Repetitive DNA and its evolution: genomic strategies to understand the processes and exploit biodiversity rapid evolution in copy number, location and sequence, with diverse turnover mechanisms. often mark the major differences between closely related species Sym038: Plant genomes: not just for models anymore 38A: 28 July 2011 Pat Heslop-Harrison and Trude Schwarzacher Farah Badakshi, Ana Claudia Guerra, Guto Kuhn, Faisal Nouroz, Mateus Mondin phh4@le.ac.uk www.molcyt.com Twitter: #IBC18
Peanut, Arachis hypogaea, 2n=4x=40 AABB genome constitution Ana Claudia Guerra Araujo, EMBRAPA, Brazil
BAC in situ individual BACs have various repetitive sequences with
Many of the repetitive sequences are retrotransposons and DNA transposons Some are microsatellite motifs Some are satellites including the most rapidly evolving sequences
ancestral High-copy number High-copy number High-copy number Low-copy number Low-copy number High copy spp: homogenized, amplification from a limited number of master copies Low copy spp: much variation
120bp repeat unit family in Triticum, Aegilops and Secale species Homology between sequences 70-100% in Triticum/Aegilops and Secale species 0.1 749 Afacer1 1000 T.mono106/42!133pt1 T.mono106/42!155pt1 996 Ae.umb106/208!1911 Ae.umb106/208!1810 1000 Ae.umb106/208!2012 S.vav147!259pt1 Pet w 25/208!088 578 Pet w 25/208!077 S.vav42!248 537 S.vav106/208!215pt1 S.vav106/208!204pt2 915 S.vav106/208!215pt2 S.vav25/208!182 S.mon42!136 Pet 22594 25/42!3324 Pet 22594 25/42!3425 623 CS/325/208!1820 Pet 2259425!315 S.vav25/208!193 981 T.tau25/147!2524pt2 CS/325/147!2322pt2 L.moll25/42!156 Pet w25/147!123 CS/325/208!1719 998 CS/325/147!2120 Pet w25/147!3231 580 616 882 564 725 Ae.umb25/147!124 Ae.umb25/208!168 L.moll25/42!167 119Repeat2 Pet w 25/208!099 S.mon147!1811 S.vav106/208!204pt1 942 119Repeat3 S.vav147!2711pt1 S.mon25/147!081 T.mono25/208!1212 119Repeat1 S.vav42!237pt1 S.vav42!237pt2 T.tau25/147!2625 T.tau25/147!2726 Ae.umb25/208!157 T.tau25/208!1517 T.tau106/42!177 Ae.umb25/42!091 L.moll25/42!189 1000 CS/325/208!1618 T.tau25/147!2524pt1 CS/325/147!2322pt1 Pet 22594 25/208!2325 Pet 22594 25/147!1918 Pet 22594 25/208!2426 T.tau25/208!1315 985 646 T.tau25/208!1416 Ae.umb25/147!135 Ae.umb25/147!146 Ae.umb25/208!179 Ae. tauschii (D genome) Irrespective of copy number in the genome S. cereale (R genome)
ancestral High-copy number High-copy number High-copy number High-copy number Low-copy number Low-copy number High copy and low copy spp: no significant homogenization
www.molcyt.com Brassica nigra (BB) 2n=16 Brassica carinata (BBCC) 2n=34 Brassica juncea (AABB) 2n=36 Brassica oleracea (CC) 2n=18 Brassica napus (AACC) 2n=38 Brassica rapa (AA) 2n=20 10
Genome Specificity of a CACTA (En/Spm) Transposon B. napus (AACC, 2n=4x=38) hybridized with C-genome CACTA element red B. oleracea (CC, 2n=2x=18) B. rapa (AA, 2n=2x=20) Alix et al. The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation. Plant Journal
Genome Specificity of a CACTA (En/Spm) Transposon B. napus AJ 245479 AC 189496 B. rapa AC 189446 AC 189655 AC 189480 B ot1-1 large insertion specific of Bot1-1 B. oleracea B ot1-2 large insertion in common between Bot1-2 and Bot1-3 B ot1-3 Bo6L1-15 1010bp Rearrangement specific of Bot1-3
