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Research Article

Development of Kompetitive Allele Specific PCR Markers for Anaerobic Germination 1 Locus in Rice

Jung-Woo Lee1, Joong Hyoun Chin2, Soo-Cheul Yoo1,3,*
Plant Breeding and Biotechnology 2021;9(1):20-31.
Published online: March 1, 2021

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*Corresponding author Soo-Cheul Yoo, scyoo@hknu.ac.kr, Tel: +82-31-670-5082, Fax: +82-31-670-5089
• Received: September 1, 2020   • Revised: November 22, 2020   • Accepted: December 10, 2020

Copyright © 2021 by 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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Development of Kompetitive Allele Specific PCR Markers for Anaerobic Germination 1 Locus in Rice
Plant Breed. Biotech.. 2021;9(1):20-31.   Published online March 1, 2021
DOI: https://doi.org/10.9787/PBB.2021.9.1.20
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Development of Kompetitive Allele Specific PCR Markers for Anaerobic Germination 1 Locus in Rice
Plant Breed. Biotech.. 2021;9(1):20-31.   Published online March 1, 2021
DOI: https://doi.org/10.9787/PBB.2021.9.1.20
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Development of Kompetitive Allele Specific PCR Markers for Anaerobic Germination 1 Locus in Rice
Image Image Image Image Image
Fig. 1 Genotyping and phenotyping of IR64-AG1 plants with control plants. (A) Schematic diagram of InDel region in AG1 locus. The yellow color represents the InDel region located in AG1 locus and the light green color represents the region of OsTPP7 (Os09g20390). The arrows indicate the primer positions of the existing gel-based marker (DFR marker, Kretzschmar et al. 2015). (B) PCR amplification of control varieties with existing AG1 gel-based marker. (C) Phenotypic screening and survival rates of the control plants for anaerobic germination.
Fig. 2 Primer design of AG1-InDel region for KASP marker development and genotyping of the control varieties. (A) Primer design of AG1-KP-InDel-1 marker in AG1-InDel region. Arrows indicate the location and direction of the primer sequences. One common primer (Common-primer-1) and three allele-specific primers (Allele-specific primer-1, Allele-specific primer-2 and Allele-specific primer-3) targeting the SNP position (G/A) were designed. (B) Genotyping results of the control varieties by the developed AG1-InDel specific KASP marker (left table) and the scatter plot of the KHO and IR64 type alleles (right panel). Allele 1 and Allele 2 are tolerance (KHO) and susceptible (IR64) types, respectively. RFU1 and RFU2 represent FAM and HEX fluorescent values, respectively (NTC = non template control).
Fig. 3 Genomic location of the four AG1 flanking KASP markers and validation of the markers with the control varieties. (A) Genomic position of the AG1 InDel and four flanking SNPs that were used to develop the flanking KASP markers, AG1-KP-SNP-1, AG1-KP-SNP-2, AG1-KP-SNP-3 and AG1-KP-SNP-4 markers. Red arrows represent the position of the KASP markers on chromosome 9. (B) Genotyping results of the control panel by the KASP markers. IR64-A, P and S represent IR64 harboring AG1, Pup1 and Sub1, respectively.
Fig. 4 Validation of the existing gel-based DFR marker and three developed KASP markers using BC3F2 segregating lines. (A) Genotyping of 27 BC3F2 segregating lines with the DFR marker. (B) Genotyping of 27 BC3F2 segregating lines with the developed KASP markers. (C) The scatter plot of the KHO, heterozygote and IR64 type alleles. Orange, blue and green dots are allele 1 (tolerant), allele 2 (sensitive) and heterozygote types, respectively. T, tolerant type; S, sensitive type; H, heterozygote.
Fig. 5 Neighbor joining tree for the profiling of the 172 rice varieties using the five KASP markers (AG1-KP-SNP-1, AG1-KP-SNP-2, AG1-KP-InDel-1, AG1-KP-SNP-3 and AG1-KP-SNP-4). Hollow and solid circles represent Korean and foreign varieties, res-pectively.&nbsp;

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Fig. 5

Development of Kompetitive Allele Specific PCR Markers for Anaerobic Germination 1 Locus in Rice

The list of the primers used in this study.

