess structural relatedness (Figure 4). As shown in Figure 3,the predicted product of the Beta vulgaris SCHULTE reversetranscriptase gene has conserved rve and Rvt2 domainsshared among highly similar domains of products of LTRretrotransposons from Medicago truncatula, Vitis vinifera,Oryza sativa japonica, Zea mays, and Glycine max. Exceptfor the Solanum demissum and Vitis vinifera accessions, theRvt2 domain evidently has a conserved YVDDIIF active site(Figure 3).

Similar LTR retrotransposon gene products in Ara-bidopsis thaliana, Solanum demissum, and particularly inPhaseolus vulgaris are structurally divergent (Figures 3 and4). A search of the TIGR plant repeat database revealedthat SCHULTE produced nucleotide sequence matches withmany different copia-like retrotransposons in all four fam-ilies: Brassicaceae, Fabaceae, Gramineae, and Solanaceae.The best PRD nucleotide sequence alignment match (E =3.8e−232) was to ToRTL1 in Lycopersicon esculentum.

Probable expression of integrase/reverse transcriptasegene(s) in active SCHULTE-like retrotransposon was shownby BLAST alignment (E = 0.0) of the ORF with BI643218,an EST, or expressed sequence tag. Expression of both LTRs

International Journal of Plant Genomics 3

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Hp6

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CK1-class

Transposon

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Figure 1: BAC physical map showing two LTR retrotransposons SCHULTE and SCHMIDT both highlighted in green and with darker greenLTRs with rounded corners. On the other hand, the previously reported CACTA transposon Coe1, highlighted in light beige, has short tanLTRs, a yellow DNA transposase gene and orange ORF1 and ORF2. Exons are rectangles, and direction of arrows indicates direction oftranscription. Exons of core plant genes are blue, and exons of hypothetical protein genes are red. Solid lines between exons depict introns.Scale is in 2 Kb increments. BAC is about 130 kilobase pairs. SCHULTE has an integrase gene in orange and a hypothetical gene in red.SCHMIDT has a single ORF retroelement reverse transcriptase polyprotein gene in orange.

was clearly evidenced by alignments, E = 0.0, with ESTsBI698297 and BI698341. Four other ESTs showing somealignment (E = e−151 to E = 9e−36) also suggest other likelyactive SCHULTE-like elements.

Another LTR retrotransposon discovered usingLTR STRUC and LTR FINDER, SCHMIDT was so namedto honor a pioneering researcher of repeat DNA elementsin Beta. SCHMIDT, 11 565 bp retroelement, encodes acomplete Ty3-gypsy-class polyprotein in a single ORF

without introns. The SCHMIDT reverse transcriptase genehas all of the domains expected of an intact retroelementpolyprotein, and the domain order is indicative of Ty3-gypsy-class. SCHMIDT has 2561 bp LTR sequences with100% identity, consistent with this transposable element stillbeing active.

EMBOSS analysis of the 130 Kb NPR1-carrying sugar-beet BAC revealed the presence of at least 24 inverted repeat(IR) sequences but, for the purposes of this report, let us


26 Kb 28 Kb 30 Kb 32 Kb 34 Kb 36 Kb

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Figure 2: A detailed schematic of Beta vulgaris retrotransposons SCHULTE and SCHMIDT in red. Various numbered inverted repeat (IR)sequences in either light or dark blue or violet have arrows indicating relative direction. Green lines show the location of the DNA sequencemotif CACTATAA. Heavily dotted ovular regions depict the size and position of the LTRs. The lightly dotted region shows the size of thewhole retroelement. A green box shows the location of the polymerase binding site. Boxes show the position and size of exons. Lines betweenexons indicate introns. A centrally located small yellow box depicts the active site of the retroelement. An orange box shows the location ofthe polypurine tract. Scale is in Kb and is located underneath the illustrations in increments of 2 Kb per tick. Names of putative genes arelocated above the illustrations.

describe only those inverted sequences associated with LTRretrotransposons SCHULTE and SCHMIDT. The followingtwo pairs of IR 8/21 inverted repeat sequences are associatedwith SCHULTE (Figure 2). The IR 8/21 inverted repeatsequences share 94% identity (18/19).

35690 cttagtttgtacctttgtt 35708| | | | | | | | | | | | | | | | | |

35781 gaatcaaacaaggaaacaa 35763

26302 aacaaaggaacaaactaag 26320| | | | | | | | | | | | | | | | | |

35708 ttgtttccatgtttgattc 35690

The following pair of IR 22 inverted sequences, associatedwith SCHULTE (Figure 2), are 9.5 Kb apart and share 80%identity (shown below), but this pair of IR 22 are also directrepeats with 96% identity.

