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Research Article

Development of SNP Marker Set to Select Varieties Tolerant to Multiple Abiotic Stresses in Rice

Jung-Woo Lee, Jung-Seok Oh, Soo-Cheul Yoo2,*
Plant Breeding and Biotechnology 2023;11(3):208-219.
Published online: September 1, 2023

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*Corresponding author Soo-Cheul Yoo, scyoo@hknu.ac.kr, Tel: +82-31-670-5082, Fax: +82-31-670-5089, †These authors contributed equally to this study.
• Received: August 16, 2023   • Revised: August 23, 2023   • Accepted: August 24, 2023

Copyright © 2023 by the Korean Society of Breeding Science

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Development of SNP Marker Set to Select Varieties Tolerant to Multiple Abiotic Stresses in Rice
Plant Breed. Biotech.. 2023;11(3):208-219.   Published online September 1, 2023
DOI: https://doi.org/10.9787/PBB.2023.11.3.208
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Development of SNP Marker Set to Select Varieties Tolerant to Multiple Abiotic Stresses in Rice
Plant Breed. Biotech.. 2023;11(3):208-219.   Published online September 1, 2023
DOI: https://doi.org/10.9787/PBB.2023.11.3.208
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Development of SNP Marker Set to Select Varieties Tolerant to Multiple Abiotic Stresses in Rice
Image Image
Fig. 1 Representative clustering patterns of genotyping. Colored plots represent the genotyping results from the Fludium markers for 172 domestic and abroad varieties. Each color code of the plots has the meaning of genotypes: homozygotes of Allele X (red; FAM fluorescence) and homozygotes of Allele Y (green; HEX fluorescence). The plots of gray color have meaning of no call genotype or NTC (non-template control). (A-Q) Genotyping result for the marker of Saltol-SNP-1 (A), Saltol-SNP-2 (B), Saltol-SNP-3 (C), Saltol-SNP-4 (D), Saltol-SNP-5 (E), DTY2-2-SNP-1 (F), DTY2-2-SNP-2 (G), DTY2-2-SNP-3 (H), DTY2-2-SNP-4 (I), DTY4-1-SNP-1 (J), DTY4- 1-SNP-2 (K), DTY4-1-SNP-3 (L), AG1-KP-SNP-1 (M), AG1-KP-SNP-2 (N), AG1-KP-SNP-3 (O), Sub1A-SNP1 (P), Sub1A-SNP2 (Q).
Fig. 2 Phylogenetic tree constructed using the UPGMA method. (A, B) The phylogenetic tree of 172 domestic and abroad varieties using the developed Fludium markers for four abiotic stresses (A) and saline stress (B). Red solid circles represent foreign varieties and the others are domestic varieties.

Fig. 1

Fig. 2

Development of SNP Marker Set to Select Varieties Tolerant to Multiple Abiotic Stresses in Rice

List of KASP markers developed in this study and relevant information

KASP marker assays ID Trait Chromosome Position (bp) Target gene/QTL SNP (phenotypea)) SNP (color of dyeb))
Saltol-SNP-1 Salinity 1 10,217,743 Saltol C(T):T(S) C(F):T(H)
Saltol-SNP-2 Tolerance 1 11,430,304 - A(T):G(S) A(F):G(H)
Saltol-SNP-3 1 11,460,305 - G(T):C(S) G(F):C(H)
Saltol-SNP-4 1 11,463,299 - A(T):T(S) A(F):T(H)
Saltol-SNP-5 1 11,463,595 - A(T):C(S) A(F):C(H)
DTY2-2-SNP-1 Drought 2 2,085,377 qDTY2.2 G(T):C(S) G(F):C(H)
DTY2-2-SNP-2 Tolerance 2 2,792,380 - A(T):G(S) A(F):G(H)
DTY2-2-SNP-3 2 3,200,039 - A(T):G(S) A(F):G(H)
DTY2-2-SNP-4 2 4,044,509 - A(T):C(S) A(F):C(H)
DTY4.1-SNP1 Drought 4 222,034 qDTY4.1 C(T):T(S) C(F):T(H)
DTY4.1-SNP2 Tolerance 4 330,380 - C(T):T(S) C(F):T(H)
DTY4.1-SNP3 4 2,281,969 - T(T):C(S) T(F):C(H)
AG1-KP-SNP-1 Anaerobic 9 12,333,777 OsTPP7 C(T):T(S) C(F):T(H)
AG1-KP-SNP-2 germination 9 12,326,525 - G(T):A(S) G(F):A(H)
AG1-KP-SNP-3 Tolerance 9 12,215,550 - A(T):G(S) A(F):G(H)
Sub1A-SNP1 Submergence 9 Sub1A gene Sub1A C(T):T(S) C(F):T(H)
Sub1A-SNP2 Tolerance 9 Sub1A gene - A(T):G(S) A(H):G(F)

