Development of Kompetitive Allele Specific PCR Markers for Submergence Tolerant Gene Sub1 in Rice

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1 Plant Breed. Biotech (March) 7(1):62~66 RAPID COMMUNICATION Online ISSN: Print ISSN: Development of Kompetitive Allele Specific PCR Markers for Submergence Tolerant Gene Sub1 in Rice Je-Hoon Moon 1, Daniel Son 2, Jung-Woo Lee 1, Soo-Cheul Yoo 1 * 1 Department of Plant Life & Environmental Science, Hankyong National University, Anseong 17579, Korea 2 Department of Agronomy & Medicinal Plant Resources, Gyeongnam National University of Science and Technology, Jinju 52725, Korea ABSTRACT The SUBMERGENCE 1 (SUB1) locus, conferring tolerance to complete inundation, was identified, and gel-based DNA markers, AEX1 and GnS2, were previously developed for marker-assisted breeding (MAB). However, a high throughput and high specific method, at low cost, is still required to detect Sub1 alleles. Kompetitive Allele Specific PCR (KASP) markers enable high throughput analysis for a large number of seeds, as well as detection of both alleles, in a single reaction. In this study, we developed KASP markers that can distinguish specific alleles at Sub1A loci based on single nucleotide polymorphisms (SNPs). Marker validations were carried out by genotyping of a segregating population with the developed KASP markers. The results from KASP assay and gel-based marker analysis were consistent for Sub1A alleles. KASP markers developed for Sub1A would be helpful due to high accuracy, low cost, and a high throughput genotyping feature in MAB. Keywords Submergence tolerance, Gel-based marker, KASP marker, Sub1A, Single-nucleotide polymorphism INTRODUCTION Submergence stress, caused by flash floods, is one of the main constraints to rice production in rainfed lowlands of Asia and Africa (Mackill et al. 1996). Sub1 locus responsible for submergence tolerance was mapped on chromosome 9 using a mapping population derived from a cross of a tolerant derivative of the FR13A cultivar and the intolerant japonica cultivar M-202 (Xu et al. 2006). Two different allele types (tolerant type named Sub1A-1 and susceptible type named Sub1A-2) have been identified in the Sub1A gene (Xu et al. 2006; Septiningsih et al. 2009). To determine the allele types of Sub1A, two markers were developed based on its single nucleotide polymorphisms (SNPs). GnS2, a cleaved amplified polymorphic sequence (CAPS) marker, can detect the SNP discriminating Sub1A-1 and Sub1A-2, with restriction sites, for AluI or PvuII (Neeraja et al. 2007). AEX1 was developed for the tolerant allele of Sub1A-1 by designing a DNA marker with the SNP at the 3 end (Septiningsih et al. 2009). However, these gel-based DNA markers are less suitable for high throughput genotyping in marker-assisted selection (MAS), because they are expensive, time consuming and labor intensive. Alternatively, Kompetitive Allele Specific PCR (KASP) of LGC Genomics ( were developed as a fluorescence-based SNP genotyping system, which can distinguish SNP-based alleles (Graves et al. 2016). Here we developed KASP markers based on the SNPs specifically targeted by two gel-based markers, GnS2 and AEX1, for Sub1A. Marker validations were also performed and compared with the genotyping results obtained from the gel-based markers. It is expected that the KASP markers developed for Sub1A in this study will contribute Received December 17, 2018; Revised December 24, 2018; Accepted December 24, 2018; Published March 1, 2019 *Corresponding author Soo-Cheul Yoo, scyoo@hknu.ac.kr, Tel: , Fax: Copyright c 2019 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 ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Development of KASP Markers for Sub1 63 to increasing of the efficiency of genotyping and facilitate high throughput genotyping in MAS for submergence tolerance rice breeding. MATERIALS AND METHODS Plant materials Tolerant (FR13A, IR49830) and susceptible (IR64, BR11, M202, Nipponbare, Komboka, Samba Mashuri and Swarna) varieties to submergence stress were used to develop the Sub1-specific KASP markers (Mackill et al. 1996; Neeraja et al. 2007; Septiningsih et al. 2009). All varieties were obtained from the International Rice Research Institute (IRRI), Philippines. In addition, a segregating population consisting of 20 F 2 plants, derived from a cross between Komboka (susceptible) and IR49830 (tolerant), was used to validate the developed KASP markers. All F 2 seeds were provided by Dr. Joong-Hyoun Chin, Sejong University, Korea. Analysis of gel-based markers for Sub1 GnS2 and AEX1 were previously designed as genespecific markers for Sub1A (Neeraja et al. 2007; Septiningsih et al. 2009). Genomic DNA was extracted from leaf samples using the cetyl trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980). The thermal cycling condition differed only in annealing temperature during PCR reaction. After initial denaturation for 4 minutes at 94 C, PCR underwent 1-minute denaturation at 94 C, 1-minute annealing at 65 C and 55 C for AEX1 and GnS2, respectively, and 1-minute extension at 72 C, with a final extension for 5 minutes at 72 C at the end of 35 cycles. The PCR products, electrophoresed in 2% agarose gel, were visualized using the gel-imager. KASP markers development and assay To develop the KASP markers, we used the sequences of Sub1A-1 and Sub1A-2, obtained from GenBank (FR for Sub1A-1, FR for Sub1A-2) (Fig. 1), and then allele-specific primers for KASP were designed by LGC genomics based on the SNPs detected between these two alleles of Sub1A. These primers are listed in Table 1. The KASP markers assay was performed in a 10 L reaction volume with 5 L of DNA template, 0.14 L of assay mix (containing a common primer and two different allele specific primers with FAM and HEX fluorophores, respectively), 5 L of master mix (containing fluorescence resonant energy transfer and taq polymerase). KASP amplification was performed according to the manual of KASP provided by LGC Genomics ( com). After amplification, the fluorescence signals from the end PCR products were read using the CFX Maestro software (Bio-Rad, USA). RESULTS AND DISCUSSION Development of allele-specific KASP marker The Sub1A gene identified from FR13A is a key gene conferring submergence tolerance in rice (Mackill et al. 1993; Xu et al. 2006). Although some varieties, such as IR64, have Sub1A, natural variants of Sub1A, called Sub1A-2, failed to show phenotypic tolerance to submergence stress (Septiningsih et al. 2009). Two gel-based allelespecific primers, AEX1 and GnS2, were previously designed based on these allele variations of Sub1A (Neeraja et al. 2007; Septiningsih et al. 2009) (Fig. 1). To develop KASP markers based on allele-specific SNPs of Sub1A-1 and Sub1A-2, we designed primers targeting the SNPs at the 556th (T/C) and 678th (A/G), on which AEX1 and GnS2 markers were designed, respectively (Fig. 1, Table 1). Newly designed KASP markers were tested with two submergence tolerant and seven susceptible varieties. The separation of tolerant and susceptible alleles was successfully performed by these two KASP markers (Fig. 2A). We further performed gel-based marker analyses to compare the genotyping results of the KASP markers with those of the gel-based markers. PCR amplification with AEX1 primers produced the expected band size of 231 bp for two varieties (FR13A and IR49830), which are known to be tolerant, but did not produce any detectable bands for the other susceptible varieties (Fig. 2B). PCR analysis with GnS2 primers produced the expected bands of 242 bp from six varieties, including the two tolerant ones (Fig. 2C, upper panel). Of the six varieties, two turned out to be