26751−> 26786aaaaagaaaatctgttttggaaaagattttattttt

| | | | | | | | | | | | | | | | | | | | | | | | | | | | |tttttattttagaaaagattttgtctaaaagaaaaa

36247


S T TK P L E L I H I DL CG PMR I QS R S GK R Y V L V I VDDY S R Y TWV I F L S S KDE T YDE F L V F AK R VQNL S GHK I MH I R S DHGK E F E NY K F DDL S RDNG L DHNF S A P R T P QQNGVV E R KNR T L E EM Beta vulgaris ABM55238 1S T S R P L E L L HMDL F G P S R T P S L GGK S Y A Y V I VDDF S R Y TWV L F L S QK S E A F Y E F S K F CNKVQNE KG F S I TC I R S DHGR E F E NF DF E E Y CNKHG I NHNF S A P R T P QQNGVV E R KNR T L QEM Vitis vinifera CAN74951 1S T S R P L E L L H I DL F G P VNT A S L Y G S K Y G L V I VDDY S RWTWVK F I K S KDY AC E V F S S F C TQ I QS E K E L K I L KVR S DHGG E F E NE P F E L F C E KHG I L HE F S S P R T P QQNGVV E R KNR T L QEM Medicago truncatula ABD32582 1MTDA P GQL L HMDTVG P AKVQS I GAKWY V L V I VDDF S R Y SWV F FMA TKDE A F KHF R G L F L QL E L E F P G S L K R I R S DNGG E F KNA S F E QF CNE R G L EHE F S S P R V P QQNGVV E R KNCV F V EM Oryza sativa japonica AAR873351T TDR P L E L L HMDL F G P I A Y I S I GG S K Y C L V I VDDY S R F TWV F F L QE K S QTQE T L KG F L R R AQNE F G L R I KK I R S DNGT E F KN S Q I E S F L E E E G I KHE F S S P Y T P QQNGVV E R KNR T L L DM Zea mays ABF67921 1T T S R V L E L L HMDLMG PMQV E S L GGK R Y A Y VVVDDF S R F TWVK F I R E K S E T F E V F K E L S L R L QR E KDCV I K R I R S DHGR E F E N S R L T E F C T S E G I THE F S AA I T P QQNG I V E R KNR T L QE A Glycine max AAO73521 1R AQK P L E L I HTDVCG P I K P K S L GK S NY F L L F I DDF S R K TWV Y F L K E K S E V F E I F KK F K AHV E K E S G L V I K TMR S DR GG E F T S K E F L K Y C E DNG I R RQL TV P R S P QQNGVA E R KNR T I L EM Arabidopsis thaliana AAG50698 1R ANQK L QL I HTDVCG P I K TD S L S GNK Y F L L F I DDY T RMCWV Y F I R L K S E V F DV F KQF K A L V E NQCNL R I K A L R S DNGG EHT S F QF V E F CN S TC I E CQL T L P Y T P QQNGV S E R KNR TVMEM Solanum demissum AAT38797 1KCDK I L G L I HTDL ADL KQTMS R GGKNY F V T F I DDF S R Y TKV Y L I KHKDE A F DMF L T Y K A E V E NQL NKK I K R I R S DR GG E Y V - - L F NDY CVK E G I I HE V T P P Y S P E S NGVA E R KNR T L K EM Phaseolus vulgaris AAR13298 1

S GTML I A S E L P RNFWA E AVNT ACH I I NR AMMR P I I NK T P Y E L Y F Beta vulgaris ABM55238 121AR TML NE NNL P K Y FWA E AVNT S C Y V L NR I L L R P I L KK T P Y E LWK Vitis vinifera CAN74951 121AR TMI HE NNL AKHFWA E AVNT S C Y I QNR I Y I R PML E K T A Y E L F K Medicago truncatula ABD32582 121AR TML DE Y K T P R K FWT E A I NT AC Y I L NR V F L R S K L GK T S Y E L R F Oryza sativa japonica AAR87335121AR TML DE Y K T P DR FWA E AVNT AC Y A I NR L Y L HR I L KK T S Y E L L T Zea mays ABF67921 121A R VML HAK E L P YNLWA E AMNT AC Y I HNR V T L R R GT P T T L Y E I WK Glycine max AAO73521 121AR SML K S K R L P K E LWA E AVAC AV Y L L NR S P TK S V S GK T P QE AWS Arabidopsis thaliana AAG50698 121A R C L L L E R K I P NQF L A E A I NT S V Y L L NR L P T K A L QDMT P Y E AWC Solanum demissum AAT38797 121MNAML I S S NA P DNLWG E S L L T AC F L QNR I PHR - K TGK T P Y E LWK Phaseolus vulgaris AAR13298 119

