Supplemental Information

for

Centromere-targeted de novo integrations of

an LTR retrotransposon of Arabidopsis lyrata

Sayuri Tsukahara, Akira Kawabe, Akie Kobayashi, Tasuku Ito, Tomoyuki Aizu, Tadasu Shin-i, Atsushi Toyoda, Asao Fujiyama,

Yoshiaki Tarutani, Tetsuji Kakutani

Contents Supplemental Tables (S1 to S3) …. 1 Supplemental Figures (S1 to S4) …. 9

1

Table S1 Flanking sequences of new Tal1 insertions in loci other than 178bp centromeric

repeats

ddm1 (family8) 5'LTR flanking sequences of Tal1 number of reads At1g74000 (a protein similar to strictosidine synthase) exon 1 At3g02640 (unkwown protein) exon 1 At4g08765 (transposable element gene) exon 1 At5g31662 (transposable element gene) exon 1 At5g57380 (VERNALIZATION INSENSITIVE 3) intron 1 between At1g33290 and At1g33300 1 between At1g40101 and At1g40103 1 between At3g44325 and At3g44326 1 between At4g05000 and At4g05010 1 between At4g33980 and At4g33985 1 multi-locus match 7 Total 17 ddm1 (family8) 3'LTR flanking sequences of Tal1 number of reads At1g04470 (InterPro DOMAIN/s: Munc13 homology 1 ) exon 1 At2g07160 (transposable element gene) exon 1 At2g07500 (transposable element gene) exon 1 At3g25490 (Protein kinase family protein) exon 1 At3g45620 (a putative protein with a DWD motif) exon 1

At4g03340 (Core-2/I-branching beta-1,6-N-acetylglucosaminyltransferase family protein) exon 2

At5g14940 (Major facilitator superfamily protein) exon 1 At5g31999 (transposable element gene) exon 1 At5g34880 (transposable element gene) exon 1 At5g40380 (a cysteine-rich receptor-like protein kinase) exon 1 between At1g33130 and At1g33140 2 between At2g02820 and At2g02830 1 between At2g10605 and At2g10606 1 between At3g22380 and At3g22400 1 between At3g47341 and At3g47342 1 between At3g48275 and At3g48340 1 between At3g56408 and At3g56420 1 between At4g04540 and At4g04545 1 between At4g14670 and At4g14690 1 between At4g16160 and At4g16165 1 between At4g47950 and At3g47965 1 multi-locus match 6 Total 29 ddm1 (family9) 5'LTR flanking sequences of Tal1 number of reads At1g19300 (PARVUS/GLZ1 gene) exon 2 At1g20080 (SYTB) 3'UTR 1 At1g23210 (glycosyl hydrolase 9B6 (GH9B6)) exon 1 At1g49260 (hypothetical protein) exon 1

At1g53650 (RNA-binding protein, putative, similar to RNA-binding protein) exon 1

At1g71690 (Protein of unknown function (DUF579)) intron 1

At2g01270 (a protein disulfide isomerase-like (PDIL) protein) exon 1

2

At2g06002 (other RNA) exon 2 At2g10605 (transposable element gene) exon 1

At2g17580 (Polynucleotide adenylyltransferase family protein) exon 4

At2g21120 (Protein of unknown function) exon 1

At2g23030 (a member of SNF1-related protein kinases (SnRK2)) intron 1

At3g12750 (A member of Zrt- and Irt-related protein (ZIP) family) exon 2

At3g23700 (Nucleic acid-binding proteins superfamily) exon 3 At3g32110 (transposable element gene) exon 1

At3g42670 (a nuclear localized SNF domain containing protein involved in RNA silencing) exon 1

At3g51310 (Homolog of yeast retromer subunit VPS35) intron 1

At3g62980 (an auxin receptor that mediates auxin-regulated transcription) intron 2

At3g63320 (Protein phosphatase 2C family protein) exon 1

At4g00150 (Belongs to one of the LOM (LOST MERISTEMS) genes) exon 2

At4g02320 (Plant invertase/pectin methylesterase inhibitor superfamily) exon 2

At4g04960 (Concanavalin A-like lectin protein kinase family protein) exon 1

At4g23130 (a receptor-like protein kinase) 2 At4g26010 (Peroxidase superfamily protein) exon 2 At4g26630 (transposable element gene) intron 1 At4g31910 (HXXXD-type acyl-transferase family) intron 1 At4g35840 (RING/U-box superfamily protein) exon 2 At5g09540 (Chaperone DnaJ-domain superfamily protein) exon 1