a)T: tolerant, S: susceptible.

b)F: FAM fluorescence, H: HEX fluorescence.

List of KASP marker assay primers used in this study.

KASP marker assays ID FAM-labelled primer (5’-3’) HEX-labelled primer (5’-3’)1) Reverse primer (5’-3’)2)
Saltol-SNP-1 TTTGGTAAGACATGAATGAACATCAG CTTTTGGTAAGACATGAATGAACATCAA CCACTGCACTACATTTTCTTTCAACTGAA
Saltol-SNP-2 TTCCAGGAACATCATAAATTTTGCTATT TCCAGGAACATCATAAATTTTGCTATC GGGCATCCTGAGACAAATGTTTTCCAT
Saltol-SNP-3 GACACGGTGGAGAGGTCGAC GACACGGTGGAGAGGTCGAG ACTCGAGGCACTCCGGTGAGAT
Saltol-SNP-4 ATAAACAACAAGTGTGAGAAAACCCT CTATAAACAACAAGTGTGAGAAAACCCA GAACTCATTTCTCTCCTATGTGTTTCGTT
Saltol-SNP-5 GTTCAACAAATAGGACTTCATATTTATGGAA CAACAAATAGGACTTCATATTTATGGAC CGCAGACACTTGTAGCATTAGATTTCATT
DTY2-2-SNP-1 GTTCTGTTTGACTTTGACTTCG GCTGTTCTGTTTGACTTTGACTTCC CCAATGTATTTTTTTGCCTTTTTGTTTCAA
DTY2-2-SNP-2 GACACTAGAATCAACAGTAATCCTGAT ACACTAGAATCAACAGTAATCCTGAC GCATATTTGTTTCACTGCTGATGACTCTA
DTY2-2-SNP-3 GATTTTCTTAGTTGCAGTATTTTCTGATTCA TTCTTAGTTGCAGTATTTTCTGATTCG GATGCCAAAACACAATGCATATAAAGGAAA
DTY2-2-SNP-4 GAGTTTTTGAGCTGGTAAATTTTGGAATTATT GTTTTTGAGCTGGTAAATTTTGGAATTATG CACTTGAAATTTGAATGACGTGGAGTACTA
DTY4.1-SNP1 CAATCGCTGATGAAGCTGTCGAC CAATCGCTGATGAAGCTGTCGAT CTGCCGACTGCATTATCCCTACTTT
DTY4.1-SNP2 GCTGATTTCATTGAATTTTGTTGC GGCTGCTGATTTCATTGAATTTTGTTGT GTTCATACTACGTGAAAACGAACTTCACAA
DTY4.1-SNP3 GCCACCGTGTGGGAGATGGAA CCACCGTGTGGGAGATGGAG TGGCCATGGCGGCGGCGAA
AG1-KP-SNP-1 ACGTTACAATGGTTTGAGTATATGG GTCTACGTTACAATGGTTTGAGTATATGA TTGTAAGGGTAAGGGTGGGACCTAT
AG1-KP-SNP-2 GAGACGGAGAAGACGGAGAAG GGAGACGGAGAAGACGGAGAAA CCATCACCGCCAAGAAGTCATCTTA
AG1-KP-SNP-3 GGATTGCAATAAATTATTGTCAAGTTTTACAA GCAATAAATTATTGTCAAGTTTTACAG GGATACAGTGTTTGAAATATGGAAATGTTA
Sub1A-SNP1 AGAAGCAGAGCGGCTGCGG CAAGAAGCAGAGCGGCTGCGA CTTCCCACCCGCCGATCTTTCTT
Sub1A-SNP2 GTCCGAGCAGCACTCCAGC CGTCCGAGCAGCACTCCAGT GTCCGGCGACGCGCGCATA

1)FAM/HEX-labeled primers are allele-specific primers.