- - - - - - - - - - - - - - WR L QK S HP V E L I I S - - - - - - - - - - - - - D I S K - - - - - - - - - - - - - - - - - - - - - - - - - AR L E A I R I L I A F AA YMG F K L YQMDVKC A F L NGY L NE DV Y V E QP P G F E NNN Beta vulgaris ABM55238 1- S T I HK I K S L KCK Y G E L V P R P S NQS V I GTKWV F RNKMDE NG I I VRNK AR L VAQGYNQE E G I DY E E T F A P VAR L E A I RML L A F AC F KDF I L YQMDVK S A F L NG F I NE E V Y V E QP P G F QS F N Vitis vinifera CAN74951 1AMQE E L NQF QRNDVWDL V P K P S QKN I I GTKWV F RNK L NE QG E V T RNK AR L VAQGY S QQE G I DY T E T F A P VAR L E A I R L L L S Y A I NHG I I L YQMDVK S A F L NGV I E E E V Y VKQP P G F E DL K Medicago truncatula ABD32582 1AMHE E L E NF E RNKVWT L V E P P F GHN I I GTKWV F KNKQNE DG L I VRNK AR L VAQS F TQV E G L DF DE T F AHVAR I E A I K L L L - - - - - - - - - - - - - - - - - A F L NG F I QE E V Y VKQS P G F E NP D Oryza sativa japonica AAR873351AMQE E L NNF T RNE VWHL V P R - P NQNVVGTKWV F RNKQDEHGVV T RNK AR L VAKGY S QV E G L DF G E T Y A P VAR L E S I R I L L A Y A T YHG F K L YQMDVK S A F L NG P I K E E V Y V E QP P G F E D S E Zea mays ABF67921 1AMQE E L E QF K RNE VWE L V P R P E GTNV I GTKWI F KNK TNE E GV I T RNK AR L VAQGY TQ I E GVDF DE T F A P VAR L E S I R L L L GVAC I L K F K L YQMDVK S A F L NGY L NE E V Y V E QP KG F ADP T Glycine max AAO73521 1AMDE E I K S I QKNDTWE L T S L P NGHK A I GVKWV Y K AKKN S KG E V E R Y K AR L VAKGY S QR AG I DYDE V F A P VAR L E TVR L I I S L AAQNKWK I HQMDVK S A F L NGDL E E E V Y I E QP QGY I VKG Arabidopsis thaliana AAG50698 1AMQDE L DV I KKNGTWQL VDR P - - - - - - - - - - - - - - - RNCK - - - - - - - - - - - - - - - - - - - - - - - - E T F A P VAR YDT I K L I L A F A S HS SWQ I HQL DVK S A F L N S L L A E E I Y V E QP DG F S I P G Solanum demissum AAT38797 1A I K T E L E S I KKNNTWT L VDL P KGAK P I GCKWI F KKK YHP DG S I E K Y K AR L VAKG F TQKHN I DY F DT F A P V T R I S S I R V L L A L A S I HK L V I HQMDVK T T F L NG E L E E E I YMTQP E GCVV L G Phaseolus vulgaris AAR13298 1

