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Research Article

Next-Generation Sequencing Based Transposon Display to Detect High-Throughput Insertion Polymorphism Markers in Brassica

Plant Breeding and Biotechnology 2016;4(3):285-296.
Published online: August 31, 2016

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*Corresponding author: Tae-Jin Yang, tjyang@snu.ac.kr, Tel: +82-2-880-4547, Fax: +82-2-8873-2056
• Received: August 11, 2016   • Revised: August 20, 2016   • Accepted: August 23, 2016

Copyright © 2016 The Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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    Horticultural Plant Journal.2021; 7(6): 539.     CrossRef
  • Miniature inverted-repeat transposable elements (MITEs), derived insertional polymorphism as a tool of marker systems for molecular plant breeding
    Venkatesh, B. Nandini
    Molecular Biology Reports.2020; 47(4): 3155.     CrossRef

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Next-Generation Sequencing Based Transposon Display to Detect High-Throughput Insertion Polymorphism Markers in Brassica
Plant Breed. Biotech.. 2016;4(3):285-296.   Published online August 31, 2016
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Next-Generation Sequencing Based Transposon Display to Detect High-Throughput Insertion Polymorphism Markers in Brassica
Plant Breed. Biotech.. 2016;4(3):285-296.   Published online August 31, 2016
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Next-Generation Sequencing Based Transposon Display to Detect High-Throughput Insertion Polymorphism Markers in Brassica
Image Image Image Image
Fig. 1 Next-generation sequencing (NGS) based transposon display. (A) Steps involved in NGS-based transposon display analysis. The target region (red dotted circle) used for the sequencing. (B) Identification of polymorphism site (presence/absence of conserved miniature inverted-repeat transposable element [MITE] sequences) by analyzing reads from the different accessions. PCR: polymerase chain reaction.
Fig. 2 Structure of the BraSto-2 (Bs2) miniature inverted-repeat transposable element (MITE) and primers for MITE- display. Primers from the Bs2 consensus region, terminal inverted repeat (TIR) and MseI restriction enzyme site shown as arrows. Target regions are shown with dotted ovals. P: primers, F: forward, R: reverse. W in primer sequence is degenerate base symbol can bind to A/T.
Fig. 3 Validation of sequencing based next-generation sequencing-based transposon display analysis of BraSto-2 (Bs2) miniature inverted-repeat transposable element (MITE) family members by insertion polymorphism survey. (A) Bs2 family member shows Brassica rapa ‘Chiifu’ specific insertion compare to ‘Kenshin’. (B) Bs2 family member shows B. rapa ‘Kenshin’ specific insertion compare to ‘Chiifu’. (C) Shared or common insertion Bs2 between ‘Chiifu’ and ‘Kenshin’. Fig. 3D–F show the corresponding gel validation of A, B, and C, respectively. Black and grey arrow head indicate the MITE insertion (full site) and non-insertion (empty site), respectively. Star indicates the polymorphism in Br-6 produced by a shared insertion of ‘Chiifu’ and ‘Kenshin’. Fig. 3D–F are based on the primers 3, 34, 88 from Table 3, respectively.
Fig. 4 In silico map of BraSto-2 (Bs2) members showing the surveyed and newly identified members on the Brassica rapa pseudo chromosome. Red bars represent the 401 Bs2 members on the B. rapa pseudo-chromosome. Black and yellow arrowheads indicate that the 90 in silico candidate members of Bs2 utilized by insertion polymorphism survey. Green and pink stars indicate the B. rapa ‘Chiifu’ and B. rapa ‘Kenshin’ specific insertions, respectively.
Next-Generation Sequencing Based Transposon Display to Detect High-Throughput Insertion Polymorphism Markers in Brassica

List of accessions used for the display and insertion survey.

