Eiko Himi

Red grain colour gene (R) of wheat is a Myb-type transcription factor

Red pigment of wheat grain was reported to be polyphenol compounds, phlobaphene or proanthocyanidin, which were synthesized through the flavonoid biosynthesis pathway. We examined the expression of four genes in the flavonoid pathway in developing wheat grains: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and dihydroflavonol 4-reductase (DFR). The expression of these genes in the white-grained wheat lines was reduced, compared with that in the red-grained wheats. These results suggest that the R genes for grain colour, located on the long arms of chromosomes 3A, 3B and 3D, are transcriptional activators of the flavonoid synthesis genes. We identified three Myb-type transcription factors (Tamyb10-A1 on 3A, Tamyb10-B1 on 3B and Tamyb10-D1 on 3D), which were also located in the same region as the R loci, and were expressed predominantly in developing grains. In wheat lines with the recessive allele r3 (3B) Tamyb10-B1 had a deletion which caused a frame-shift in its open reading frame. Tamyb10-A1, and -D1 were not expressed in wheats with r2 (3A) and r1 (3D).

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

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.

Ant28 gene for proanthocyanidin synthesis encoding the R2R3 MYB domain protein (Hvmyb10) highly affects grain dormancy in barley

A number of anthocyanin- and proanthocyanidin-free mutants (ant mutants) in barley were induced and selected because of breeding interest to reduce proanthocyanidins, which could cause haze and degrade the quality of beer. Ant loci, known as anthocyanin or proanthocyanidin synthesis genes, are classified into Ant1 to Ant30 through allelism tests. However, only the Ant18 gene has been molecularly shown to encode dihydroflavonol 4-reductase (DFR), which is involved in both anthocyanin and proanthocyanidin synthesis. In this study, an R2R3 MYB gene of barley was isolated by PCR and named Hvmyb10 due to its similarity to Tamyb10 of wheat, which is a candidate for the R-1 gene grain color regulator. The predicted amino acid sequences of Hvmyb10 showed high similarity not only to Tamyb10 but also to TT2, the proanthocyanidin regulator of Arabidopsis. Non-synonymous nucleotide substitutions in the Hvmyb10 gene were found in all six ant28 mutants tested. Mapping showed that a polymorphism in Hvmyb10 perfectly cosegregated with the ant 28 phenotype on the distal region of the long arm of chromosome 3H. These results demonstrate that ant28 encodes Hvmyb10, the R2R3 MYB domain protein that regulates proanthocyanidin accumulation in developing grains. The reduced grain dormancy of ant28 mutants compared with those of the respective wild types indicates that Hvmyb10 is a key factor in grain dormancy in barley.

Duplicate polyphenol oxidase genes on barley chromosome 2H and their functional differentiation in the phenol reaction of spikes and grains

Polyphenol oxidases (PPOs) are copper-containing metalloenzymes encoded in the nucleus and transported into the plastids. Reportedly, PPOs cause time-dependent discoloration (browning) of end-products of wheat and barley, which impairs their appearance quality. For this study, two barley PPO homologues were amplified using PCR with a primer pair designed in the copper binding domains of the wheat PPO genes. The full-lengths of the respective PPO genes were cloned using a BAC library, inverse-PCR, and 3′-RACE. Linkage analysis showed that the polymorphisms in PPO1 and PPO2 co-segregated with the phenol reaction phenotype of awns. Subsequent RT-PCR experiments showed that PPO1 was expressed in hulls and awns, and that PPO2 was expressed in the caryopses. Allelic variation of PPO1 and PPO2 was analysed in 51 barley accessions with the negative phenol reaction of awns. In PPO1, amino acid substitutions of five types affecting functionally important motif(s) or C-terminal region(s) were identified in 40 of the 51 accessions tested. In PPO2, only one mutant allele with a precocious stop codon resulting from an 8 bp insertion in the first exon was found in three of the 51 accessions tested. These observations demonstrate that PPO1 is the major determinant controlling the phenol reaction of awns. Comparisons of PPO1 single mutants and the PPO1PPO2 double mutant indicate that PPO2 controls the phenol reaction in the crease on the ventral side of caryopses. An insertion of a hAT-family transposon in the promoter region of PPO2 may be responsible for different expression patterns of the duplicate PPO genes in barley.

