Characterization and expression analysis of the starch synthase gene family was performed from grain amaranth. Extensive searches for sequence data for amaranth starch synthase genes were done in the literature and in GenBank, and identified four genes encoding GBSSI, SSSI, SSSII, and SBE. Alignment of predicted amino acid sequences showed that there were low levels of sequence identity among these four genes (10.6–34.5%). There was higher homology in the C-terminal region and lower homology in the N-terminal region of the amaranth starch synthases. Starch synthases in plants can be classified into six groups, and phylogenetic analyses showed that the starch synthases from amaranth were in four of these groups; GBSSs, SSSIs, SSSIIs, and SBEs of dicot plants. Interestingly, all amaranth starch synthases were sister to the subgroup consisting of the rest of the dicot plants, clustering more closely with dicots than with monocots. Analysis of the expression patterns of starch synthase genes in storage and non-storage tissues of amaranth revealed two types of expression patterns; (1) GBSSI was rapidly expressed at the middle or mid-late stages of seed development; and (2) SSSI, SSSII and SBE genes were expressed constitutively during seed maturation. These four genes were expressed in non-storage tissues as well as in storage tissue, suggesting that non-storage and storage tissues of amaranth share a common mechanism for expression of starch synthase genes. This summary of the basic characteristics of starch synthase genes will contribute further studies on amaranth starch.

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TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that can be employed to generate allelic series of induced mutations in targeted genes for functional analyses. To date, TILLING resources in Arabidopsis thaliana are only available in accessions Columbia and Landsberg erecta. Here, we extended the Arabidopsis TILLING resources by developing a new population of ethyl methanesulfonate (EMS)-induced mutant lines in another commonly used A. thaliana accession C24. A permanent collection of 3,509 independent EMS mutagenized M2 lines was developed in A. thaliana accession C24, and designated C24TILL. Using the TILLING method to search C24TILL for mutations in four selected genes identified a total of 73 mutations, comprising 69.6% missense, 29.0% sense, and 1.4% nonsense mutations. Consistent with the propensity of EMS to induce guanine alkylation, 98.4% of the observed mutations were G/C to A/T transitions. Based on the mutations identified in the four target genes, the overall mutation density in the C24TILL collection was estimated to be 1/345 kb. TILLING the DUO POLLEN 1 (DUO1) gene from the C24TILL collection identified a truncation mutation leading to a deficiency in sperm cell differentiation. Taken together, a new TILLING resource, the C24TILL collection, was generated for A. thaliana accession C24. The C24TILL collection provides an allelic series of induced point mutations that will serve as a useful alternative reverse genetic resource for functional genetic studies in A. thaliana.

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Grain shape and size are involved in the main components of the domestication syndrome in cereals. Wheat grain shape has been dramatically altered at each stage of the domestication of tetraploid wheat and through common wheat speciation. To elucidate the evolutionary change of wheat grain shape, principal component (PC) analysis of grain shape-related traits was first conducted using wild and cultivated tetraploid, synthetic hexaploid, and common wheat accessions. The synthetic hexaploid wheat lines were previously produced through interspecific crosses between two common wheat progenitors, tetraploid wheat and Aegilops tauschii, and produced grains similar to those of cultivated tetraploid wheat. To identify genetic loci related to the difference in grain shape between common wheat and the synthetic wheat, the 15 traits related to grain and spikelet shape were measured in 108 F₂ individuals between Norin 61 and a synthetic wheat line, and the first three PC values for the 15 traits, PC1, PC2 and PC3, were mapped as quantitative traits in the F₂ population. In total, six QTLs, found on chromosomes 1A, 5A, 1D, 2D and 7D, showed significant LOD scores. Among them, a QTL for PC2, located on the 2DS chromosomal region near the Ppd-D1 locus, mainly contributed to the phenotypic difference in grain shape. Tg-D1, controlling tenacious glume phenotype, was located at a similar region to the 2DS QTL, which suggested that the Tg-D1 locus pleiotropically affects not only glume toughness but also spikelet and grain shape in hexaploid wheat. Therefore, it was predicted that wheat grains were rapidly improved toward a shorter and rounder phenotype accompanied with free-threshing wheat formation.