No. ID Species Accession no. Reference
1 Br1 Brassica rapa ‘Chiifu’ (C) (Wang et al. 2011)
2 Br2 B. rapa ‘Kenshin’ (K) (Sampath et al. 2013)
3 Br3 B. rapa OC 1 (Lee et al. 2014)
4 Br4 B. rapa OC 2 (Lee et al. 2014)
5 Br5 B. rapa YE 1 (Lee et al. 2014)
6 Br6 B. rapa YE 2 (Lee et al. 2014)
7 Bo1 Brassica oleracea C1234 (Lee et al. 2015)
8 Bo2 B. oleracea C1184 (Lee et al. 2015)
9 Bo3 B. oleracea C1235 (Lee et al. 2015)
10 Bo4 B. oleracea C1176 (Lee et al. 2015)

Summary of reads analysis from NGS-based transposon display of Bs2 MITE family against two Brassica rapa accessions.

Accession Insertion sites based on in silico mapping PCR validation


Total Accession specificz) Success IP-Bs2 among Br1, Br2 IP-Bs2 among 10 accessions
Br1 127 83 75 27 (36) 59 (78)
Br2 60 16 15 7 (46) 10 (66)

Values are presented as number only or number (%).

z)Bs-2 sites specific to Br1 and Br2.

NGS: next-generation sequencing, MITE: miniature inverted-repeat transposable element, PCR: polymerase chain reaction, IP-Bs2: Insertion polymorphism of Brasto-2 (bs2) members.

Insertion polymorphisms survey of Bs2 candidates based on NGS-based transposon display analysis against 10 Brassica accessions.

No. Primer sequence Product size (bp) Temperature (°C) Primer source In silico specificity PCR validation Gel profilez)