Isolation of candidate genes for the barley Ant1 and wheat Rc genes controlling anthocyanin pigmentation in different vegetative tissues

MYB transcription factors exist in a large copy number and control various plant phenotypes. We cloned R2R3 MYB-type transcription factors that determine the coloration of basal sheaths in barley and wheat coleoptiles. These genes are highly homologous to maize C1 and rice OsC1, regulators for anthocyanin biosynthesis, but they control seed pigmentation in maize and rice. On the basis of high homology, barley and wheat counterparts are designated HvC1 and TaC1, respectively. HvC1 gene is located on the short arm of chromosome 7H, and TaC1 genes are located on the short arms of chromosomes 7A, 7B, and 7D (TaC1-A1, B1, and D1, respectively). HvC1 is a strong candidate for Ant1 because of (1) complete co-segregation of anthocyanin pigmentation phenotype of the basal sheath with the HvC1 genotype in genetic mapping, and (2) complete deletion of the HvCl gene in two anthocyanin-decreased allelic mutants (ant1.1 and ant1.2) that were induced by irradiation. In contrast, colorless coleoptile wheat lines had lesions in all three genomes consisting of a single-nucleotide substitution or a 1-bp deletion of TaC1-A1, a 1.7-kb insertion of TaC1-B1, and a 2.0-kb insertion of TaC1-D1. At least one normal TaC1 gene appears to be sufficient to produce anthocyanin pigments in wheat coleoptiles. Previous crossing experiments localized Rc (red coleoptile) genes to homoeologous group 7 chromosomes and deduced Rc genotypes of several wheat lines. Their TaC1 gene sequence variation coincided with deduced Rc genotypes; therefore, the present molecular genetic study demonstrates that TaC1 is a strong candidate for Rc in wheat.

Differential Expression of Three Flavanone 3-Hydroxylase Genes in Grains and Coleoptiles of Wheat

Flavonoid pigments are known to accumulate in red grains and coleoptiles of wheat and are synthesized through the flavonoid biosynthetic pathway. Flavanone 3-hydroxylase (F3H) is a key enzyme at a diverging point of the flavonoid pathway leading to production of different pigments: phlobaphene, proanthocyanidin, and anthocyanin. We isolated three F3H genes from wheat and examined a relationship between their expression and tissue pigmentation. Three F3Hs are located on the telomeric region of the long arm of chromosomes 2A, 2B, and 2D, respectively, designated as F3H-A1, F3H-B1, and F3H-D1. The telomeric regions of the long arms of the chromosomes of homoeologous group 2 of wheat showed a syntenic relationship to the telomeric region of the long arm of rice chromosome 4, on which rice F3H gene was also located. All three genes were highly activated in the red grains and coleoptiles and appeared to be controlled by flavonoid regulators in each tissue.

Identification of QTLs for yield-related traits in RILs derived from the cross between pLIA-1 carrying Oryza longistaminata chromosome segments and Norin 18 in rice

To improve rice yield, a wide genetic pool is necessary. It is therefore important to explore wild rice relatives. Oryza longistaminata is a distantly related wild rice relative that carries the AA genome. Its potential for improving agronomic traits is not well studied. Introgression line (pLIA-1) that carries Oryza longistaminata’s chromosome segments, showed high performance in yield-related traits under non-fertilized conditions. Therefore, to illustrate Oryza longistaminata’s potential for improving yield-related traits, RILs from the F1 of a cross between pLIA-1 and Norin 18 were developed and QTL analysis was done using the RAD-Seq method. In total, 36 QTLs for yield-related traits were identified on chromosomes 1, 2, 3, 5, 6, 7, 8, 10, and 11. Clusters of QTLs for strongly correlated traits were also identified on chromosomes 1, 3, 6, and 8. Phenotypic data from recombinant plants for chromosomes 1 and 8 QTL clusters revealed that the pLIA-1 genotype on chromosome 1 region was more important for panicle-related traits and a combination of pLIA-1 genotypes on chromosomes 1 and 8 showed a favorable phenotype under non-fertilized conditions. These results suggest that Oryza longistaminata’s chromosome segments carry important alleles that can be used to improve yield-related traits of rice.