At5g13320 (an enzyme capable of conjugating amino acids to 4-substituted benzoates) exon 1

At5g13600 (Phototropic-responsive NPH3 family protein) exon 1 At5g24150 (squalene monooxygenase gene homolog) exon 1 At5g26775 (transposable element gene (LINE)) exon 2 At5g30440 (CACTA-like transposase family) 2 At5g42230 (serine carboxypeptidase-like 41 (scpl41)) exon 1 At5g45530 (Protein of unknown function (DUF594)) exon 3

At5g56960 (basic helix-loop-helix (bHLH) DNA-binding family protein) exon 1

At5g59640 (CACTA) exon 1 between At1g02260 and At1g02280 1 between At1g18090 and At1g18120 1 between At2g05115 and At2g05117 1 between At3g17850 and At3g17860 1 between At4g02710 and At4g02715 2 between At4g38410 and At4g38420 2 between At5g03360 and At5g03370 2 multi-locus match 33 Total 98 ddm1 (family9) 3'LTR flanking sequences of Tal1 number of reads At1g25300 (Octicosapeptide/Phox/Bem1p family protein) exon 1 At1g43745 (transposable element gene (Athila)) exon 1 At1g55560 (SKU5 similar 14 (sks14)) exon 1 At1g55740 (seed imbibition 1 (SIP1)) exon 1 At1g70450 (Protein kinase superfamily protein) intron 1 At1g75090 (DNA glycosylase superfamily protein) intron 2 At2g11040 (transposable element gene) exon 1 At2g29060 (GRAS family transcription factor) exon 1

3

At3g09270 (glutathione transferase belonging to the tau class of GSTs) exon 1

At3g13090 (member of MRP subfamily) exon 1 At3g44175 (transposable element gene) exon 1 At3g53200 (Member of the R2R3 factor gene family) exon 1

At3g54970 (D-aminoacid aminotransferase-like PLP-dependent enzymes superfamily protein) intron 1

At4g06477 (transposable element gene) exon 1 At4g06489 (pseudogene) exon 1 At4g07725 (transposable element gene (Ty3/gypsy)) exon 2 At4g23130 (a receptor-like protein kinase) exon 1 At4g28640 (Auxin induced gene, IAA11 (IAA11)) intron 1

At4g34510 (KCS17, a member of the 3-ketoacyl-CoA synthase family) exon 1

At5g02660 (zinc ion binding) exon 1

At5g17330 (one of two isoforms of glutamate decarboxylase) exon 2

At5g24780 (an acid phosphatase similar to soybean vegetative storage proteins) intron 1

At5g29337 (transposable element gene (Athila)) exon 1 At5g38500 (unknown protein) exon 1 At5g45540 (unknwon protein) exon 1

At5g49150 (a transmembrane domain containing protein expressed in sperm cells) exon 1

At5g56500 (TCP-1/cpn60 chaperonin family protein) exon 2 between At1g13970 and At1g13980 1 between At2g02690 and At2g02700 1 between At2g10150 and At2g10160 3 between At2g11465 and At2g11480 1 between At2g28310 and At2g28315 1 between At2g34500 and At2g34510 1 between At2g35658 and At2g35670 1 between At4g02990 and At4g03010 1 between At4g06547 and At4g06548 1 between At5g31650 and At5g31685 1 between At5g31685 and At5g31702 1 between At5g31804 and At5g31821 2 multi-locus match 18 Total 64

4

Table S2 Number of reads of Tal1 insertion for each position within 178bp centromeric repeat

WT (family2) WT (family6) ddm1 (family8) ddm1 (family9) position nucleotide forward reverse forward reverse Forward reverse forward reverse

1 A 0 0 0 0 9 10 6 13 2 A 0 0 3 4 10 10 9 10 3 S 0 0 0 0 0 0 0 0 4 C 0 0 0 0 1 0 0 0 5 T 1 0 6 0 16 10 8 10 6 T 0 0 0 0 0 0 0 1 7 C 0 0 0 0 0 0 0 0 8 T 0 0 0 0 2 1 1 0 9 T 0 0 8 0 15 24 40 33