Gel-based marker Primer name Sequence (5ʹ-3ʹ)
DFR DFR_F2 CCACCATGATGTAGTTCAGTTGTGAAC
DFR_R2 CACCGTTAAAATCGGCCGTTAG
DFR_LB2 CGGCTTCGTCTTCACCTGAAC
KASP marker Primer name Sequence (5ʹ-3ʹ)
AG1-KP-InDel-1 Allele-specific primer-1 TAATTTCTTCGTTTTGTTCTCGATGTTTC
Allele-specific primer-2 AATTTCTTCGTTTTGTTCTCGATGTTTT
Allele-specific primer-3 GGCTTCGTCTTCACCTGAACGAAA
Common-primer-1 GAAGGATTACTTATATGACACCTAGCCTT
AG1-KP-SNP-1 Allele-specific primer ACGTTACAATGGTTTGAGTATATGG
Allele-specific primer GTCTACGTTACAATGGTTTGAGTATATGA
Common-primer TTGTAAGGGTAAGGGTGGGACCTAT
AG1-KP-SNP-2 Allele-specific primer GAGACGGAGAAGACGGAGAAG
Allele-specific primer GGAGACGGAGAAGACGGAGAAA
Common-primer CCATCACCGCCAAGAAGTCATCTTA

Agreement of the markers calculated by Cohen’s kappa method.

Markers % of consistency with the DFR marker Cohen’s Kappa coefficient (k) Strength of agreementz)
AG1-KP-InDel-1 100 1.00 Very good
AG1-KP-SNP-1 70.3 0.54 Moderate
AG1-KP-SNP-2 66.7 0.48 Moderate

Profiling of 172 domestic and foreign varieties using developed KASP markers in this study.