Forward (5′–3′) Reverse (5′–3′) Chr# Start End Br1 Br2 Br3 Br 4 Br 5 Br 6 Bo1 Bo2 Bo3 Bo4
1 TTGTTTGATCAGGGGAGCAT CCAGGGTACCTATCCGCTTT 858 58.45 1 21801279 21802374 C C 1 2 - 2 2 2 2 2 2 2
2 TTGCAGATTTGATGTTGTGAA GCGCGGGTTATTACCTAGTG 713 57 2 23504716 23506305 C C 1 2 3 1 3 1 1 1 1 1
3 CTACCGCCAAGTTCAAGCA TAAGGGACCCCAAAGCATAA 773 56.85 3 742954 744043 C C 1 2 3 3 3 3 1 1 1 1
4 TTGAGCCTAGTCCGAGCAAT CTCATCTCCAAACCCCATTC 749 58.4 3 10933621 10934710 C C 1 2 1 3 3 1 1 1 1 1
5 ACCGTGGTGAGGTAAAAGGA TGTTCTTTGCCTTGGAACCT 751 57.4 3 18816045 18817634 C C 1 2 1 3 3 1 2 2 2 2
6 TGCAGATCTCTTTGCTCATCA CTGTTCTCTGCGATGCTCAC 746 58.95 3 20935930 20936992 C C 1 2 2 2 2 2 2 2 2 2
7 GCATCTCTGAGCTGGTTTCC GTCCTCGTTGACGGAGAAAG 976 60.5 3 17138648 17140237 C C 1 2 - - - - - - - -
8 GCAAATTATGCACAATCTTACAA TGGATATATGATGCTGTCAAAAA 751 55.7 3 23886171 23887233 C C 1 2 - 2 1 2 - - - -
9 GGAATCGAATGGGATCAAAA TCTAAAAACGCTGGCTCCAT 886 55.35 5 11493271 11494360 C C 1 2 3 3 3 3 - - - -
10 CCGGCTGATTGCTCTAATGT CAACATATGCCTCCACCACA 816 58.4 5 104661 105738 C C 1 2 - 2 2 2 - - - -
11 TGAAACAAAACGCTTTCTCG TCAAAATAGTCACCAATCGGAGT 988 56.8 5 3281961 3283550 C C 1 2 - 1 1 1 - - - -
13 CAATGCAAGCCTCACGTATG CTTATTGGCCATGCCTGACT 647 58.4 5 22026785 22027585 C C 1 2 - - - - - - - -
12 CGCAAAAGTGTACAAAATCTCAA TGGAGTAGACCTGGCGGTAA 644 59 5 2018577 2019377 C C 1 2 - - - - - - - -
14 GGCACAACCAAGCCAATAAT TTACACGCACCGAATTTGAC 735 56.4 6 22061648 22062736 C C 1 2 2 2 2 2 2 2 2 2
15 AAACGGCAATTCGTCTTTTC TTGCCTCGTAGCACTTTTCTC 755 56.85 6 18749148 18750231 C C 1 2 - 3 2 2 2 2 2 2
16 AAAGAAAGCTTTGGCTTAGCTG ACCCATATCACCCGACCATA 710 58.4 6 23123270 23124363 C C 1 2 - 3 3 1 2 2 2 2
17 GAAGAAGCGAGCGAGAAGAA CTTGCCTTCTGATCCCAATC 910 58.4 7 22424472 22426064 C C 1 2 - 3 1 1 2 2 2 2
18 ATGTCGCAACTGAACCAAAA CAAATTACATTCGGGGCCTA 723 55.35 8 3233906 3235467 C C 1 2 2 3 3 2 - - - -
19 CCAGCTCACCACTTCACAAA CAAAACAATCGGTTGGGAAT 712 56.35 8 16701066 16702155 C C 1 2 2 2 2 1 - - - -
20 TGTACGTACGTGAGAATGAGATAAT ACCCTCATGATGCATGGTTT 726 58.65 8 2388601 2389689 C C 1 - - - - - 2 - - 2
21 ATAATAGGCGGCAAGAGCAC AAACCATCGAAATGCTCACC 705 57.4 9 3996150 3997235 C C 3 2 2 3 3 2 - - - -
22 CGATGGTACATCAAAAACAAACA CCATATGGTCCAAGGAAGGA 824 57.95 9 23729398 23730487 C C 1 2 3 3 1 2 2 2 2 2
23 AATTGGGACGAAAAGGGATT CTTTCGGAAACAGAGGGTGT 768 56.35 9 22776 23864 C C 1 2 - 2 2 2 - - - -
24 AGCCTACCGCTTAATGCAAA TGTACAATGTATTTTCCTAACCAAAG 778 58.2 9 6049964 6051065 C C 1 2 - 1 1 1 - - - -
25 ACAACGCACTTTCAAAAGCA CACCGAAGTTTTCTTTTGCTG 814 55.85 9 6435580 6436680 C C 1 2 - - - - - - - -
26 TGAGAAGCGTTTTCTGAGCA CGGGTGTTTTTATAAGTTACACGTT 827 58.65 10 15070338 15071427 C C 1 2 1 3 1 1 2 2 2 2
27 CTCACCAGCAGGGACACATA TGGGCCACATTTTCTTAGGT 805 58.45 10 15382535 15383623 C C 1 2 - 3 3 2 2 2 2 2
28 TGTTTACGGCAAGAACAAGA GGTGATCATGAAAGATGCAA 842 54.3 3 12382669 12383669 K K 2 3 - - - - 1 1 1 1
29 CACCTCCTTCTCGCAGTATT GAGGAAGGAAAAGGTTCGAG 897 58.4 5 3096661 3097721 K K 2 1 - - - - - - - -
30 TTGGATCAGATTCGGTTTTT ACGGCCAAAGATTACAACAT 795 53.3 5 4793442 4794518 K K 2 3 1 1 1 1 - - - -
