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Development of PCR markers for Tamyb10 related to R-1, red grain color gene in wheat

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Abstract

The grain color of wheat affects not only the brightness of flour, but also tolerance to preharvest sprouting. Grain color is controlled by dominant R-1 genes located on the long arm of hexaploid wheat chromosomes 3A, 3B, and 3D (R-A1, R-B1, and R-D1, respectively). The red pigment of the grain coat is composed of catechin and proanthocyanidin (PA), which are synthesized via the flavonoid biosynthetic pathway. We isolated the Tamyb10-A1, Tamyb10-B1, and Tamyb10-D1 genes, located on chromosomes 3A, 3B, and 3D, respectively. These genes encode R2R3-type MYB domain proteins, similar to TT2 of Arabidopsis, which controls PA synthesis in testa. In recessive R-A1 lines, two types of Tamyb10-A1 genes: (1) deletion of the first half of the R2-repeat of the MYB region and (2) insertion of a 2.2-kb transposon belonging to the hAT family. The Tamyb10-B1 genes of recessive R-B1 lines had 19-bp deletion, which caused a frame shift in the middle part of the open reading frame. With a transient assay using wheat coleoptiles, we revealed that the Tamyb10 gene in the dominant R-1 allele activated the flavonoid biosynthetic genes. We developed PCR-based markers to detect the dominant/recessive alleles of R-A1, R-B1, and R-D1. These markers proved to be correlated to known R-1 genotypes of 33 varieties except for a mutant with a single nucleotide substitution. Furthermore, double-haploid (DH) lines derived from the cross between red- and white-grained lines were found to necessarily carry functional Tamyb10 gene(s). Thus, PCR-based markers for Tamyb10 genes are very useful to detect R-1 alleles.

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Acknowledgments

We thank Dr. Flintham (John Innes Centre) for providing Novosibirskaya 67 and its NIL; Dr. Mares (The University of Adelaide) for the gift of AUS1490 and EMS-AUS; Dr. Sorrells (Cornell University) for giving Cornell 595; and Dr. Tanio (National Agricultural Research Center, Japan) for providing SZF. This work was supported by grants from the Iijima Memorial Foundation for the Promotion of Food Science and Technology and the Ministry of Agriculture, Forestry, and Fisheries of Japan.

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Authors and Affiliations

  1. Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama, Japan

    Eiko Himi, Masahiko Maekawa & Kazuhiko Noda

  2. Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan

    Hideho Miura

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  1. Eiko Himi
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  2. Masahiko Maekawa
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Correspondence to Eiko Himi.

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Communicated by M. Sorrells.

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122_2011_1555_MOESM1_ESM.ppt

Fig. S1 Positions of primers in this study. Three exons of Tamyb10 gene are described with rectangles. Black-painted regions represent the MYB consensus region. Untranslated regions and introns are described with horizontal lines. Primers are shown with triangles. Right-directed triangles indicate sense primers, and left-directed triangles indicate antisense primers. Primers shown with white triangles represent the primers designed on the basis of the maize P gene. Primers shown with black triangles are designed on the basis of Tamyb10 genes. (PPT 112 kb)

122_2011_1555_MOESM2_ESM.ppt

Fig. S2 Genome-determined primers for Tamyb10. a Fragments of the Tamyb10-A1 gene were amplified with Tamyb10-LP1 and Tamyb10-RP1. b Fragments of the Tamyb10-B1 gene were amplified with Tamyb10-LP2 and Tamyb10-RP1. c Fragments of the Tamyb10-D1 gene were amplified with Tamyb10-LP3 and Tamyb10-RP3. (PPT 126 kb)

122_2011_1555_MOESM3_ESM.ppt

Fig. S3 Primers and PCR conditions for Tamyb10 amplification. PCR conditions are described in boxes below the structures of each Tamyb10 gene. The expected sizes of amplification fragments using each set of primers are listed in the tables. a-c Tamyb10-A1 genes of Norin 61 (R-A1b), CS (R-A1a), and Norin 17 (R-A1a). The red and black boxes indicate exons. The arrows indicate primer positions. A grey region with a black box in Tamyb10-A1 of CS represents an unknown sequence. 2.2 kb of GeST was inserted into second intron of Tamyb10-A1 of Norin 17. b Tamyb10-B1 gene of CS (R-B1a). The green and black boxes indicate exons. A white triangle represents the site of a 19-bp deletion. Amino acid sequences are altered by a frame shift. c Tamyb10-D1 gene of CS (R-D1b). The blue boxes indicate exons.(PPT 462 kb)

122_2011_1555_MOESM4_ESM.ppt

Fig. S4 Deduced amino acid sequences encoded by Tamyb10-A1 of AUS1490 (R-A1b), Tamyb10-B1 of Norin 61 (R-B1b), and Tamyb10-D1 of CS (R-D1b). The R2 and R3 repeats of the MYB consensus region are shaded in black and gray, respectively. Tamyb10-A1 of CS (R-A1a) lacked the N-terminal sequences shown in blue letters of Tamyb10-A1 of AUS1490 (R-A1b). A white rectangle indicates the substituted residue from glycine (G) to glutamic acid (E) in EMS-AUS. A white triangle indicates the 19-bp deletion site of Tamyb10-B1 in CS. Essential residues of the conserved amino acid signature (D/ELx2R/Kx3Lx6Lx3R) as the structural basis for interaction between MYB and bHLH proteins are shown with a dotted black circle. The conserved sequences (IRTKAL/IRC) among Tamyb10, Arabidopsis TT2, and rice OsMYB3 are shown in bold letters, and the KAxRC sequences in the motif also seen in C1 protein are shown in red letters. (PPT 124 kb)

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Himi, E., Maekawa, M., Miura, H. et al. Development of PCR markers for Tamyb10 related to R-1, red grain color gene in wheat. Theor Appl Genet 122, 1561–1576 (2011). https://doi.org/10.1007/s00122-011-1555-2

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