10 C 0 0 0 0 0 0 0 0 11 T 0 0 0 0 0 0 0 0 12 T 0 0 2 0 11 5 22 19 13 G 0 0 0 0 0 0 0 0 14 C 0 0 0 0 0 0 0 0 15 T 0 0 0 0 0 0 0 0 16 T 0 0 0 0 0 0 0 0 17 C 0 0 0 0 0 0 0 0 18 T 0 0 0 0 1 0 0 0 19 C 0 0 2 0 5 4 16 7 20 A 0 0 0 0 0 0 0 1 21 A 0 0 0 0 0 0 0 0 22 A 0 0 0 0 0 3 0 0 23 G 0 0 0 0 0 1 0 0 24 C 0 0 0 0 0 0 0 0 25 T 0 0 0 0 0 0 0 0 26 T 0 0 1 0 12 8 5 5 27 T 0 0 0 0 0 1 1 0 28 S 0 0 2 1 3 7 22 1 29 A 0 0 3 3 9 16 11 9 30 T 0 0 0 0 3 2 0 1 31 G 0 0 0 0 0 0 0 0 32 G 0 0 0 0 0 0 0 0 33 T 0 0 0 0 0 0 1 0 34 G 0 0 0 0 0 0 0 1 35 T 0 0 0 0 0 0 0 0 36 A 0 0 0 0 4 6 4 8 37 G 0 0 9 8 20 27 31 15 38 C 0 0 1 0 1 0 1 0 39 C 0 0 0 0 3 2 3 2 40 A 0 0 5 0 15 4 4 6 41 A 2 0 1 0 9 13 15 55 42 A 0 0 0 0 0 2 5 2 43 G 0 0 2 0 3 0 0 2 44 T 1 0 0 0 2 0 2 3 45 C 0 0 0 0 0 0 0 0 46 C 0 0 0 0 0 0 0 0 47 R 0 0 0 0 0 0 0 0 48 T 0 1 0 5 25 29 68 32 49 A 0 0 7 0 0 0 2 0 50 T 0 0 0 0 3 2 0 6 51 G 0 0 5 0 6 1 2 4 52 A 0 4 0 0 0 0 0 3 53 G 0 0 0 2 3 1 1 3

5

54 T 0 0 0 0 2 0 4 3 55 C 0 0 0 0 0 0 0 0 56 T 5 0 1 5 18 19 15 17 57 T 0 0 0 0 0 0 0 0 58 T 2 0 0 0 1 4 3 2 59 G 0 0 0 0 0 0 0 0 60 K 0 0 0 0 0 0 2 4 61 C 0 0 3 0 2 5 6 3 62 T 0 0 0 0 0 0 0 1 63 T 0 0 3 0 4 3 4 4 64 T 0 2 1 3 10 9 5 6 65 G 0 0 0 0 0 0 0 1 66 T 0 0 0 0 0 0 0 0 67 R 0 0 0 0 3 2 1 6 68 T 0 0 0 0 0 0 0 0 69 C 0 0 0 0 0 1 0 0 70 T 0 0 0 0 4 2 6 2 71 T 0 3 33 14 21 19 54 15 72 C 0 0 0 0 0 0 0 0 73 T 0 0 0 0 0 0 0 0 74 A 0 0 11 5 55 15 42 20 75 A 0 0 0 0 0 0 0 2 76 C 0 0 0 0 0 0 1 0 77 A 0 0 0 0 0 2 2 3 78 A 0 0 0 0 1 0 0 0 79 G 0 0 0 0 2 1 0 0 80 G 0 0 0 0 0 0 1 1 81 A 2 3 11 8 42 41 60 46 82 W 0 0 0 4 1 2 0 0 83 A 0 0 0 0 0 0 0 0 84 C 0 0 6 152 20 19 25 34 85 A 0 0 0 2 2 2 0 3 86 M 0 0 0 0 1 0 1 1 87 T 0 0 0 0 0 1 3 0 88 A 0 0 0 1 1 3 2 1 89 C 0 0 0 0 0 0 0 0 90 T 0 0 4 0 3 2 2 4 91 T 0 0 1 0 11 5 5 13 92 A 0 0 0 2 1 0 1 2 93 G 0 0 0 0 0 0 0 0 94 G 0 0 0 0 1 1 2 1 95 C 0 0 0 0 0 0 0 0 96 T 0 0 0 0 0 0 0 0 97 T 0 0 0 0 2 3 0 2 98 T 0 0 0 0 0 0 0 2 99 T 0 0 0 0 6 5 8 4