2)Reverse primers are common primers.

Validation of KASP markers with the control varieties.

Sample Saltol-SNP-1 Saltol-SNP-2 Saltol-SNP-3 Saltol-SNP-4 Saltol-SNP-5
Allele RFU_1 RFU_2 Allele RFU_1 RFU_2 Allele RFU_1 RFU_2 Allele RFU_1 RFU_2 Allele RFU_1 RFU_2
IR64 Allele2 3765.08 4968.89 Allele2 2612.31 2545.97 Allele2 3812.58 6702.01 Allele2 2646.61 2499.42 Allele2 2669.36 2660.83
IR64-Saltol Allele1 6514.09 2882.8 Allele1 3041.22 2049.5 Allele1 7919.24 3105.35 Allele1 3097.16 2023.94 Allele1 3408.14 2111.12
Pokkali Allele2 3813.43 6124.81 Allele1 2831.07 1998.89 Allele1 7277.74 2992.35 Allele1 3145.07 2028.88 Allele2 2700.2 2704.92
NTC No Call 3708.49 1896.96 No Call 2576.03 1874.63 No Call 3555.29 1889.66 No Call 2557.01 1854.77 No Call 2655.05 1856.12
Sample DTY2.2-SNP-1 DTY2.2-SNP-2 DTY2.2-SNP-3 DTY2.2-SNP-4
Allele RFU_1 RFU_2 Allele RFU_1 RFU_2 Allele RFU_1 RFU_2 Allele RFU_1 RFU_2
IR64 Allele2 2629.21 2630.05 Allele2 3726.04 5402.83 Allele2 3765.29 5668.48 Allele2 3908.31 6165.91
IR64-DTY2.2+4.1 Allele1 3257.18 2053.37 Allele1 7525.46 3127.95 Allele1 6976.09 2990.59 Allele1 7652.61 3160.55
Addey sel Allele1 3265.54 2011.19 Allele1 7301.05 3169.9 Allele1 7151.86 3058.99 Allele1 7323.76 3030.61
NTC No Call 2581.52 1854.69 No Call 3375.49 1890.55 No Call 3473.84 1928.7 No Call 3621.66 1897.18
Sample DTY4.1-SNP1 DTY4.1-SNP2 DTY4.1-SNP3
Allele RFU_1 RFU_2 Allele RFU_1 RFU_2 Allele RFU_1 RFU_2
IR64 Allele2 2648.43 2507.94 Allele2 2630.02 2586.76 Allele2 2630.83 2636.64
IR64-DTY2.2+4.1 Allele1 3161.85 2040.5 Allele1 3171.15 2062.87 Allele1 2771.05 2604.9
Addey sel Allele1 3199.2 2051.37 Allele1 3232.52 2074.23 Allele1 3236.89 2063.25
NTC No Call 2560.05 1859.41 No Call 2560.05 1859.41 No Call 2560.05 1859.41

RFU_1: FAM fluorescent values, RFU_2: HEX fluorescent values, NTC: non-template control.

Table 1 List of KASP markers developed in this study and relevant information

a)T: tolerant, S: susceptible.

b)F: FAM fluorescence, H: HEX fluorescence.

Table 2 List of KASP marker assay primers used in this study.

1)FAM/HEX-labeled primers are allele-specific primers.

2)Reverse primers are common primers.

Table 3 Validation of KASP markers with the control varieties.

RFU_1: FAM fluorescent values, RFU_2: HEX fluorescent values, NTC: non-template control.

Table 1

Table 2

Table 3

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