L P NHV Y K L DK A L Y GL KQA P R SWY E R L S K F L L E NNF K R GKVDK T L F L K S KGTD I L L VQ I Y VDD I I F GA TNE T L CK E F S R L V S NE F EMSMMG E L NF F L G L Q I KQT E KG I I VHQQK Y I K E L L K Beta vulgaris ABM55238 69F P NHV F K L KK A L Y GL KQA P R AWY E R L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N - - - - F S KCMHS E F EMSMMG E L NY F L G L Q I KQL K E GT F I NQAK Y I KDL L K Vitis vinifera CAN74951 120HP DHV Y K L KK S L Y GL KQA P R AWYDR L S NF L I KNDF E R GQVDT T L F R R T L KKD I L I VQ I Y VDD I I F G S TNA S L CK E F S K LMQDE F EMSMMG E L K F F L G I Q I NQS K E GV Y VHQTK Y TK E L L K Medicago truncatula ABD32582 121F P NHV F K L S K A L Y GL KQA P R AWYDR L KNF L L AKGF TMGKVDK T L F V L KHGDNQL F VK I YMDD I I F CC S THA L VVDF A E NMR R E F EMSMMG E L S Y F L G L Q I KQT P QGT F VHQTK Y TNDL L R Oryza sativa japonica AAR87335104Y P NHV Y R L S K A L Y GL KQA P R AWY E C L RDF L I ANGF KVGK ADP T L F TK T L E NDL F VCQ I Y VDD I I F G S TNE S TC E E F S R I MTQK F EMSMMG E L K Y F L G F QVKQL QE GT F I S QTK Y TQD I L A Zea mays ABF67921 120HP DHV Y R L KK A L Y GL KQA P R AWY E R L T E F L TQQGY R KGG I DK T L F VKQDA E NLMI AQ I Y VDD I V F GGMS NEML RHF VQQMQS E F EMS L VG E L T Y F L G L QVKQME D S I F L S QS R Y AKN I VK Glycine max AAO73521 121E E DKV L R L KK A L Y GL KQA P R AWNT R I DK Y F K E KDF I KC P Y EHA L Y I K I QK E D I L I AC L Y VDDL I F TGNNP SMF E E F KK EMTK E F EMTD I G LMS Y Y L G I E VKQE DNG I F I TQE GY AK E V L K Arabidopsis thaliana AAG50698 121K E DQV Y L L TK A L Y GL KQS P R AWY E RMDNHL I QL GF S R S QS E A T L Y VKV T - - - - - - - - - - - - - - - - AG S K I E L I QR F KDEME K I F EMTDL GVMK Y F L GME V L QS S DG I F I CQQK Y I L D I L N Solanum demissum AAT38797 82QK E KVCK L L K S L Y GL KQA P KQWHE K L DNV L L C E GF S TNDADKCV Y S R S E NG E Y V I I C L Y VDDML I F GTCND I V F K TK L F L G S K F EMKDMG E A S V I L GVK I I R KGD S I L L S QE K Y T E K L L K Phaseolus vulgaris AAR13298 121

K Y G L E N S K I NHT PMGT S Beta vulgaris ABM55238 189R F NME E AKVMK T PMS S S Vitis vinifera CAN74951 192K F K L E DCKVMNT PMHP T Medicago truncatula ABD32582 241R F KME NCK P I S T P I G S T Oryza sativa japonica AAR87335224K F GMKDAK P I K T PMGTN Zea mays ABF67921 240K F GME NA S HK R T P A P TH Glycine max AAO73521 241K F KMDD S NP VC T PME CG Arabidopsis thaliana AAG50698 241R F KMQDCK P V S T P I S TG Solanum demissum AAT38797 186K F GY YDF K S V S T P YDAN Phaseolus vulgaris AAR13298 241

Figure 3: Amino acid residue alignment of SCHULTE’s integrase (top) and reverse transcriptase (bottom) domains. The active site ofthe RT domain from Beta vulgaris is boxed in along with corresponding sections of other reverse transcriptase proteins from differentplants. Amino acids matching the consensus sequence are shaded. Numbers indicate cumulative amino acid positions; Arabidopsis thaliana(AAG50698), Beta vulgaris (ABM55238), Glycine max (AAO73521), Medicago truncatula (ABD32582), Oryza sativa japonica (AAR87335),Phaseolus vulgaris (AAR13298), Solanum demissum (AAT38797), Vitis vinifera (CAN74951), Zea mays (ABF67921).

Beta vulgaris ABM55238

Vitis vinifera CAN74951 Medicago truncatula ABD32582

Oryza sativa japonica AAR87335 Zea mays ABF67921

Glycine max AAO73521 Arabidopsis thaliana AAG50698

Solanum demissum AAT38797 Phaseolus vulgaris AAR13298

99 84

88 100

74

98

0.1

Ajellomyces capsulatus NAml XP_001543176

Figure 4: Similarity tree constructed by neighbor joining method of the reverse transcriptase domain of SCHULTE from Beta vulgarisalong with corresponding domains of other reverse transcriptase proteins from different plants. The numbers on the branches are bootstrap(confidence) values. Genbank accession numbers of amino acid sequences are given following plant names.

sequence identity which matches primarily with Prem1-and Xilon1-like gypsy-like RTRs in Zea, Oryza, Sorghum,and Triticum. This finding suggests divergent evolution,where a SCHMIDT-like ancestor originated in monocots,then, upon lateral transfer to sugarbeet, natural selectionfor structural similarity or convergence in a new geneticbackground resulted in a high degree of amino acidsimilarity of the protein product with other gypsy-likeretrotransposons in eudicots. The predictions of conver-gent evolution are structurally similar proteins encoded byphylogenetically distinct retrotransposons. Whether similar

sequences arose through convergent or divergent evolu-tion, it is interesting to simply note that SCHMIDT hasa significant degree of nucleotide sequence identity pri-marily with certain gypsy-like retrotransposons found inmonocots.

Expression of retroelements similar to SCHMIDT insugarbeets is suggested by the finding that SCHMIDTgave BLAST alignments with the following ESTs: BI643170,BI643158, BI698360, and BI643246 (E = 10−161, 3E = 10−79,E = 10−68, and 5E = 10−59, resp.). These four BLAST hitsrepresent only about 0.02% of the ESTs in the collection.