31 CGAAACAAGAACCCAAAAAC GCCCAATGACCACTCTAAAC 883 56.35 5 19979539 19980595 K K 2 3 - - - - - - - -
32 CCTTTGTGGGGTTTACTGTC TCTGATTACCAAACCTTGCAT 843 56.95 6 17041904 17042957 K K 2 1 - - - - 2 2 2 2
33 GGTTTCCTTTGGTGGTGATA AATAACCGGATCAAACCTGA 784 55.35 6 24368107 24369107 K K 2 3 - - - - 1 1 1 1
34 TGGTTGGAGATGGAAAATAAA TCGAAATTCAGCTCAATCAA 808 52.9 9 27626655 27627536 K K 2 1 2 2 2 2 2 2 2 2
35 GTGTAGCCTATGGGGACGAA CGCCCCTAAAGACAGCTAAA 701 59 1 963071 964133 C Shared 1 - 1 1 - - - - - -
36 GCTGTCCCTGAAAAAGGAGA TCCGGTTGGCTTAAAAATTG 843 56.35 1 13502760 13503861 C Shared 3 3 3 3 3 3 1 1 1 1
37 AGCTCATACACCTCGGGAGA ATGCAGCTCGTGTCTGAGAA 759 59.45 1 19445593 19446681 C Shared 1 1 - 1 1 1 2 2 2 2
38 AGGTACGGTTTCTCGGATCA CGTGCATAGCTGTAAAACGA 620 57.4 1 2584681 2585481 C Shared 3 3 - - - - - - - -
39 ACAGAGGACCATACCGGAAC AAGTCCTAAATACTCCCTCCGTTT 694 61.15 1 2961011 2961811 C Shared 1 1 - - - - - - - -
40 TTCACCACGAGTTGTCTTCG GCGTTTCTCAATTCCTCTGC 611 58.4 1 9276641 9277441 C Shared 1 1 - - - - - - - -
41 TTGCATTAGCCGACAATGAT GGAGCTTCTTCTTTCCACCA 630 56.35 1 10697577 10698377 C Shared 1 1 - - - - 2 2 2 2
42 TGGGAATCGTAGGAATGGAG CCATCAGAGTCTCACCGACA 619 59.45 1 14754268 14755068 C Shared 1 1 - - - - 1 1 1 1
43 TCAAGCAACTTCTCCAGCAA AGGTCAGTGCAAGCAAAGGT 432 57.4 1 18275269 18276069 C Shared 1 1 - - - - 1 1 1 1
44 ATCCGATCAACATTGCCTTC CATTTTGTGGTGAAGCTGGA 652 56.4 1 22118044 22118844 C Shared 1 1 - - - - 2 2 2 2
45 GAAGCCACTACTGGTGTGTATGA TGACATCATTCTTGCTATGATCC 661 61.1 1 23625986 23626786 C Shared 1 1 - - - - - - - -
46 GCAATTGTCAACAATACGTAAGC TGTCGTTTTCGGTTTTCTATG 659 57.4 1 27587965 27588765 C Shared 3 1 - - - - - - - -
47 TTGGGGACCAATACCAATGT GAGTGTTGGCCTTCGTCTTC 857 58.45 2 8839647 8841236 C Shared 1 1 1 1 1 1 2 2 2 2
48 AAAAGCCTAAGGGCATCTCC AATGCCTGCCCGTTACTCTA 856 58.4 2 20825514 20826587 C Shared 1 1 1 1 1 1 2 2 2 2
49 TGGTCTGATTGGTTCATTGG CTGCAAAATAACCGGTTTGA 719 55.35 2 532895 533984 C Shared 1 1 - 1 1 1 2 2 2 2
50 GCTGTTGATATCGAAGAATGTGA AAAACCGGAAGGAGTAACAAAA 707 57.95 2 6905529 6906618 C Shared 1 1 - 1 - 1 - - - -
51 CCGTAGAATGTGTGGGTGAA AGAAGGCAAAGGCAAAGACA 809 57.4 2 25921691 25922779 C Shared 3 3 - 3 3 2 - - - -
52 CAAAGCCAGCTTCGTCTTTC TTTTGGAAACGAGGGAGTACA 831 57.9 2 27426840 27427899 C Shared 1 1 - 1 1 1 - - - -
53 CCAAGGGTGTTAGGGATATTT CCCATACCTTTTCAAACCAG 812 56.9 2 7699471 7700522 K Shared 1 1 - - - - - - - -
54 CAGGTTGTTGTGGGTTTTGA ACAGTCGCCATTTCTCACCT 705 57.4 3 2817737 2818831 C Shared 1 1 1 1 1 1 - - - -
55 AACGTGTGTGGGTGAAAGTG TGTGTACATGGCATTTGCTG 886 57.4 3 24785163 24786251 C Shared 1 1 2 2 2 2 - - - -
56 CCCGATAAAAATTTATGGTAGCAC ACGCAAGTCAGAGCTGGTTA 876 59.25 3 29733660 29734749 C Shared 1 1 1 1 1 1 - - - -
57 TGCTGCAAATGCAACTTTTT CCTGCCCCAACTGTATTTTC 934 55.35 3 22824441 22825982 C Shared 1 1 - 1 - - - - - -
58 CGAATATGGACACGTGAAAA GTCCATAGAGGCATCCAAAC 778 56.35 3 8150869 8151901 K Shared 3 3 - - - - 2 2 2 2
59 AAATGTCGCCACTGAATCTG AACCGAATCAAACCAACCAG 869 56.4 4 15959699 15960723 C Shared 1 1 1 1 1 1 2 2 2 2
60 TGAATTGAAGCCACAAGCTA CACGTGTTGTTTTGTTCGTT 842 54.3 4 13346608 13347631 K Shared 1 1 - - - - 1 1 1 1