Sequence level at scale of 100s to 10000s of bp analysis by dotplot Faisal Nouroz
Dot-plots of genomic sequence from homologous pairs of BACs Region of high homology between A and C sequence kb Brassica rapa (A genome) sequence Region of low homology 4kb Insertion-gap pair: present in C genome 500bp Insertion-gap pair: present in A Microsatellite Transposed (moved) sequence Sequence level at scale of 100s to 1000s of bp analysis by dotplot Dotter plot of Brassica oleracea var. alboglabra clone BoB028L01 x Brassica rapa subsp. pekinensis An inversion clone KBrB073F16 with transposable elements. 28/07/2011 14 gi 195970379 vs. gi 199580153 Brassica oleracea (C genome) sequence
AAGTGAATGGATGCTCGCATTAGTTACTATGAGCCGATTCTCGCTCTTGCGAAAGCTAAAGAGGAAAAGGCCTTCGCATTGCAGAAG AGCTGGCTGCCAGCGAGCAAGAGGTTTTCAATATTGGCTTGTGGAAAATTTGTTGCCACTTTTGCTTTACTAAGGAATGAAATAATAC TTGTTTTTTTTTTTCATGGTTAATATTAGAAGATATAATTTCCTTTGAAGTTAGATTACGTTTCTTTATGTCGACGAAGTGAAGAAATATT GTCTTGTTTATGGTTCCTTCTAGTCCCAACCTTTTTTCAAGAAGGTACAGTACGTGTCAGGATTTATATGGATATACACA TATCCTATTGCGCAATTGTCAATAATAGCACTTTTTGAAGTTTATGTCTCAAAATAGCACTAGAAGGAGAAAGTCACAAAAATGATATT CATTAAAGGGTAAAATATCTCTTATATCCTTGGTTTAAAATTAAATAAACAAACAAAAATAAATAAAAATAAATAAAAAAAATGAAAAAA AAGAAATTTTTTTTATAGTTTCAGATTATATGTTTTCAGATTCGATTTTTTTTTTATTTTTTTATTTTTTTCGAAATTTTTTTTTTATTTTTTTTCA AATTTTCTTTTTATAATTTAAAAATACTTTTTGAAACTGTTTTTTTAATTTTTATTTTTTATTTTAGTATTTATTTTTTATAAAATTTTAAACCCT AATTCCTAAACCCCCACCCCTTAACTCTAAACCCTAAGGTTTGGATTAATTAACCCAATGGATATAAGTGTATATTTACCTCTTTAATGA AACCTATTTTTGTGACTTTGAATCTTGAGTGCTACTTTGGGAACAAAAACTTGGTTTGGTGCTATCCTAGTCTTTTTCTCTATCCTATT 28/07/2011 TACCACCCTTCTTTGTTCAATACTTTTTACAGTTTTTGGAAAGGACATGTTTCTTCTATCATCACTTAATGGTTATATATGTATGAGAAG TTTGAAAGAGATTACACTGTTTTGGAATATTAAAAAAAAAAGATATTACAAGATCTGATTTTGTTTGTATTTTAAAATTCTACCAAATC TCTCCTCAAAATCTTGGTCAAAGTCCAAAAATCCAAATATCTCAGTTAAATTCCACCAAATATGAAATCCTAAAACTTTTCCAAAATA GTTCAATAAGCCCTTAGTGTTTGGTG TSD TIR 551-bp BART1 TE 9-bp TSD (TATCCTATT) 6-bp TIR and 66-bp imperfect sub-tir TIR TSD Brassica rapa with inserted 542bp sequence not present in B. oleracea. 9bp TSD (red letters and arrow) and TIR (blue). Flanking primers used in PCR as blue arrows on sequence Faisal Nouroz 2011 16
B. rapa AA 2n=20 B. juncea AABB 2n=36 B. nigra BB 2n=16 B. carinata BBCC 2n=34 B. oleracea CC 2n=18 B. napus AACC 2n=38
GACACTCTTCCCAATCGTTCATTCCTGACGTCATTAGGCAACCACCTCTGTTTTTCCCCACCACAAACAGTGAATACATCTCTCCTATCTCTC TCAGAATCGTCAGTGTTTGCTCTCCGTTGCTTACTCGCTTCTCTATGAATCCAACTTGCCCCGTCGTTACAAATCTGCCAAAAATAAACCAAA ACCAGTCCGGTCAATGAAAAAAATGCCAATGTTTCAGGTCTAGAAATTATCCACAACCCTAGTACTAAGATCTGAAATTTATGAGGGAGATAA ACATTTTTAGGTTAATTGTAAGAAAAAATATTTATAATTTTTGGGCCATGCAGCAAATACATAATATTTCCTTAAAATTTGGATTGTAAGAC TAATAGTGTTTGAGTATTTGATATTTGATATCTTTTAAAAAAGGAAACAAAATTGAATTTCTAAATAAGATTATATTTTTAAAATAAAACAAT AAAAATACATAAAAATAGTTACAAAAAAAAATATATATATTGTTAAACCGTTAGCAAATTAAATACTAAATCCTATACCCTAAATCCTAAACT CCAAACCCTAAATGATAAACCTTAAATCTTGGATAAACCGTAAACCATTGGAAAATTTTAAAACCTAATCATACATTAAAAACTAAAATTTAA TAACACTAAACCCTAAACCCTAATCACTAAACCCTAAACCCTTAGATAAATCATGAACCCTTGGATAAATCATAAACTCTAAATCAAAAATAT TTAAAATTAAACCCTAAAATATATAATTTATCCAAGGGCTCAGAGTTTACCCAAGGGTTTAGGGTTTAGTGATTAGGATTTAGGGTTTAGTGT