1 - - - - - - - - - - - S PWGA P V L - - - - - - F VK K KDG S - MR L C I DY R E L NNV T I KNK Y P L P R I DD L F DQL NGA S V F S K I D L R S GYHQL R V ADKDV P K T A F R T R Y GHY E F T VMP F G L TNA P A I FMD Beta vulgaris ABM55240 1 - - - - - - - - - - - S PWGA P V I - - - - - - F V E K KDHT - QRMC VDY R A L NE V T I KNK Y P L P R I DD L F DQL E GA T V F S K I D L R S GYHQL R I R E E D I P K T A F T T R Y G L F E C T VMS F G L TNA P A F FMN Oryza sativa japonica ABA922331 - - - - - - - - - - - S PWGA P V I - - - - - - F VDK KDG S - QRMC VDY R S L NE V T I KNK Y P L P R I DD L F DQL R GAC V F S K I D L R S GYHQL K I RN S D I P K T A F T T R Y G L Y E Y T VMS F G L TNA P A Y FMY Sorghum bicolor AAD22153 1 - - - - - - - - - - - S PWGA P V L - - - - - - F VNK KDG S - R RMC VDY R S L NE V T I KNK Y P L P R I E D L F DQMK GAK V F S K I D L R S GYHQL K I R A E DV P K T A F T T R Y G L Y E F L VMS F G L TNA P A Y FMN Zea mays AAL59229 1 - - - - - - - - - - - S P F S L P I L - - - - - - L VK K KDG S - WR F C TDY R A L NA I T VKD S F PMP T VD E L L D E L HGAQY F S K L D L R S GYHQ I L VQP E DR E K T A F R THHGHY EWL VMP F G L TNA P A T F QC Glycine max AAO23078 1 - - - - - - - - - - - S P F S S P V I - - - - - - L VK K S DG S - WRMC VDY R A L NK V T I KDK F P I P VVD E L L D E L NGAK L F S K L D L R S GYHQ I KMHANDV S K T A F R THE GQY E F L VMP L V L TNA P A T F Q S Primula vulgaris ABD78322 1 - - - - - - - - - - - S P C GV P A L - - - - - - L T P K KDG S - WRMC VD S R A I NK I T I K Y R F P I P R L DDML DMMVG S V I F S K I D L R S GYHQ I R I R P GD EWK T S F K T KDG L Y EWL VMP F G L TNA P S T FMR Vitis vinifera CAN79321 1 - - - - - - - - - - - S P C S V P V L - - - - - - L V P K KDGT - WRMC VDC R A I NK I T VK Y RHP I P R L DDML DQL C G S K I F S K I D L K S GYHQ I R L NP GD EWK T A F K T K Y G L Y EWL VMP F G L TNA P S T FMR Solanum demissum AAW28577 1 - - - - - - - - - - - S P C AV P I I - - - - - - L V P K KDGT - S RMC VDC R G I NN I T I R Y RHP I P R L DDML D E L S G S I I F S K VD L R S GYHQ I RMK L GD EWK T A F K T K F G L Y EWL VMP F G L TNA P S T FMR Hordeum vulgare AAK94517 1 - - - - - - - - - - - S P C AV P V I - - - - - - L V P K KDGT - WRMC VDC R A I NN I T I R Y RHP I P R L DDML D E L S GA I V F S K VD L R S GYHQ I RMK L GD EWK T A F K T K F G L Y EWL VMP F G L TNA P S T FMR Zea mays AAM94350 1 - - - - - - - - - V P K K S G I T V I QNE ANE L I P T R I QT GWR VC I DY R K L NL A T R KDHF P L P F I DQML E R L AGHE F Y C F L DGY S GY NQ I P I A P E DQE K T T F T C P Y GT F A Y R RMP F G L CNA P A T F QR Asparagus officinalis ABD631421 S TWV S P VHY V P K K GGMT VVKN S KD E L I P T R T T T GHRMC I DY R K L NA A S R KDHF P L P F I DQML E R L ANHP Y Y C F L DGY S G F F Q I P I HP NDQE K T T F T C P Y GT F A Y K RMP F G L CNA P A T F QR Arabidopsis thaliana BAB10790

