Genetic dissection of a genomic region for a quantitative trait locus, Hd3, into two loci, Hd3a and Hd3b, controlling heading date in rice.

The rice photoperiod sensitivity gene Hd3 was originally detected as a heading date-related quantitative trait locus localized on chromosome 6 of rice. High-resolution linkage mapping of Hd3 was performed using a large segregating population derived from advanced backcross progeny between a japonica variety, Nipponbare, and an indica variety, Kasalath. To determine the genotype of Hd3, we employed progeny testing under natural field and short-day conditions. As a result, two tightly linked loci, Hd3a and Hd3b, were identified in the Hd3 region. Nearly-isogenic lines for Hd3a and Hd3b were selected from progeny using marker-assisted selection. The inheritance mode of both Hd3a and Hd3b was found to be additive. Analysis of daylength response in nearly-isogenic lines of Hd3a and Hd3b showed that the Kasalath allele at Hd3a promotes heading under short-day conditions while that at Hd3b causes late heading under long-day and natural field conditions.

Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS.

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes.

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map-based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine-rich repeats (LRRs); thus, Pib is a member of the NBS-LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N-terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.

Characterization and genetic mapping of simple sequence repeats in the rice genome.

We searched partial sequences of over 22,706 rice cDNA and 1220 genomic DNA clones to find and characterize simple sequence repeats (SSRs) in the rice genome. The most frequently found repeated SSR motif in both cDNA and genomic DNA sequences was d(CCG/CGG)n. The second most frequently found SSR was d(AG/CT)n. In contrast with mammalian genomes, in which d(AC/GT)n sequences are the most abundant, d(AG/GT)n sequences were not frequently observed in rice. Sequences containing d(CCG/CGG)n, d(AG/CT)n repeats, and other SSRs were chosen for polymorphism detection. It was predicted that 17 of 20 SSRs in cDNA sequences were located in 5'-untranslated regions near initiation codons. Twenty-two loci can be mapped on our RFLP linkage map by these SSRs. Six markers were tested with 16 japonica rice varieties as templates for PCR. Two markers exhibited amplified fragment length polymorphism among these rice varieties, implying that SSRs are polymorphic among rice varieties which have similar genetic backgrounds. Even these polymorphic SSRs are located within or around genes which code ubiquitous proteins.

Screening of RAPD markers linked to the photoperiod-sensitivity gene in rice chromosome 6 using bulked segregant analysis.

Bulked segregant analysis was used to determine randomly amplified polymorphic DNA (RAPD) markers in a specific interval in the middle of chromosome 6 of rice for tagging the photoperiod sensitivity gene. Two pools of F2 individuals (japonica cv. Nipponbare and indica cv. Kasalath) were constructed according to the genotypes of three restriction fragment length polymorphism (RFLP) markers located at both ends and the middle of the targeted interval. Then another pair of pools were constructed based on the "graphical genotype," which was made with our high density linkage map. RAPD analysis was performed using these DNA pools as templates, and polymorphic fragments were detected and mapped. Using 80 primers, either singly or pairwise, we tested 2,404 primer pairs and established 14 markers tightly linked to the photoperiod sensitivity gene. The obtained RAPD markers were converted into sequence-tagged sites by cloning and sequencing of the polymorphic fragments and they can be used directly for construction of physical maps. This bulked segregant method can be applied for any species and any region of interest in which detailed linkage maps or physical maps are needed.

Determination of RAPD markers in rice and their conversion into sequence tagged sites (STSs) and STS-specific primers.

We produced 102 randomly amplified polymorphic DNA (RAPD) markers mapped on all 12 chromosomes of rice using DNAs of cultivars Nipponbare (japonica) and Kasalath (indica) and of F2 population generated by a single cross of these parents. Sixty random primers 10 nucleotides long were used both singly and in random pairs and about 1,400 primer-pairs were tested. Using both agarose gel and polyacrylamide gel electrophoresis enabled us to detect polymorphisms appearing in the range from < 100 bp to 2 kb. The loci of the RAPD markers were determined onto the framework of our RFLP linkage map and some of these markers were mapped to regions with few markers. Out of the 102 RAPD markers, 20 STSs (sequence-tagged sites) and STS-specific primer pairs were determined by cloning, identifying and sequencing of the mapped polymorphic fragments.

