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Photograph showing the typical bioassay conducted using CR2711â 76, Jaya, Rathu Heenati and TN-1 along with other test lines. (PPTX 542 KB)

Photograph showing the bioassay result of negative and positive BPH31 BC2F2s of marker validation experiment at maximum lethal stage (ninth day after infestation) for the Laguna BPH population. (PPTX 296 kb)

Graphical representation of area of honeydew secretion of (a) first experiment, (b) repeated experiment for Isabela BPH colony, (c) for Bicol BPH colony, and (d) for Nueva Ecijia BPH colony; among the test genotypes including parental lines, Jaya and CR2711–76; P1, P2, 13 t, RH, and F1 are Jaya, CR2711–76, BPH13 donor line, Rathu Heenati, and F1 is the hybrid of Jaya × CR2711–76, respectively; RH and TN-1 are resistant and susceptible checks. (PPTX 178 kb)

Rice molecular linkage map obtained from 107 SSR and STS markers of 151Â F2:3 mapping population of the cross combination Jaya Ă CR2711â 76. Chromosome numbers are presented on the top of each linkage group, marker names are presented on the right side of each linkage group, and their intervals in cM are presented on the left side of the linkage group. This linkage group was generated by QTL IciMapping software considering the marker position and order retrieved from MAPMAKER ver. 2.0. (PPTX 160 kb)

Schematic diagram showing material generation for the identification and introgression of BPH31. (PPTX 71 kb)

(a) Raw signal intensity bar graph (in silico gene expression analysis as available in public domain, http://ricexpro.dna.affrc.go.jp/GGEP/graph-view.php?featurenum=29277 ) showing the expression intensity of LOC_Os03g46550 gene. (b) Preferential gene expression at different developmental stages of the rice plant. (c) Tissue-specific differential expression of LOC_Os03g46550 (based on information available at http://www.ricearray.org/expression/meta_analysis.shtml ). (PPTX 706 kb)

List of newly designed InDel markers for fine mapping of BPH31. (PPTX 77 kb)

The schematic diagram of primary mapped BPH resistance locus BPH31 on chromosome 3 flanked by RM251 and RM2334 with 48.40 LOD and 80.71% phenotypic variance explained derived from 151Â F2:3 mapping population in the whole-genome view. This result was retrieved from the linkage analysis of (a) QTL IciMapping ver. 4.0 (Meng et al. 2015) and (b) WinQTL cartographer ver. 2.5 (Wang 2012) software package. (PPTX 658 kb)

Graphical representation of 7 years BPH (biotype 4) bioassay result of parental lines and checks. (PPTX 171 KB)

Photograph depecting the area of honeydew secreated when Lagun colony of BPH fed on TN-1, Rathu Heenati, CR2711â 76 and Jaya testlines. (PPTX 287 kb)

Haplotype analysis of the NAL1 gene from six varieties using WGS data according to the previously analyzed NAL1 haplotypes (Takai et al. 2013). The nucleotide position was calculated based on the previous report (1â =â the starting nucleotide of 5â UTR in Takanari) and newly identified polymorphisms in this study were highlighted by red characters. The 1640-position nucleotide (G/A) and the 2884-position nucleotide (G/A) were used for designing the SPIKE-01SNP marker and SPIKE-03 SNP marker, respectively. S.S., synonymous nucleotide substitution. (DOC 78 kb)

Summary of whole-genome sequencing of six varieties. (DOC 47 kb)

Screening of DNA polymorphisms in the Gn1a gene using WGS data. Sequencing reads were aligned to the reference genome sequence (IRGSP-1.0). Note that the Gn1a gene lay on the opposite strand of the reference sequence. Screen-captured images of IGV software showed nucleotide variations that were used for marker development. (A) Three SNPs located in the Gn1a promoter region were shown with their genomic location on chromosome 1. Chr1: 5276405, Chr1: 5276521, and Chr1: 5276591 SNPs were used for Gn1a-17SNP/Gn1a-17SNP-FD, Gn1a-18SNP-FD, and Gn1a-19SNP-FD markers, respectively. (B) About a 70-bp deletion (red circles) near the 3â UTR of Gn1a was found in varieties NSIC Rc158 and NSIC Rc238. This indel was used for designing the Gn1a-indel3 marker. (DOC 178 kb)

Marker designing schemes for SNP-type polymorphisms. (A) Schematic presentation of the tetra-primer PCR method for designing Gn1a-17SNP marker. The target SNPs, G and A, are highlighted in each genome and its surrounding sequences are represented with the allele-specific primers (pink, G allele-specific primer; green, A allele-specific primer). Actually, the SNP is determined by the last nucleotide (filled triangle) of the allele-specific primer. Proper annealing of the last nucleotide of the primer (the 3’ end) is very important for PCR amplification because Taq DNA polymerase start polymerization at that nucleotide through adding dNTP. For instance, the A allele-specific primer (green) can be annealed to the G allele genome but the efficiency of DNA polymerization will be very low because of no annealing of the 3’ end of the primer, resulting in no PCR band or a very weak band. To increase allele specificity, we gave an artificial mismatched nucleotide near the 3’ end (second or third nucleotide from the 3’ end) of the allele-specific primer (lowercase with underlined nucleotide in Figure). Primer combination of the Gn1a-17SNP marker, its predicted band size, and deduced gel image depending on genotypes were presented. (B) Schematic presentation of the separated allele-specific PCR method for designing the SPIKE-01SNP marker. To discriminate G/A SNP, the SPIKE-01SNP marker consisted of two PCRs (PCR #1, SPIKE-01SNP-GF/R; PCR #2, SPIKE-01SNP-AF/R). Between the G allele-specific primer (pink) and A allele-specific primer (green), only the last nucleotide of each allele-specific primer (filled triangle) is different. To increase allele specificity, the artificial mismatched nucleotide near the 3’ end was given in the allele-specific primer. PCRs were performed with each allele-specific primer and common reverse primer. Primer combinations of the SPIKE-01SNP marker, its predicted band size, and deduced gel image depending on genotypes were presented. (C) The effect of artificial mismatched nucleotide near the 3’ end of the allele-specific primer. As an example, the SPIKE-01SNP marker was designed without the artificial mismatched nucleotide in allele-specific primers that were shown on the gel image. Non-allele-specific PCR amplifications were obtained with these primers. Using artificial mismatch, we obtained allele-specific PCR amplifications (Fig. 6a). (DOC 240 kb)

Primers for preparation of PCR products and PCR product sequencing. (DOC 35 kb)

Summary of Fluidigm SNP genotyping results. (DOC 90 kb)

Markers for Fluidigm SNP genotyping platform. (DOC 36 kb)