Supplemental Note on Gene Specific Markers

Markers associated with major genes for rice blast resistance and for the grain quality traits amylose content, gelatinization temperature, and aroma were used to screen thetwo BC3ILs evaluated in the Uniform Regional Rice Nursery (URRN). For these gene-specific assays, DNA was extracted from 20 mg of brown rice using the modified CTAB method described in Fjellstrom et al. (2004). Five markers were used to detect known blast resistance genes: AP5659-1 and AP5659-5 associated with the presence of Pi-z (Fjellstrom et al. 2006), RM208 associated with the presence of Pi-b, RM224 associated with the presence of Pi-k (Fjellstrom et al. 2004), and Pi-indica, a marker indicative of the resistant Pi-ta allele (Wang et al. 2010). RM190, an SSR marker associated with the granule bound starch synthase gene, was used to screen for amylose content (Chen et al. 2008; Bao et al. 2006) along with a marker associated with the WxEx6 functional SNP in the Waxy gene (Chen et al. 2010). A perfect marker detecting the 8 bp deletion in the BADH2 gene was used to screen for grain aroma (Kovach et al. 2009; Bradbury et al. 2005) and the GC/TT SNP in the Alk gene was used to screen for gelatinization temperature (Bao et al. 2006). For each marker, controls were included for each known allele.

PCR reactions for the gene-specific assays were performed in 25l reaction volumes consisting of 20 ng of DNA, 10 mMTris–HCl pH 8.3, 50 mMKCl, 2.5 mMMgCl2, 300 nM of each primer, 1 U of Taq DNA polymerase (Promega, Madison, WI, USA). Reverse primers were unlabeled in order to reduce the cost, and the forward primers were labeled with either 6FAM, Tamra, or Hex (Integrated DNA Technologies,Coralville, IA, USA). DNA was amplified with MJ Research Tetrad PCR machines (Waltham, MA, USA) under the following conditions: initial denaturation at 94°C for 5 minutes; then 30 cycles of 94°C for 30 seconds, 55–67°C (dependent on the marker) for 30 seconds, and 72°C for 1 minute; 5 minute final extension at72°C. PCR products were pooled based on sizes of amplified fragments (typically three markers per run along with ROX-labeled size standard) to reduce the cost, and the DNA was denatured by heating samples at 94°C for 5 minutes. The samples were separated on an ABI Prism 3100 DNA analyzer using methods as described by the manufacturer (Applied Biosystems,Foster City, CA, USA).

References

Bao J, Corke H, Sun M (2006) Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice (Oryzasativa L.). Theor Appl Genet 113 (7):1171-1183. doi:10.1007/s00122-006-0355-6

Bradbury L, Henry R, Jin Q, Reinke RF, Waters DLE (2005) A perfect marker for fragrance genotyping in rice. Mol Breed 16 (4):279-283. doi:10.1007/s11032-005-0776-y

Chen M-H, Bergman C, Pinson S, Fjellstrom R (2008) Waxy gene haplotypes: Associations with apparent amylose content and the effect by the environment in an international rice germplasm collection. Journal of Cereal Science 47 (3):536-545. doi:10.1016/j.jcs.2007.06.013

Chen M-H, Fjellstrom R, Christensen E, Bergman C (2010) Development of three allele-specific codominant rice Waxy gene PCR markers suitable for marker-assisted selection of amylose content and paste viscosity. Mol Breed 26 (3):513-523. doi:10.1007/s11032-010-9419-z

Fjellstrom R, Conaway-Bormans C, McClung AM, Marchetti MA, Shank AR, Park WD (2004) Development of DNA markers suitable for marker assisted selection of three genes conferring resistance to multiple pathotypes. Crop Sci 44 (5):1790-1798

Fjellstrom R, McClung A, Shank A (2006) SSR markers closely linked to the Pi-z locus are useful for selection of blast resistance in a broad array of rice germplasm. Mol Breed 17 (2):149-157. doi:10.1007/s11032-005-4735-4

Kovach MJ, Calingacion MN, Fitzgerald MA, McCouch SR (2009) The origin and evolution of fragrance in rice (Oryzasativa L.). Proc Natl Acad Sci USA 106 (34):14444-14449. doi:10.1073/pnas.0904077106

Wang X, Fjellstrom R, Jia Y, Yan WG, Jia MH, Scheffler BE, Wu D, Shu Q, McClung A (2010) Characterization of Pi-ta blast resistance gene in an international rice core collection. Plant Breeding 129 (5):491-501. doi:10.1111/j.1439-0523.2009.01706.x