No Variety Ori Marker name No Variety Ori Marker name
M1 M2 M3 M4 M5 M1 M2 M3 M4 M5
1 APO FR S T T S S 87 IR29 FR T T T T T
2 Akitakomachi FR S T T T T 88 IR8 FR S S S S S
3 MS11 FR S T T T T 89 IR56 FR S S S S S
4 Nipponbare FR S T T T T 90 IR36 FR S T T S S
5 Koshihikari FR S T T T T 91 IR24 FR S T T S S
6 Hitomebore FR S T T T T 92 IR64 FR S S S S S
7 Azucena FR S T T T T 93 IR72 FR S T T T T
8 Ausboro FR S T T S S 94 IR49830 FR T S S S S
9 Aus299 FR S T T S S 95 Chucheongbyeo KO S T T T T
10 ARC11544 FR S T T T T 96 Yeonghojinmi KO S T T T T
11 Aswina FR T T T S S 97 Junam KO S T T T T
12 Aus261 FR S T T T T 98 Ilmi KO S T T T T
13 CO39 FR S T T S S 99 Nampyeong KO S T T T T
14 Chahora 144 FR S T T T T 100 Samkwang KO S T T T T
15 BR11 FR S T T S S 101 Ilpum KO S T T T T
16 Basmati 370 FR S T T S S 102 Hopum KO S T T T T
17 Basmati bahar FR T T T T T 103 Haiami KO S T T T T
18 Binadhan10 FR T T T S S 104 Dongjin 1 KO S T T T T
19 Fedearroz50 FR T T T T T 105 Sindongjin KO S T T T T
20 Dular FR T T T T T 106 Dongjin KO S T T T T
21 Dom sofid FR S T T T S 107 Gopum KO S T T T T
22 ColombiaXXI FR S S S S S 108 Seonong 1 KO S T T T T
23 Crdhan 405 FR S T T NC NC 109 Seonong 2 KO S T T T T
24 Cypress FR S T T T T 110 Seonong 5 KO S T T T T
25 Kalarata 1-24 FR T T T T T 111 Seonong 4 KO S T T T T
26 IRBB66 FR S T T S S 112 Seonong 3 KO S T T T T
27 Inia Tacuri FR S T T T T 113 Seonong 6 KO S T T T T
28 FL478 FR T T T T T 114 Seonong 7 KO S T T T T
29 Giza 178 FR T T T T T 115 Seonong 8 KO S T T T T
30 IAC 165 FR S T T T T 116 Seonong 11 KO S T T T T
31 Komboka FR T T T S S 117 Seonong 10 KO S T T T T
32 Kharsu 80A FR T T T T S 118 Seonong 9 KO S T T T T
33 Kasalath FR S T T S S 119 Seonong 12 KO S T T T T
34 Khaiyan FR S NC T NC NC 120 Seonong 13 KO S T T T T
35 Khao Lhan On FR S T T T T 121 Seonong 14 KO S T T T T
36 Minghui 63 FR T T T T T 122 Seonong 17 KO S T T T T
37 MatatAG1 FR T T T T T 123 Seonong 16 KO S T T T T
38 Makassane FR S T T T T 124 Seonong 15 KO S T T T T
39 Lijiangxin-tuanheigu FR S T T T T 125 Seonong 18 KO S T T T T
40 M202 FR S T T T T 126 Seonong 19 KO S T T T T
41 Madabaru FR T T T T S 127 Seonong 20 KO S T T T T
42 Phka Rumdoul FR T T T T T 128 Hwayong KO S T T T T
43 Osgovka FR S T T T T 129 Chilbo KO S T T T T
44 NSIC RC 238 FR T S S S S 130 Seonong 21 KO S T T T T
45 Moroberekan FR S T T T T 131 Odae KO S T T T T
46 N22 (heat) FR T T T T T 132 Unkwang KO S T T T T
47 NSIC RC 222 FR T S S S S 133 Obong KO S T T T T
48 Rinaldo bersani FR S T T T T 134 Milyang 23 KO S S S S S
49 Rayada FR T T T S S 135 Jinmi KO S T T T T
50 PSB RC 82 FR S T S S S 136 Hwacheongbyeo KO S T T T T
51 Sabri FR S T T T T 137 Dasan KO S S S S S
52 Saducho FR S T T S S 138 Hanmaeum KO S T T T T
53 Samba Mahsuri FR S S S S S 139 Boramchan KO S T T T T
54 Taducan FR T T T S S 140 Milkyqueen KO S T T T T
55 Swarna FR S T T T S 141 Bakjinju KO S T T T T
56 San-Huang-Zhau No2 FR T T T T T 142 Boseogchal KO S T T T T
57 Tai FR T S S S S 143 Dongjinchal KO S T T T T
58 Tainung 67 FR S T T T T 144 Baegokchal KO S T T T T
59 Tong 88-7 FR S T T T T 145 Aromi KO S T T T T
60 Vandana FR S T T T T 146 Heugkwang KO S T T T T
61 Utri Merah FR T T T T T 147 Daerip 1 KO S T T T T
62 Vuninzara FR S S S S S 148 Geunnun KO S T T T T
63 WAB 56-125 FR T T T T T 149 Jeogjinju KO S T T T T
64 Way Rarem FR S T T S T 150 Miho KO S T T T T
65 Kusahonami FR S T T T T 151 Sobi KO S S S S S
66 Phka Rumduol FR T T T T T 152 Shinongheugchal KO S T T T T
67 G Zhenshan 97B FR T T T T T 153 Mipum KO S T T T T
68 Hoshiaoba FR S T T T T 154 Haedamssal KO S T T T T
69 Be Ko Aoba FR S T T T T 155 Joami KO S T T T T
70 Hukuhibiki FR S T T T T 156 Hongjinju KO S T T T T
71 TR22183 FR S T T T T 157 Heugjinju KO S T T T T
72 Inia tacuari FR S T T T T 158 Saenuri KO S T T T T
73 Adday sel FR T T T S S 159 Jeogjinjuchal KO S T T T T
74 Nona Bokra FR T T T T S 160 Seilmi KO S T T T T
75 A69-1 FR S S S S S 161 Cheongwoo KO T T T T T
76 Hasawi FR T T T T T 162 Yeongwoo KO T T T T T
77 IR64-SPIKE FR S S S S S 163 Jonong KO S T T T T
78 IR64-Saltol FR S S S S S 164 Nokwoo KO S T T T T
79 Pokkali FR T T T T T 165 Mokyang KO S S S S S
80 IR64-EMF FR S S S S S 166 Nokyang KO S T T T T
81 IR64-AG1 FR S S T T T 167 Sanggol KO S T T T T
82 IR64-Pup1 FR S S S S S 168 Hanareum 2 KO S T T T T
83 IR64-HTSF4.1(73) FR T T T T T 169 Mogwoo KO T T T T T
84 IR64-DTY2.2+DTY4.1 FR S S S S S 170 Namwon1 KO S T T T T
85 IR64-Sub1 FR S S S S S 171 Jinbubyeo KO S T T T T
86 IR28 FR T T T T T 172 Koryeong13 KO S T T T S
Table 1 The list of the primers used in this study.
Table 2 Agreement of the markers calculated by Cohen’s kappa method.

z)Strength of agreement based on Altman (1990). Poor, k < 0.20; fair, k = 0.21-0.40; moderate, k = 0.41-0.60; good, k = 0.61-0.80; very good, k = 0.81-1.00.

Table 3 Profiling of 172 domestic and foreign varieties using developed KASP markers in this study.

Ori, origin; M1, AG1-KP-SNP-4; M2, AG1-KP-SNP-3; M3, AG1-KP-InDel-1; M4, AG1-KP-SNP-2; M5, AG1-KP-SNP-1; T, tolerant; S, susceptible; FR, foreign; KO, Korean.

Table 1

Table 2

Table 3

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