103 LMNR I F HE F L DK F VVV F I DD I L I Y S RNE T E HD E HL R I I L E T L R KNQL Y AK F S K C E F R L E K V A F L GHF V Beta vulgaris ABM55240 103 LMNK V FME Y L DK F VVV F I DD I L I Y S K T K E E HE E HL R L A L E K L R E HQL Y AK F S K C E FWL S E VK F L GHV I Oryza sativa japonica ABA92233103 MMNK V FME Y L DK F VVV F I DD I L V F S K T K E E HA E HL R L V L QK L R E HK L Y AK R S K C E FWL E E V S F L GHVV Sorghum bicolor AAD22153 103 LMNK V FME Y L DQF VVV F I DD I L I Y S S NE E AHE DHL R L V L QK L RDNQL Y AK F S K CD FWL K E V A F L GH I V Zea mays AAL59229 103 LMNK I F QF A L R K F V L V F F DD I L I Y S A SWKDHL KHL E S V L QT L KQHQL F A R L S K C S F GDT E VDY L GHK V Glycine max AAO23078 103 AMN S V F K P F L E NL C L F F F DD I L V Y S K TND E H I CHL E AV L K KMS E HK F F AK S S K C K F F QK E I DY L GHL I Primula vulgaris ABD78322 103 I MTQV L K P F I GR F VVV Y F DD I L I Y S R S C E DHE E HL KQVMR T L R A E K F Y I NL K K C T FMS P S VV F L G F VV Vitis vinifera CAN79321 103 LMNHV F KD F HGK F I VV Y F DD I L I F S QNL D E HL E HL K K V F E V L RNQR L F ANL K K C T F C VDR VV F L G F VV Solanum demissum AAW28577 103 LMNE V L R A F I GR F VVV Y F DD I L I Y S R S L E DHL DHL R AV F T A L RDA R L F GNL GK C T F C TDR V S F L GY VV Hordeum vulgare AAK94517 103 LMNE V L R A F I GK F VVV Y F DD I L I Y S K S MD E HVDHMR AV F NA L RDA R L F GNL E K C T F C TDR V S F L GY VV Zea mays AAM94350 112 CM I S I F S DMV E R F L E I FMDD F S I F GDT F S QC L HHL K L V L E R C R E KNL T L NWE K CHFMVKQG I V L GHVV Asparagus officinalis ABD63142121 CMT S I F S D L I E EMV E V FMDD F S V Y G S S F S S C L L NL C R V L K R C E E TNL V L NWE K CHFMV R E G I V L GHK I Arabidopsis thaliana BAB10790

1 V P EWKWD S I S MD F V T S L P NT P R GNDA I WV I VDR L T K S AHF L P I N I S F P V AQL A E I Y I K E I VK L HGV P S S I V S DRD P R F T S R FWK S L QE A L G S K L R L S S A YHP QTDGQ S E R T I Q S L E D L L R Medicago truncatula ABO81153 1 I P EWKWD S I S MD F I T G L P K T R R KND S I WV I VDR L T K S AHF L P V R T T Y K VDQL T E I Y I A E I V R L HGV P S S I V S DRD P K F T S HFWGA L HE A L GT K L R L S S A YHP QTDGQT E R TNQ S L E D L L R Cicer arientinum CAC44142 1 V P GWKWD S I S MD F V T A L P K S R S GNDT I WV I VDR L T K S AV F I P I K E TWK K KQL A T T Y I KHVV R L HGV P KD I I S DRD S R F L S K FWK K VQAN L GT T L KMS T A F HP A TDGQT E R TNQTME DML R Beta vulgaris ABM55240 1 I P EWKWE E I GMD F I T G L P R T S AGHD S I WVVVDR L T K V AHF I P VK T T Y T GNK L A E L YMA R VVC L HGV P K K I V S DR G S QF T S K FWQK L QL EMGT R L NF S T A YHP QTDGQT E R I NQ I L E DML R Oryza sativa japonica ABA922331 I P EWKWE E VGMD F I VG L P R TQR GY D S I WV I VDR L T K V AHF I P VK T S Y S GDR L A E L YME R I VC L HGV P K K I V S DR GTQF T S HFWK AVHD S L GT K L NF S T A YHP QTDGQT E R I NQ I L E DML R Sorghum bicolor AAD22153 1 V P EWKWE E I S MD F I VG L P R T RDGY D S I WV I VDR L T K V AHF I P VK T T Y S GAQL A E L YMS R I VC L HGV P K K I V S DR GTQF T S R FWK R L HE S MDT K L NF S S A YHP QTDGQT E R TNQV L E DML R Zea mays AAL59229 1 I P QQVWE DV AMD F I T G L P N S - F G L S V I MVV I DR L T K Y AHF I P L K ADY N S K VV A E A FMS H I VK L HG I P R S I V S DRDR V F T S T FWQHL F K L QGT T L AMS S A YHP Q S DGQ S E V L NK C L EMY L R Glycine max AAO23078 1 MP E QTWS E I S MD F I NG L P T S - KNY NC I WVVVDR L T K Y AHF I P L KHP F GAK E L ANE F L QN I F K L HG L P K K I I S DRDT I F T S D FWK E L F HL L GT K L L L S T A F HP QTDGQT E I VNK S L E T Y L R Primula vulgaris ABD78322 1 V P S K PWE D L S MD F V L G L P R TQR G F D S I F VVVDR F S KMAHF I P C K K A S DA S Y V A A L F F K E VV R L HG L P Q S I V S DRD - - - - - - - - - - - - - K L - - - - - - - S - - - - - - - - - - - - - NR S L GNL L R Vitis vinifera CAN79321 1 V S NF PWI D I S MD F I L G L P R T K Y GKD S I F VVVDR F S KMA R F I P C K K TNDA S HV AD L F VK E VVK L HG I P R T I V S DRDAK F L S HFWR I LWGK L GT K L L F S T S CHP QTDGQT E VVNR T L GNML R Solanum demissum AAW28577 1 V P S V PWE D I S MD F V L G L P R T K K GRD S I F VVVDR F S KMAHF I P CHK S DDA ANV AD L F F R E I I R L HGV P NT I V S DRDAK F L S HFWR C LWAK L GT K L L F S T T CHP QTDGQT E VVNR S L S TML R Hordeum vulgare AAK94517 1 V P S A PWE D I S MD F V L G L P R T R K GRD S V F VVVDR F S KMAHF I P CHK TDDA TH I AD L F F R E I V R L HGV P NT I V S DRDAK F L S HFWR T LWAK L GT K L L F S T T CHP QTDGQT E VVNR T L S TML R Zea mays AAM94350 1 L S V E L F D LWG I D FMG P F P N S - F GNV Y I L V AV E YMS KWV E AV AC K TN - DNK VVVK F L K E N I F A R F GV P R A I I S DNGTHF CNR S F E A LMR K Y S I THK L S T P YHP QT S GQV E V TNRQ I KQ I L E Asparagus officinalis ABD631421 L E V E I F DVWG I D FMG P F P S S - Y GNK Y I L V AVDY V S KWV E A I A S P TN - DA R VV L K L F K T I I F P R F GV P R I M I S DGGKHF I NK V F E NL L K KHGVKHK V A T P YHP QT S GQV E I S NR E I K A I L E Arabidopsis thaliana BAB10790