64 Plant Breed. Biotech. 2019 (March) 7(1):62~66 Fig. 1. DNA sequence alignment of Sub1A-1 and Sub1A-2 for KASP marker development.

3 64 Plant Breed. Biotech (March) 7(1):62~66 Fig. 1. DNA sequence alignment of Sub1A-1 and Sub1A-2 for KASP marker development. The alleles of Sub1A-1 and Sub1A-2 are distinguished by two SNP sites located at 556th (T/C) and 678th (A/G). AEX1 primers amplify Sub1A-1 allele only at SNP1 (556th, T/C), while GnS2 amplifies both alleles of Sub1A-1 and Sub1A-2 at SNP2 (678th, A/G), which includes the restriction site. Two KASP markers were designed to target both SNP1 and SNP2 sites, respectively. Red and blue boxes represent genomic locations of forward and reverse primers designed for AEX1 and GnS2 markers, respectively; dotted line represents restriction site of AluI or PvuII. Table 1. Primer sequences of the markers for Sub1. Marker Primer name Gel-based marker AEX1 GnS2 (CAPS) Marker KASP marker Primer name Forward primer 5ʹ-AGGCGGAGCTACGAGTACCA-3ʹ 5ʹ-CTTCTTGCTCAACGACAACG-3ʹ Reverse primer 5ʹ-GCAGAGCGGCTGCGA-3ʹ 5ʹ-TCGATGGGGTCTTGATCTCT-3ʹ Common primer Allele-specific primer Sub1A-SNP1 (T/C) 5ʹ-CTTCCCACCCGCCGATCTTTCTT-3ʹ 5ʹ-CAAGAAGCAGAGCGGCTGCGA-3ʹ 5ʹ-AGAAGCAGAGCGGCTGCGG-3ʹ Sub1A-SNP2 (A/G) 5ʹ-GTCCGGCGACGCGCGCATA-3ʹ 5ʹ-CGTCCGAGCAGCACTCCAGT-3ʹ 5ʹ-GTCCGAGCAGCACTCCAGC-3ʹ Sub1A-1 types and the other four, Sub1A-2 types, after treatment with the restriction enzyme, PvuII, which produced two different sizes of bands, 132 and 110 bps (Fig. 2C, lower panel). As a result, the genotyping result of the KASP markers assay was consistent with that of the gel-based markers. Validation of the KASP markers To validate the developed KASP markers, we also applied both gel-based and KASP markers to the seg-