61 TGTCGTTTTGGTTTTCAATG GTGCCAGATTTTTAGCGACT 802 54.3 4 15212718 15213750 K Shared 3 3 - - - - 1 1 1 1
62 AGCAAGTGCCTCTCGAGTCT TCAAAATAGTCACCAATCGGAGT 851 59.9 5 3281461 3283050 C Shared 1 1 1 1 1 2 - - - -
63 TTACGGAGGGAAAGCAGAGA CGTAAATGCTCTCCAAATGC 776 57.4 5 10244950 10246022 C Shared 1 1 3 3 3 1 - - - -
64 TCGATTTCTTCCCATCAACC TTGGAAGTAGCTCCGCAAAT 883 56.4 5 12834818 12835901 C Shared 3 3 3 3 3 3 2 2 2 2
65 CTCTTCCGCTCTACCAACTGA TCCTTCAACCTCCAACATGA 891 58.85 5 18108358 18109422 C Shared 1 1 1 1 1 1 1 1 1 1
66 CTGCTTGAATCGGCTACAAA CGGGCATCCAAATACTCTGT 826 57.4 5 18163408 18164496 C Shared 1 1 1 1 1 1 3 2 2 3
67 GGAATGGTGAAGGACCTGAA CCTAGCTCGACCATGGAGAC 786 60.45 5 19371747 19372834 C Shared 1 1 1 1 1 1 - - - -
68 TTTGCACCTAATTGATTTCCTTT TGTCACGTGTGAAACATACTCC 811 58 5 21905619 21906708 C Shared 1 1 1 1 1 1 - - - -
69 GGGTGGTTAACGAGCCAGTA TGGAAAACCATGGCAAAAA 879 55.7 5 23188047 23189647 C Shared 1 1 1 1 1 1 2 2 3 2
70 GCGTGGTTACCTTCAATTCC AGACTCGAGAGGCACTTGCT 614 59.45 5 3282051 3282851 C Shared 1 1 - - - - 1 1 1 1
71 TTTTTGGAGATGCATTTAGTGG ACGCCAAAACTGAAAAGGAA 465 55.45 5 7674505 7675305 C Shared 1 1 - - - - 1 1 1 1
72 GGAGTTGCCATATTGGAAGG GCCTCATACAGGAGGTGAGC 683 60.45 5 12835307 12836107 C Shared 1 1 - - - - 1 1 1 1
74 CAAAATCCACCGTCAAACTG CCATTCAACCCGCTGTTACT 454 57.4 5 16754371 16755171 C Shared 2 3 - - - - 3 3 3 3
75 CGTGCATAGCTGTAAAACGAC TGCATCTGCTGCTTTCATTT 622 56.85 5 18109146 18109946 C Shared 2 3 - - - - - - - -
73 AGGCCAACACAATAGGATCG AAACGGCTACCACATCCAAG 442 58.4 5 13116126 13116926 C Shared 1 1 - - - - 1 1 1 1
76 CACGGTTGTGTGACAGATTG GCCTAGGCTAGTGACCTCCA 713 60.45 6 18132269 18133370 C Shared 1 1 2 2 2 2 2 2 2 2
77 ACACTGTGCGGACAAAAATG TCTTTCTGCAAACCCCTAGC 841 57.4 6 22195497 22196585 C Shared 1 1 3 3 1 2 - - - -
78 TAAGGTGGGCCGTAACGTAG GTCTCCGATGAAACGATGCT 845 59.45 6 15006054 15007142 C Shared 1 1 - 3 2 1 - - - -
79 TTGGGATGACAAGGATTTCT CGACAAGCACAGAGACAAAG 885 56.35 6 2249171 2250222 K Shared 1 1 - - - - - - - -
80 CACATGGAACCTTTCTCCTC TATCGGGTAAAGCCAATGAT 774 56.35 6 20148620 20149708 K Shared 1 1 - - - - 1 1 1 1
81 GGGGTTAGAATCGTCCTTTT TTCTTGCGTGTTGGTATCAC 858 56.4 6 21234008 21235039 K Shared 1 3 - - - - 2 2 2 2
82 TGTAGACTCCTCCCAACGTCT GGAAGTGGTGGATGCTGTTT 810 59.85 8 13609744 13610827 C Shared 1 1 1 3 3 1 - 2 - -
83 GGCAGAGAGCAGTTTCGATT CTTCTCTGCCCAAACCTACC 897 59.45 8 17589879 17591468 C Shared 1 1 2 3 1 2 - - - -
84 TTGAAAAGCAAACCCTTCTC ATTTTGGTTGGTTCATACCG 779 54.3 8 19318537 19319537 K Shared 1 1 - - - - - - - -
85 GCACTGAGCTTTGTCATGGA TCTCACGTCCCTCTCCATCT 742 59.45 9 6120652 6121741 C Shared 1 - - - - - 3 2 3 3
86 CGAGGAATGTGGTGATGATG CGAGGAATGTGGTGATGATG 810 58.4 9 6223231 6224320 C Shared 1 1 - - - - - - - -
87 AGCAGCTATTGCATGGTCAC TTGAGCTCTATTGGCAAGCA 722 57.4 9 19954499 19955588 C Shared 1 1 1 1 1 1 2 2 2 2
88 CCACCCTTCAAGCATCAAAT AAACAGAGACAACGCTGCTG 876 57.4 9 26437989 26439077 C Shared 1 1 3 3 1 2 3 1 3 2
89 GGTTTGCTACCCACAAAATCA CGCTCCTCTATTCGGACACT 898 58.95 9 26805934 26807535 C Shared 1 1 1 1 1 2 - - - -
90 AGGCCCCAATTTCCCTATAA CCTTTTTCACCATTTACATTCG 877 56.5 10 7137934 7139011 C Shared 1 1 1 3 3 1 - - - -