121 I C V L E QGGTWD S HL P L I E F T Y NN S YH S S I GMA P F E A L Y Medicago truncatula ABO81153 121 AC V L DDR G SWDHV L P L I E F T Y NN S F HT S I GMA P Y QA L Y Cicer arientinum CAC44142 121 AC A I D F QG SWE DQL D L I E F S Y NN S YHA S I KMA P F E A L Y Beta vulgaris ABM55240 121 AC V L D F GG SWDKNL P Y A E F S Y NN S Y QA S L QMA P Y E A L Y Oryza sativa japonica ABA92233121 AC A L QY GT SWDK S L P Y A E F S Y NN S Y QQ S L KMA P F E A L Y Sorghum bicolor AAD22153 121 AC A L KHGR SWDK S L P Y A E F S Y NN S Y QA S L KMA P F E A L Y Zea mays AAL59229 120 C F T Y E HP K GWVK A L PWA E FWY NT A YHMS L GMT P F R A L Y Glycine max AAO23078 120 C Y T S QY P KNWAKWI Y L A E FWY N S T THT S I KMP P F K A L Y Primula vulgaris ABD78322 88 C I V RDQL R KWDNX L P QA E F A F N S S TNR T T GY S P F E V A Y Vitis vinifera CAN79321

121 A I L K GK L T SWE DY L P I V E F A Y NR T F H S S T GK T P F E VV Y Solanum demissum AAW28577 121 AV L K TNL K LWE E C L P H I E F A Y NR S L H S T T KMC P F E I V Y Hordeum vulgare AAK94517 121 AV L K KN I KMWE DC L P H I E F A Y NR S L H S T T KMC P F Q I V Y Zea mays AAM94350 119 K T VNHNR KDWS VK L CDA LWA Y R T A F K ANL GMS P Y R L V F Asparagus officinalis ABD63142119 K I VG S T R KDWS AK L DDA LWA Y R T A F K T P I GT T P F NL L Y Arabidopsis thaliana BAB10790

Figure 5: Amino acid residue alignment of the reverse transcriptase (top) and integrase (bottom) domains of SCHMIDT from Betavulgaris, with its active site boxed in, along with corresponding sections of other reverse transcriptase proteins from different plants.Amino acids matching the consensus sequence are shaded. Numbers indicate cumulative amino acid positions; Arabidopsis thaliana(BAB10790), Asparagus officinalis (ABD63142), Beta vulgaris (ABM55240), Cicer arietinum (CAC44142), Glycine max (AAO23078),Hordeum vulgare (AAK94517), Medicago truncatula (ABO81153), Oryza sativa japonica (ABA92233), Primula vulgaris (ABD78322),Solanum demissum (AAW28577), Sorghum bicolor (AAD22153), Vitis vinifera (CAN79321), Zea mays (AAM94350), Zea mays(AAL59229).

