Tandem repeats in a group II intron provide resolution in phylogenetic and phylogeografic studies of the genus Populus.
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1 Tandem repeats in a group II intron provide resolution in phylogenetic and phylogeografic studies of the genus Populus. Barbara Fussi Federal Research entre for Forests, Vienna (STRI)
2 Thanks to all colleagues who provided us with samples, or supported our own collection trips: Nica M.S (IS raiova, RO) Ipati. (IS Tulcea, RO) vrčková H., Máchová P. (FMRI, Z) Bartha D. (Sopron, H) Benke. (ERTI Sárvár, H) Kaijba D., Bogdan S. (Faculty of Forestry, Zagreb, RO) astiglione S. (Salerno, I) onzales-martinez S. (Madrid, E) Supported by the STRIN DEMY OF SIENE (ÖW)
3 The idea behind To study the structure of a chloroplast group II intron (rpl16) in Populus To find out how different numbers of tandem repeats affect the folding of the intron To help us understand how different foldings influence the functioning of the intron during the splicing process To resolve phylogenetic relationships based on tandem repeats
4 Methods DN was extracted from dry leaves of 15 poplar species and hybrids Sequencing was performed directly on PR products on an automated sequencer Obtained sequences were editied using Sequence Investigator and VectorNTI (Invitrogen) For determining the secondary structures we used the Vienna RN package (thermodynamic structure prediction and phylogenentic comparision)
5 Methods Sequences were aligned using the software MSLE and lustalx The repeat structure was visualized using the software Tandem Repeat Finder ap/indel coding using SeqState and subsequent phylogenetic analysis were performed in PP.
6 Tandem repeat structure Stepwise length variation of 16bp in P. alba and P. tremula on garose gels
7 Sequencing revealed tandem repeats in 5 locations
8 Secondary structure of rpl16 group II intron in P. alba 0 1 VI V IV 16bp repeats differences in domain IV Folding without 16bp repeat
9 0 1 Secondary structure of rpl16 V IV group II intron in P. alba VI Folding without 16bp repeat bp repeats differences in domain IV one hairpin is missing, if 16bp sequence is repeated Folding contains 6 tandem repeats of 16bp indicated by the arrow
10 Self-splicing introns selfsplicing introns require help of an additional protein domain IV can be an ORF for the maturase domainiv can interact with additional splicing factors
11 11S5 rpl16 Repeat nit 1 Repeat nit 1 Eco RI (271) Repeat nit 2 Eco RI (310) Repeat nit 2 1 rpl16 Pop40 sp meadp SW 386 bp Eco RI (271) 425 bp Pop27 tremuloides anterbury NewEng 394 bp Repeat nit 1 Repeat nit 1 Repeat nit 2 Eco RI (310) Repeat nit 2 Pop32 balsamifera Klagenfurt Eco RI (291) 375 bp Eco RI (271) Pop15 x berolinensis 6 Spittelau 425 bp Pop29 tremuloides NewEng 400 bp Pop34 trichocarpa Taschelbach bp Repeat nit 1 Repeat nit 2 Eco RI (310) Repeat nit 1 Repeat nit 2 Eco RI (271) Pst I (53) Eco RI (272) Pop16 x berolinensis KH K Pst I (68) Pop35 trichocarpa Taschelbach bp Eco RI (280) 425 bp Repeat nit 1 Repeat nit 2 Eco RI (310) I19 rpl bp Repeat nit 1 Repeat nit 2 Pop36 trichocarpa Taschelbach 2-3 Pop02 laurif Dahlem 384 bp 425 bp Pop21 x canescens ablenzgasse Eco RI (271) 400 bp Repeat nit 1 Repeat nit 2 Eco RI (310) Repeat nit 1 Repeat nit 2 Pop05 balsamif NewEng 375 bp Eco RI (271) Pop14 x berolinensis Dahlem 425 bp Pop24 alba x grandidentata Dahlem 400 bp Pop09 balsam LungauM 375 bp Repeat nit 1 Repeat nit 2 Eco RI (310) Repeat nit 1 Repeat nit 2 Eco RI (271) Pop44 x rasumowskyana Dahlem 425 bp Pop20 trichocarpa LungauM 375 bp Repeat nit 1 Repeat nit 2 Eco RI (310) Pop37 grandidentata SzombathelyH 400 bp Repeat nit 1 Pop39 x charkowiensis measw Repeat nit 2 Repeat nit bp Repeat nit 2 Repeat nit 1 Pop26 alba-canescens WienDK Repeat nit bp Pop13 simonii W03 Eco RI (303) Repeat nit 1 Pst I (65) 389 bp Repeat nit 1 Repeat nit 2 Eco RI (284) Pop04 Balsam ForsthDI 418 bp Repeat nit 2 Repeat nit 1 Pop25 tremuloides NewEng Pop12 simonii W bp Pst I (65) Repeat nit 2 Eco RI (290) 394 bp Repeat nit 4 Repeat nit 1 Repeat nit 3 Repeat nit 2 Repeat nit 2 Pop18 trichocarpa hastata Dahlem Repeat nit bp Eco RI (301) Pop11 simonii W bp Repeat nit 4 Repeat nit 1 Repeat nit bp Repeat nit 3 Eco RI (333) Repeat nit 1 Repeat nit 1 Repeat nit 2 Repeat nit 2 Repeat nit 3 Repeat nit 4 Pop22 grandidentata Dahlem Eco RI (301) 448 bp Pop30 euphratica Isfahan 402 bp Pst I (64) Repeat nit 4 Repeat nit 1 Repeat nit 2 Repeat nit 3 Eco RI (331) Pop42 tremula VarzelonTK 416 bp Repeat nit 2 Repeat nit 3 Repeat nit 1 Repeat nit 2 Eco RI (319) Repeat nit 4 Repeat nit 5 Pop28 grandidentata-tremuloides LakeTownshendVT Pst I (50) Repeat nit 1 Eco RI (301) 457 bp Pop31 nigra SavsatTK 434 bp 86 rpl16 Repeat nit 1 Repeat nit 2 Repeat nit 3 Repeat nit 4 Eco RI (313) Repeat nit 1 Repeat nit 4 Repeat nit 2 Repeat nit 5 Repeat nit bp Repeat nit 2 Repeat nit 3 Repeat nit 4 Repeat nit 5 Repeat nit 3 Eco RI (333) Repeat nit 1 Eco RI (317) Pop10 hybr LungauF 424 bp Pop41 tremula ZiganaTK 12S5 rpl bp Repeat nit bp Repeat nit 2 Repeat nit 6 Repeat nit 3 Repeat nit 3 Repeat nit 4 Repeat nit 5 Pst I (86) Eco RI (333) Repeat nit 2 Repeat nit 1 Repeat nit 4 Eco RI (333) Pop19 x generosa Dahlem 444 bp Pop43 tremula SavsatTK 448 bp
12 Phylogenetic relationship of 64 accessions of the genus Populus Two groups within Section Tacamahaca Populus as a separate section (with interference of P. nigra and P. simonii Pop05 balsamifera New England Pop09 balsamifera* Pop20 trichocarpa* Pop32 balsamifera* Pop34 trichocarpa* Pop35 trichocarpa* Pop02 laurifolia* Pop14 x berolinensis* Pop15 x berolinensis* Pop16 x berolinensis* Pop39 x charkowiensis* Pop40 sp* Pop44 x rasumowskyana* Pop04 sp ex sect. Tacamahaca* Pop30 euphratica Iran Pop22 grandidentata* Pop28 grandidentata or tremuloides* Pop21 x canescens* Pop24 alba x grandidentata* Pop31 nigra Turkey Pop12 simonii* Pop11 simonii* Pop13 simonii* Pop26 alba or canescens Pop37 grandidentata* Pop29 tremuloides New England Pop27 tremuloides New England Pop25 tremuloides New England Pop41 tremula Turkey Pop43 tremula Turkey Pop42 tremula Turkey big leaves small leaves 60 13S5 tremula Sweden M4 tremula ustria Bootstrap values of more than 50 are given above branches S1-5 tremula Sweden 76 tremula roatia 12S5 tremula ustria S2-5 tremula Sweden 24 tremula Hungary 86 tremula roatia 29M6 tremula zech K165 tremula ustria 11S5 tremula ustria K001 tremula ustria S3-4 Sweden K170 ustria Ma1 alba Morocco T122 alba Tunisia T alba Tunisia H3 alba Spain 4 alba reece 1 alba reece Populus R20 alba Romania K004 alba ustria * cultivated, hybrids and ornamentals 98 PO2-3 alba Spain 3 alba Hungary R21 alba Romania 314 alba ustria ERF306 alba Hungary P83 alba Spain P5 alba Spain 28M6 alba zech Republic Pop19 x generosa* Pop10 x balsamifera* Pop18 trichocarpa var. hastata*
13 Phylogeographic relationship of 30 accessions of the alba-tremula complex North frica reece
14 Summary High variable tandem repeats are located in the domain IV of the intron Maturase aided splicing in the chloroplast: require an ORF in the intron or a nuclear encoded maturase Within rpl16: domain IV has lost its maturase function thus evolves fast Different numbers of repeats of a 16bp fragment only influenced the domain IV - other domains appeared stable The formation of certain hairpin structures with high base pair probabilities within the domain IV may indicate the interaction with additional splicing factors Phylogenetic analysis revealed deeper resolution when modified complex indel coding (MI) was applied 50% majority rule consensus tree using the MI supported 3 main groups: Populus and 2 groups within section Tacamahaca
15 onclusions Tandem repeats influence the folding, but may not influence the function of the intron Introns are useful for phylogenetic studies Indels should not be excluded in the analysis Future plans: adding more species and basepairs for a phylogeny in Populus
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