4 Development of KASP Markers for Sub1 65 Fig. 2. Genotyping of the varieties using KASP and gel-based markers. Genotyping of varieties both tolerant and susceptible to submergence was performed by two developed KASP markers, Sub1A_SNP1 and Sub1A_SNP2 (A). Genotyping with two gel-based markers, AEX1 (B) and GnS2 (C) was performed. Gel images before (upper panel) and after (lower panel) treatment of restriction enzyme are shown in (C). T: tolerance, S: susceptible, N.A: not amplified, R.E: restriction enzyme, Ladder: DNA ladder. regation population. Twenty Komboka/IR49830 F 2 individuals were used for genotyping. Gel-based marker analysis with AEX1 and GnS2 showed that 13 individuals harbored the tolerance allele Sub1A-1 and the other seven plants had the susceptible Sub1A-2 allele (Fig. 3B, C). KASP analyses with Sub1A_SNP1 and Sub1A_SNP2 markers produced two different alleles, Sub1A-1 and Sub1A-2, which are consistent with those of the gel-based markers (Fig. 3A). This indicates that the two newly developed KASP markers can replace the two gel-based markers. Sub1A_SNP1 is more efficient than AEX1, because AEX1, a dominant marker, can produce the tolerant allele only; however, the Sub1A_SNP1 can produce two different alleles in single reaction. The KASP Sub1A_SNP2 is also more efficient than GnS2, because GnS2 needs an additional enzyme reaction, although it produces two different alleles. In conclusion, the KASP markers developed in this study would be good replacements for the two existing gel-based markers. Application of the two KASP markers can facilitate highthroughput analysis by reducing cost and labor intensity, as well as increasing accuracy in genotyping for the breeding of submergence tolerant rice. ACKNOWLEDGEMENTS This work was supported by the Next-Generation

66 Plant Breed. Biotech. 2019 (March) 7(1):62~66 Fig. 3. Application of the KASP and gel-based markers to genotyping of the segregating F2 population.

5 66 Plant Breed. Biotech (March) 7(1):62~66 Fig. 3. Application of the KASP and gel-based markers to genotyping of the segregating F2 population. Genotyping of 20 Komboka/IR49830 F2 plants were performed with Sub1A_SNP1 and Sub1A_SNP2 markers (A). Two gel-based markers, AEX1 (B) and GnS2 (C), were used to genotype F2 plants. Gel images before (upper panel) and after (lower panel) treatment of restriction enzyme are shown in (C). T: tolerance, S: susceptible, H: heterozygous, R.E: restriction enzyme, L: DNA ladder. BioGreen 21 Program grant (No. PJ ). Murray MG, Thompson WF Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: REFERENCES Neeraja CN, Maghirang-Rodriguez R, Pamplona A, Heuer S, Graves H, Rayburn AL, Gonzalez-Hernandez JL, Nah G, Kim Collard BC, Septiningsih EM, et al A markerassisted backcross approach for developing submergence- DS, Lee DK Validating DNA polymorphisms using tolerant rice cultivars. Theor. Appl. Genet. 115: KASP assay in prairie cordgrass (Spartina pectinata Link) populations in the US. Front. Plant Sci. 6: Septiningsih EM, Pamplona AM, Sanchez DL, Neeraja CN, Vergara GV, Heuer S, et al Development of Mackill DJ, Amante MM, Vergara BS, Sarkarung S submergence-tolerant rice cultivars: the Sub1 locus and Improved semidwarf rice lines with tolerance to submergence of seedlings. Crop Sci. 33: beyond. Ann. Bot. 103: Xu K, Xu X, Fukao T, Canlas P, Maghirang-Rodriguez R, Mackill D, Coffman WR, Garrity DP Rainfed Lowland Heuer S, et al Sub1A is an ethylene-response- Rice Improvement. International Rice Research Institute, Manila, Philippines. factor-like gene that confers submergence tolerance to rice. Nature 442:

GENOTYPING BY PCR PROTOCOL FORM MUTANT MOUSE REGIONAL RESOURCE CENTER North America, International

GENOTYPING BY PCR PROTOCOL FORM MUTANT MOUSE REGIONAL RESOURCE CENTER North America, International Please provide the following information required for genetic analysis of your mutant mice. Please fill in form electronically by tabbing through the text fields. The first 2 pages are protected with gray