z)Scores of gel profile were followed by previous report (Murukarthick et al. 2014) as 1, full site; 2, empty site; 3, full and empty site; and -, no amplification. Bold used as a representative for each group in Fig. 3.

Bs2: BraSto-2, Chr#: chromosome numbers, NGS: next-generation sequencing, PCR: polymerase chain reaction, C: ‘Chiifu’, K: ‘Kenshin’.

Table 1 List of accessions used for the display and insertion survey.
Table 2 Summary of reads analysis from NGS-based transposon display of Bs2 MITE family against two Brassica rapa accessions.

Values are presented as number only or number (%).

Bs-2 sites specific to Br1 and Br2.

NGS: next-generation sequencing, MITE: miniature inverted-repeat transposable element, PCR: polymerase chain reaction, IP-Bs2: Insertion polymorphism of Brasto-2 (bs2) members.

Table 3 Insertion polymorphisms survey of Bs2 candidates based on NGS-based transposon display analysis against 10 Brassica accessions.

Scores of gel profile were followed by previous report (Murukarthick et al. 2014) as 1, full site; 2, empty site; 3, full and empty site; and -, no amplification. Bold used as a representative for each group in Fig. 3.

Bs2: BraSto-2, Chr#: chromosome numbers, NGS: next-generation sequencing, PCR: polymerase chain reaction, C: ‘Chiifu’, K: ‘Kenshin’.