100 A 0 2 0 4 14 16 7 16 101 R 0 0 0 0 2 2 1 2 102 G 0 0 3 0 3 2 2 0 103 A 0 0 0 0 0 0 0 0 104 T 0 0 0 0 0 0 0 0 105 C 0 0 0 0 0 4 2 1 106 V 0 0 0 0 1 0 0 2 107 G 0 1 0 0 0 2 3 3 108 G 0 0 0 0 2 0 6 0 109 T 0 0 0 0 0 0 0 0 110 T 0 0 0 0 0 0 3 0 111 G 0 0 0 0 0 0 0 0 112 C 0 0 0 0 0 0 1 2

6

113 G 1 3 4 0 1 3 4 3 114 G 0 0 0 0 0 1 0 0 115 T 0 0 0 0 1 1 2 2 116 T 0 0 5 0 1 0 0 0 117 Y 0 0 0 0 0 0 0 0 118 W 0 0 0 0 2 1 2 2 119 A 0 0 3 0 1 0 0 0 120 G 0 1 0 0 2 3 8 2 121 T 0 0 0 0 1 1 6 1 122 T 0 0 0 0 1 3 1 0 123 C 0 0 0 0 0 0 0 0 124 T 0 0 0 0 0 0 3 0 125 T 0 0 9 1 19 23 34 22 126 A 0 0 0 0 5 2 2 3 127 T 0 0 0 0 0 0 0 0 128 A 0 0 0 3 8 5 10 10 129 C 0 0 0 0 1 1 0 4 130 T 0 0 0 0 0 1 0 1 131 C 0 0 0 0 2 0 1 1 132 A 0 0 0 0 0 0 2 4 133 A 0 0 0 0 1 0 0 0 134 T 0 0 1 9 11 15 17 25 135 C 0 0 0 1 0 0 1 0 136 A 0 0 7 2 17 19 24 14 137 T 0 0 0 0 0 2 1 4 138 A 0 0 2 0 4 2 3 4 139 C 0 0 0 0 0 0 0 0 140 A 0 0 0 0 1 0 0 1 141 C 2 0 113 5 5 12 13 15 142 A 0 0 0 0 0 0 0 0 143 T 0 0 0 0 2 2 4 0 144 G 0 0 5 8 19 56 15 23 145 A 0 0 0 0 0 0 0 0 146 C 0 0 0 1 4 2 7 2 147 A 0 0 7 2 28 22 18 17 148 T 0 0 0 0 0 0 0 2 149 C 4 0 11 27 39 47 34 47 150 W 0 0 0 0 0 0 1 0 151 A 0 0 1 3 0 4 1 1 152 G 0 0 0 0 0 0 0 0 153 T 0 0 0 0 1 0 0 0 154 C 0 0 0 0 0 2 0 3 155 A 0 0 0 0 0 0 0 0 156 T 0 0 0 0 1 2 1 0 157 A 2 0 4 12 38 42 56 75 158 T 0 0 5 12 11 10 4 50 159 T 0 0 0 0 0 0 0 0 160 Y 0 0 0 0 0 0 0 1 161 G 0 0 0 0 0 0 0 0 162 A 0 0 0 0 0 0 0 0 163 C 0 0 0 0 0 0 0 0 164 T 0 0 0 0 0 1 0 0 165 C 0 0 0 0 0 0 3 0 166 C 0 0 0 0 3 1 2 6 167 A 0 0 0 0 0 0 1 0 168 A 0 0 14 5 7 14 11 11 169 A 0 0 0 0 3 0 2 1 170 A 0 0 0 0 0 0 2 0 171 C 0 0 0 2 0 1 0 1

7

172 A 0 0 0 0 1 0 3 1 173 C 0 0 0 0 0 0 0 0 174 T 0 0 0 0 0 0 0 0 175 A 0 0 0 0 1 4 2 5 176 A 1 0 3 4 22 16 32 55 177 C 0 0 0 0 0 0 0 1 178 C 0 0 0 73 1 0 0 0

For many of the insertions, read numbers are much lower than average depth of the sequencing

(50<), most likely reflecting somatic neo-insertions of this TE.

8

Table S3 . Primers used for cloning Tal1, RT-PCR, and generating probes for Southern

analyses.