Medicago truncatula ABO81153

Cicer arientinum CAC44142Beta vulgaris CAC44142

Oryza sativa japonica ABA92233

Sorghum bicolor AAD22153

Zea mays AAL59229Glycine max AAO23078

Primula vulgaris ABD78322Vitis vinifera CAN79321

Solanum demissum AAW28577Hordeum vulgare AAK94517

Zea mays AAM94350

Asparagus officinalis ABD63142

Arabidopsis thaliana BAB10790

Phytophthora infestans AAV92918

10076

100

10094

100

100100

100

100

78

65

0.1

Figure 6: Similarity tree constructed by neighbor joining method of the reverse transcriptase domain of SCHMIDT from Beta vulgarisalong with corresponding domains of other reverse transcriptase proteins from different plants. The numbers on the branches are bootstrap(confidence) values. Genbank accession numbers of amino acid sequences are given following plant names.


Table 1: Several LTR retrotransposons discovered within a TE nest, in addition to Coe1 [7], by LTR STRUC analysis of a sugarbeet genomicBAC carrying NPR1.

Feature SCHULTE SCHMIDT Older LTR-RTR Coe1 [7]

Active site YVDDIIL FIDDILI SCDDVLL YVDDIIL

Length of RTR 10 833 bp 11 565 bp5395 bp (before twoTE insertions)

14 531 bp

Length of LTR 1372 bp 2561 bp 780 bp 169 bp

5′ LTR-3′ LTR Identity (%) 99.3% 100.0% 99.0% 96.4%

Number of open readingframes (ORFs)

2 1 2 3

5′ beginning and 3′ end offlanking region (duplication)

ATTTT CGCTC GCTTG CTACT

Class & domains presentCopia-likeIntegrase andRTase domains

All domains expected ofa complete Gypsy-likeretrotransposon

Putative RT domainsCoe1 [7], class II within a classI, DNA transposase/Copia-likeRTase pseudogene

An older LTR retrotransposon, which had been in-terrupted by subsequent insertions of SCHULTE andSCHMIDT, became evident (Table 1) when LTR STRUCanalysis was performed on a sequence having an in sil-ico deletion of the LTR retrotransposons SCHULTE andSCHMIDT. Although very degraded and unclassifiable, theolder LTR retrotransposon was deduced to be 5395 bp with780 bp LTRs sharing 99% identity.

In conclusion, the relatively small repetitive DNAsequences previously described as “pDRVs” can now be seenas a part of the LTRs of SCHMIDT-like retrotransposons.

Planned research will address possible effects of retro-transposons on the expression of core plant genes includ-ing the NPR1 disease resistance-priming gene immediatelydownstream of the LTR retrotransposon nest.

4. Conclusions

An LTR retrotransposon nest consisting of an older retroele-ment into which both a gypsy-like SCHMIDT and a copia-like SCHULTE inserted was identified, and properties ofthe retrotransposons were described. Since LTR retrotrans-posons are driving forces in plant genome evolution (seereviews [1, 2]), they may have tremendous potential useful-ness in genetic manipulation and genome modification toenhance agricultural profitability and sustainability.

References

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[12] Z. Xu and H. Wang, “LTR FINDER: an efficient tool for theprediction of full-length LTR retrotransposons,” Nucleic AcidsResearch, vol. 35, web server issue, pp. 265–268, 2007.

[13] P. Rice, L. Longden, and A. Bleasby, “EMBOSS: the europeanmolecular biology open software suite,” Trends in Genetics, vol.16, no. 6, pp. 276–277, 2000.

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[15] D. Schulte, D. Cai, M. Kleine, L. Fan, S. Wang, and C. Jung,“A complete physical map of a wild beet (Beta procumbens)translocation in sugar beet,” Molecular Genetics and Genomics,vol. 275, no. 5, pp. 504–511, 2006.

[16] P. A. Reeves, Y. He, R. J. Schmitz, R. M. Amasino, L. W.Panella, and C. M. Richards, “Evolutionary conservation ofthe FLOWERING LOCUS C-mediated vernalization response:evidence from the sugar beet (Beta vulgaris),” Genetics, vol.176, no. 1, pp. 295–307, 2007.

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