TATTAAAATTTAGTTTTTAATGTATGATCTAAGGTTTAAGAGTTTCCAATGGTTTAGAGTTTATCCAAAGTTTAAGGTTTAACGTTTAGGGTT TAGGATTTAGGATTTAAGGTATAGGGTTTAGTATTTTGCTGAAGATTTAACAATATTAATTAATTTATTTTTTGTAACTATTTTTATATATTT TTATTATTTTATTTTTAAAATATAATATAATTTGGATATTCAATTTTATTTTCTTTTTTAAAAAATATCAAATATCAAATACTCAAACACTAT GGTTGGTGAACTTCTAGGTGTGAACCCAAGAATTACTCTTAATGTTTCATCCGATTGTGCTCAAAACCTTTCATGAACTGGCTAAAGCTGGAA ACATAGGATTAGTAAGAAGTAGAATCTTGTAAAGTACCTGTTATAGTATTCCTCTAAGAAAGTTCGATCAGTTTCGTCGTTTGTCTGATCGTT ACCAACAATCTCCATCAAAACATCGTTGTTTTCTTTGGTCACCGCGTCTCCGACAAGATTCTCTGTCTCCGAGCCATAAGCGACAAACTGTAT GATAGTGAGGTGAATCTGAGAGTTATTGATAAGCCACTGGCACAAGGACAGAGCCTCTCGATCATCAGGACCACCAAAGAACAATGCAGCGAC GTGTTGTACCGACTCAAACCCGTGAAGCTGGTGGAACCCGGTTATGTTTCTATCCACATAGATACCGATCG 28/07/2011 790-bp TE TAAT, 4-bp TSD AGTGT/ACTCT, 5-bp TIRs & 370-bp IR Insertion sequence present in B. oleracea, missing from B. rapa. TSD (red); green and blue boxes shows remarkable internal structure with 370-bp inverted repeat near-filling the insert. 18
Rapa141F Rapa185F Rapa8002 Rapa177R B. rapa (47186-48200) B. oleracea (66,350-66750) 1 246 TSD TIR 542-bp TE TIR TSD 790 1000... 28/07/2011 B. rapa (AA) B. juncea (AABB) B. napus (AACC) Hexaploid Brassica (carinata x rapa) B. nigra (BB) B. oleracea (CC) B. carinata (BBCC) B. oleracea (GK97361) =A =T =C =G............ Schematic representation of insertion in Brassica rapa and other Brassica genomes. Green, red, blue and black boxes showing DNA motifs. 19
28/07/2011 Dotplot of 790bp insertion element showing inverted repeat structure. 20
Terminal LTR retrotransposon in B.oleracea and B.napus. b) B. juncea with mariner like transposon e)
524 bp SINE670 bp hat 2781 bp MuDR Like 216 bp SINE Brassica oleracea (C genome; EU642504.1) TEs in B. oleracea Brassica rapa (A genome; AC189298.1) 505 bp Unknown 1655 bp in B.rapa Microsatellite 1189 bp Mariner 380 in B.rapa Like 700 bp hat 715 bp hat 914 bp LINE 1016 bp in LINE 3869 bp TE 402 bp hat 1080 bp LINE 1284 bp 258 MITE Unknown 819 bp Unknown 237 bp Mariner Like Inversion 242, 644 & 274 bp SINEs
Whole genome analysis From morphology, cytology, repeats, genes and whole genome sequences The most abundant transposable elements identified in XXX, LTR retrotransposons of the Copia and Gypsy superfamilies, were found to occur 50-fold and 25-fold less frequently in assembled reads than in shotgun reads, respectively... Because only predicted protein homologies were used to identify transposable elements and because all transposable elements contain extensive noncoding DNA, we expect that the vast majority of the transposable element related DNA in the XXX genome assembly was missed by this approach
phh4@le.ac.uk www.molcyt.com Repetitive DNA and its evolution: genomic strategies to understand the processes and exploit biodiversity Repeats are the greatest part of most genomes Repeats amplify, homogenize, move and deleted They are the most rapidly changing genome component They drive genome evolution, diversification and chromatin structure Sym038: Plant genomes: not just for models anymore 38A: 28 July 2011 Pat Heslop-Harrison and Trude Schwarzacher Farah Badakshi, Ana Claudia Guerra, Guto Kuhn, Faisal Nouroz, Mateus Mondin