Purpose Primer sequence Cloning of 5’ half of Tal1 5’-TAGGACTTTGTCCATATTGAT-3’ + (1st PCR) 5’-TGTGGTTACTATGAGCTCTTTC-3’ Cloning of 5’ half of Tal1 5’CGCCTGCAGTATTGATCAAGAAACGAAGAG-3’ + (2nd PCR) 5’-TGTGGTTACTATGAGCTCTTTC-3’ Cloning of 3’ half of Tal1 5’-ACATTGCAAATCAGTTTTCCC-3’ + (1st PCR) 5’-TTTGTGGGTGTGGACTGTAAG-3’ Cloning of 3’ half of Tal1 5’-ACATTGCAAATCAGTTTTCCC-3’ + (2nd PCR) 5’-GGCGAATTCTGTAAGAATATGCGGAAGAT-3’

5’-TGACAAGAAAGATTGGGAAAG-3’ + RT-PCR of Tal1 5’-GGCTTGTTGGTTGAAATAAAC-3’ 5’-CCTGTTGTCGCCAACGAAGTC-3’ + RT-PCR of GAPC 5’-CACTTGAAGGGTGGTGCCAAG-3’ 5’-TGACAAGAAAGATTGGGAAAG- 3’ + Probe for Tal1 (probe a) 5’-GGCTTGTTGGTTGAAATAAAC-3’ 5’-TCTGGATCGTTTCGCATGATTG-3’ + Probe for T-DNA LB

(probe b) 5’-GGCGTCCCGGAAAACGATTCCG-3’ 5’-GGAGAAGCTACCAAGTCTCG-3’ + Probe for Tal1 (probe c) 5’-CTTTGGAGTTCTTATCAAATAC-3’ 5’-TGTAAGAATATGCGGAAGAT-3’ + Probe for Tal1 (probe d) 5’-TATTGATCAAGAAACGAAGAG-3’ 5’-GGCGTCCCGGAAAACGATTCCG-3’ + Probe for T-DNA RB

(probe e) * 5’-GCCAATATATCCTGTCAAACAC-3’

* The PCR product was digested by EcoRI for obtaining the probe.

ddm1(family 8)

ddm1(family 9)

ddm1 (line 1) ddm1 (line 4)

WT(family 2)

WT(family 6)

ATCOPIA93

Tal1

Random

Tsukahara_Fig S1

Figure S1. Integration of Tal1 and ATCOPIA93.

Results of Tal1 insertions in four transgenic lines (two ddm1 and two wild type lines),and ATCOPIA93 insertions in five self-pollinated ddm1 lines. Score for random readsfrom the same individual is shown below. Vertical bars represent 300 reads.

ddm1WT

Random

ddm1 (line 2) ddm1 (line 3) ddm1 (line 5)

0% 100% 0% 100% 0% 100% 0% 100%

0% 100% 0% 100% 0% 100% 0% 100%

0% 100% 0% 100% 0% 100% 0% 100% 0% 100%

0% 100% 0% 100% 0% 100% 0% 100% 0% 100%

Tsukahara_FigS2

1kb WT

ddm1

5’ LTR 3’ LTR 　　　Tal1

Figure S2. Nested integrations of Tal1 in A. thaliana.

Flanking sequences of Tal1 terminal sequences often matches internal sequence of Tal1, suggesting nested integrations. The integration positions were determined using the flanking 20nt that were 100% identical to Tal1. Blue and red arrows indicate Tal1 insertions in two lines of wild-type and two lines of ddm1 mutants, respectively. Top and bottom arrows indicate insertions with 5’ to 3’ and 3’ to 5’ orientations of Tal1, respectively. Insertions into two LTRs were not distinguished, resulting in twice-overrepresented insertions there.

WT V T2 T3 T4 T5

Tal1-WT (Family2)

Tsukahara_Fig S3

Figure S3. Morphology of transgenic A. thaliana with Tal1 transgene. (A) One transgenic line with Tal1 transgene from T2 to T5 generations. Parental line and control transgenic line with empty vector are shown in left. All plants are 26 days old. (B) Tal1 transcription after self-pollination of wild type transgenic plants. Results of T2 and T5 generations are shown.

A

B

WT V T2 T5 T2 T5

Tal1-WT

GAPC RT(+)

GAPC RT(-)

Tal1 RT(-)

Tal1 RT(+)

Family2 Family3

Tsukahara_Fig S4

Figure S4. Comparison of predicted amino acid sequences of proteins encoded

in the COPIA93/20 family members. Colored letters indicate significant homology to

known domain structures by Pfam search. Black: Zinc knuckle, Blue: conserved

protease region, Green: integrase core domain, Red: reverse transcriptase.