Mapping of sequence-tagged sites in rice by single strand conformation polymorphism.

The conditions for efficient single-strand conformation polymorphism (SSCP) detection were examined for its application to mapping of DNA regions in the rice genome. Temperature for electrophoresis and glycerol concentrations in gel affected SSCP patterns significantly. The optimal detection conditions for SSCP also depends on the nucleotide sequences of fragments analyzed. Fragments over 300 bp show complicated patterns depending on their nucleotide sequences and were not suitable for SSCP analysis. Seventy primer pairs were designed from the sequence data available to amplify DNA regions as sequence tagged sites (STSs), and 39 of these STSs were found to generate SSCP between japonica rice (Nipponbare) and indica rice (Kasalath) in at least one of the experimental conditions. The maps of DNA fragments amplified from 186 F2-plant DNAs with 17 primer pairs were successfully determined. This direct mapping method of the amplified DNA fragments with PCR is simple and quite sensitive, and can be used to set markers in the gap regions of a genetic linkage map.

Sequence-tagged sites (STSs) as standard landmarkers in the rice genome.

Generating sequence-tagged sites (STSs) is a prerequisite to convert a genetic map to a physical map. With the help of sequence information from these STSs one can also isolate specific genes. For these purposes, we have designed PCR primer sets, of 20 bases each, by reference to sequences of restriction fragment length polymorphism (RFLP) landmarkers consisting of rice genomic clones. These markers were evenly distributed over the 12 chromosomes and were shown to be single copy by Southern-blot analysis. With improved PCR protocols, 63 standard STS landmarkers in the rice genome were generated. Similarity searches of all partial sequences of RFLP landmarkers by the FASTA algorithm showed that 2 of the 63 RFLP landmarkers, G357 and G385, contained part of the ORFs of aspartate aminotransferase and protein kinase, respectively.

Microbial degradation of dibenzofuran, fluorene, and dibenzo-p-dioxin by Staphylococcus auriculans DBF63.

Staphylococcus auriculans DBF63, which can grow on dibenzofuran (DBF) or fluorene (FN) as the sole source of carbon and energy, was isolated. Salicylic acid and gentisic acid accumulated in the culture broth of this strain when DBF was supplied as a growth substrate. Also, the formation of 9-fluorenol, 9-fluorenone, 4-hydroxy-9-fluorenone, and 1-hydroxy-9-fluorenone was demonstrated, and accumulation of 1,1a-dihydroxy-1-hydro-9-fluorenone was observed when this strain grew on FN. On the basis of these results, the degradation pathways of DBF and FN were proposed. The analogous oxidation products of dibenzo-p-dioxin were obtained by incubation with DBF-grown S. auriculans DBF63 cells.

Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1.

Strain SY1, identified as a Corynebacterium sp., was isolated on the basis of the ability to utilize dibenzothiophene (DBT) as a sole source of sulfur. Strain SY1 could utilize a wide range of organic and inorganic sulfur compounds, such as DBT sulfone, dimethyl sulfide, dimethyl sulfoxide, dimethyl sulfone, CS2, FeS2, and even elemental sulfur. Strain SY1 metabolized DBT to dibenzothiophene-5-oxide, DBT sulfone, and 2-hydroxybiphenyl, which was subsequently nitrated to produce at least two different hydroxynitrobiphenyls during cultivation. These metabolites were separated by silica gel column chromatography and identified by nuclear magnetic resonance, UV, and mass spectral techniques. Resting cells of SY1 desulfurized toluenesulfonic acid and released sulfite anion. On the basis of these results, a new DBT degradation pathway is proposed.