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An in-situ conservation site in Laos for a mixture of annual and perennial wild rice, LV27, which is a single swamp with an observation pier has been developed. In order to develop a strategy for evaluation of natural resources, systematic leaf sampling has been conducted and their genetic characteristics measured with 16 SSR loci. In order to determine population structure, a small number of individuals localized together were regarded as sub-populations belonging to a single mother population. Annual individuals were clustered at particular peripheral areas of the pond. Perennial individuals were close by and growing within deeper pond water. Scores of observed heterozygosity (Ho) were not significantly different between annual and perennial sub-populations, but relatively lower in annual ones. Genetic distance among annual and perennial sub-populations in close juxtaposition at peripheral sites showed that annuals were clustered against perennials. In addition, comparison of perennial sub-populations peripheral areas and inside the swamp, found they clustered together and were some distance from annual ones. When the genetic components were compared in detail, private alleles were frequently found in annual plants, suggesting there might be restriction of gene flow between annual and perennial types. Partitions of deep water perennial sub-populations identified private alleles in particular areas, suggesting there were some areas with unique polymorphisms. Combining peripheral perennial sub-populations led to the disappearance of most private alleles which implied there is frequent gene flow among perennial sub-populations. This in-situ conservation site allowed us to observe the succession of populations and also to research detailed population structure of a typical wild population and this found wild rice genetic structure in this single swamp is complex. The data obtained will provide valuable insight about how to evaluate wild populations genetically and how to deal with such populations as field collections.

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Insect odorant-binding proteins function in the sensing of odors, tastes, and pheromones. Genes encoding two odorant-binding proteins, Obp57d and Obp57e, were identified to be involved in the behavioral adaptation of Drosophila sechellia to its host plant. The two genes are expressed in cells associated with taste sensilla on the legs, and the expression pattern in the legs is conserved among closely related species. To identify the cis-regulatory elements necessary for the expression in the leg sensilla, the promoter sequences of Obp57d and Obp57e were compared among species. Two types of conserved sequence-motifs were found as candidate cis-regulatory elements. Functions of these conserved elements in the promoters of D. melanogaster Obp57d and Obp57e were examined by using a newly constructed vector that combines the advantages of φC31 integrase-based transformation and gypsy transposable-element-derived insulators. By GFP-reporter assay using the new vector, it was confirmed that these conserved elements are necessary for the expression in the legs, working synergistically with each other to affect the expression level. Single-nucleotide substitutions in these elements dramatically changed the promoter activity. These results provide insight into the molecular mechanism for evolution of adaptive behavior via modulation of OBP expression levels.

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We analyzed PCR-amplified carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene fragments from 146 Bombyx mori native strains and found extremely low levels of DNA polymorphism. Two haplotypes were identified, one of which was predominant. CAD haplotype analysis of 42 samples of Japanese B. mandarina revealed four haplotypes. No common haplotype was shared between the two species and at least five base substitutions were detected. This result was suggestive of low levels of gene flow between the two species. The nucleotide diversity (π) scores of the two samples differed markedly: lower π values were estimated for B. mori native strains than Japanese B. mandarina. We further analyzed 12 Chinese B. mandarina derived from seven areas of China, including Taiwan. The results clearly indicated that the π score was ~80-fold greater in Chinese B. mandarina than in B. mori. The extremely low level of DNA polymorphism in B. mori compared to its wild relatives suggested that the CAD gene itself or its tightly linked regions are possible targets for silkworm domestication.

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We have previously identified four significant quantitative trait loci (QTL) affecting plasma glucose concentrations on F₂ progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and non-diabetic A/J mice, one of which on chromosome 15 named Dbm4 (diabetic modifier QTL 4) was shown to affect fasting plasma glucose concentrations with a maximum LOD score of 6.17. To estimate the influence of Dbm4 itself to the diabetes-related phenotypes, we constructed congenic strain with heterozygous Ins2 mutation using the Akita allele as donor of Dbm4 locus in the A/J genetic background, and measured quantitative traits including plasma glucose concentrations in glucose tolerance test (GTT). In this study, we found the Akita allele in Dbm4 was associated with higher fasting plasma glucose concentrations as in previous QTL analysis. According to gene expression assay, key enzymes of hepatic gluconeogenesis were expressed to the more increased degree in the liver of congenic mice compared to the A/J allele based control mice. Based on these results, we concluded that diabetic modifier gene(s) exist on Dbm4 locus affecting fasting plasma glucose concentrations via regulation of gluconeogenic gene expression in the hypoinsulinemic diabetic condition. Identification of the modifier gene responsible for Dbm4 would provide new drug development targets for human type 2 diabetes with hepatic insulin resistance.

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