Barley Ant17, encoding flavanone 3-hydroxylase (F3H), is a promising target locus for attaining anthocyanin/proanthocyanidin-free plants without pleiotropic reduction of grain dormancy

Preharvest sprouting is a serious problem in grain crop production because it causes quality deterioration and economic losses. It is well known that grain colour is closely associated with grain dormancy in wheat; white-grained lines without accumulating proanthocyanidins in testa tend to be more susceptible to preharvest sprouting than red ones. All available white-grained wheat lines are restricted to triple recessive mutations at the R loci (R-A1, R-B1, and R-D1), but barley is known to have 11 independent loci conferring the proanthocyanidin-free grain phenotype. In this study, we evaluated the dormancy levels of anthocyanin/proanthocyanidin-free ant17 mutants. Three ant17 mutants showed the same levels of dormancy as their respective wild types. Sequencing of three independent ant17 alleles detected a point mutation within the coding regions of flavanone-3-hydroxylase (F3H), which are predicted to cause a premature stop codon at different sites. The F3H locus completely cosegregated with the Ant17 position on the chromosome arm 2HL. Expression of the barley F3H gene was observed in pigmented tissues, but not in nonpigmented roots and stems. This result indicates that wheat F3H may be a promising new target locus for breeding white-grained lines with a practical level of preharvest sprouting resistance.

Real-time PCR-mediated diagnosis of hemizygosity at the Tamyb10-D1 locus controlling grain color in wheat

Grain color of wheat is one of the most important economic traits, affecting both flour quality and grain dormancy. As grain color reflects the phenotype of the seed coat, which is a maternal tissue, genotyping of this trait in segregating F2 plants requires an F3 progeny test. Consequently, the development of co-dominant molecular markers for grain color enables genotyping on a single grain basis and would expedite wheat breeding. R-1 genes control grain color and have been identified as MYB-type transcription factors, denoted as Tamyb10, which reside on the 3A, 3B, and 3D chromosomes, respectively. In an earlier study we developed co-dominant markers for R-A1 and R-B1 on chromosomes 3A and 3B, but could only develop one dominant marker for R-D1 because the mutant allele deleted the entire Tamyb10-D1 fragment with an unknown deletion size. DNA of individuals of the F2 population derived from crossing the white-grained line, 1–117, and the red-grained line, OW104, was isolated and used for real-time PCR. Relative amplification ratios of Tamyb10-D1 to Tamyb10-B1 in each of 96 individuals showed a bimodal distribution without overlapping, which allowed unequivocal classification of the F2 plants into a 1.0 (dominant homozygous) or a 0.5 (heterozygous) class, in accordance with a 1:2 expected ratio. Genotypes with the R-D1 grain color (4 plants per each class) were tested by an F3 progeny test. The results perfectly coincided with the genotypes deduced by the real-time PCR diagnosis. Consequently, the hemizygous status at the Tamyb10-D1 locus controlling grain color in wheat can now be diagnosed with the real-time PCR.

Characterization and QTL Analysis of Oryza longistaminata IntrogressionLine, pLIA-1, derived from a Cross between Oryza longistaminata andOryza sativa (Taichung 65) under Non-fertilized Conditions

To meet and sustain the food demands of an ever-increasing world population, improving the yield of major cereal crops such as rice is necessary with sustainable cultivation harmonized with the environment. It is useful to utilize wild rice species as reservoirs of novel traits for breeding low-input adaptable (LIA) crops. Oryza longistaminata, a wild species of rice native only to Africa, possesses the vigorous biomass needed under low-input conditions. Thus, a potential LIA (pLIA) candidate, pLIA-1, showing large biomass, tall culm, large panicle with many primary and secondary branches and thick culms was selected from a selfed progeny of the cross between O. longistaminata and Taichung 65 (T-65), a japonica variety, under non-fertilized conditions. The pLIA-1 performance was superior to that of Koshihikari, Norin 18, T-65 and Nipponbare under fertilized and non-fertilized conditions suggesting that pLIA-1’s characteristics might be useful for breeding low-input adaptable varieties. QTL analysis in F2 of the cross between pLIA-1 and Norin 18 detected 31 QTLs for yield-elated traits under non-fertilized conditions. The pLIA-1 allele had a positive contribution in 20 of the QTLs detected. Importantly, many of the QTLs were identified around regions where O. longistaminata chromosome segments were introgressed into pLIA-1. These results suggests that the QTLs detected in the F2 are important to improve modern varieties for adaptability to low-input conditions.