Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes.

Blast resistance is one of the most important traits in rice breeding, and application of molecular markers for blast resistance breeding is likely to allow the rapid screening for the trait during early growth stages, without the need for inoculation of pathogen and phenotyping. Allele-specific PCR markers and insertion/deletion (InDel) markers, which genotype single-nucleotide polymorphisms and InDel polymorphisms, respectively, are useful tools for marker-assisted selections. We developed sets of allele-specific PCR and InDel markers for nine rice blast resistance genes -- Piz, Piz-t, Pit, Pik, Pik-m, Pik-p, Pita, Pita-2, and Pib -- which are commonly used in Japanese blast resistance rice breeding programs. For each resistance gene, we used the segregation information from thousands of progeny in several crosses or published gene locations to generate a marker that cosegregated with the gene and markers that closely flanked the gene on either side. The developed cosegregating markers uniquely discriminated among each of the lines with the individual resistance genes (except for Pita and Pita-2). Therefore, these markers will likely facilitate the development of multiline cultivars carrying one or a combination of these nine blast resistance genes. In addition, the systems we developed may be valuable tools in the quality control of seed production from blast-resistant multiline cultivars.

Segmental distribution of genes harboring a CpG island-like region on rice chromosomes.

In plant genomes, there exist discrete regions rich in CpG dinucleotides, namely CpG clusters. In rice, most of these CpG clusters are associated with genes. Rice genes are grouped into one of the five classes according to the position of an associated CpG cluster. Among them, class 1 genes, which harbor a CpG cluster at the 5'-terminus, share similarities with human genes having CpG islands. In the present study, by analyzing plant genome sequence data, primarily from rice, we investigated the chromosomal distribution of genes of each class, mainly class 1 genes. Class 1 genes were not uniformly distributed across the rice genome, but were clustered into discrete chromosomal segments. EST-based analysis of the distribution of expressed genes indicates that this segmental distribution of class 1 genes caused a preferential distribution of expressed genes within class 1 gene-rich segments. We then compared the methylation status of genes of each class to examine the possibility that differential DNA methylation, if any, is relevant to the observed differential expression level of genes inside and outside the class 1 segments. The difference in the methylation level between these genes was revealed to be fairly small, which does not support the above-mentioned possibility.

Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus.

We assessed the utility of single-nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms (InDels) as DNA markers in genetic analysis and breeding of rice. Toward this end, we surveyed SNPs and InDels in the chromosomal region containing the Piz and Piz-t rice blast resistance genes and developed PCR-based markers for typing the SNPs. Analysis of sequences from a blast-susceptible Japanese cultivar and two cultivars each containing one of these genes revealed that SNPs are abundant in the Piz and Piz-t regions (on average, one SNP every 248 bp), but the number of InDels was much lower. The dense distribution of SNPs facilitated the generation of SNP markers in the vicinity of the genes. For typing these SNPs, we used a modified allele-specific PCR method. Of the 49 candidate allele-specific markers, 33 unambiguously and reproducibly discriminated between the two alleles. We used the markers for mapping the Piz and Piz-t genes and evaluating the size of DNA segments introgressed from the Piz donor cultivar in Japanese near-isogenic lines containing Piz. Our findings suggest that, because of its ability to generate numerous markers within a target region and its simplicity in assaying genotypes, SNP genotyping with allele-specific PCR is a valuable tool for gene mapping, map-based cloning, and marker-assisted selection in crops, especially rice.

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers.

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3-4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.

Gene-associated CpG islands in plants as revealed by analyses of genomic sequences.

We screened plant genome sequences, primarily from rice and Arabidopsis thaliana, for CpG islands, and identified DNA segments rich in CpG dinucleotides within these sequences. These CpG-rich clusters appeared in the analysed sequences as discrete peaks and occurred at the frequencies of one per 4.7 kb in rice and one per 4.0 kb in A. thaliana. In rice and A. thaliana, most of the CpG-rich clusters were associated with genes, which suggests that these clusters are useful landmarks in genome sequences for identifying genes in plants with small genomes. In contrast, in plants with larger genomes, only a few of the clusters were associated with genes. These plant CpG-rich clusters satisfied the criteria used for identifying human CpG islands, which suggests that these CpG clusters may be regarded as plant CpG islands. The position of each island relative to the 5'-end of its associated gene varied considerably. Genes in the analysed sequences were grouped into five classes according to the position of the CpG islands within their associated genes. A large proportion of the genes belonged to one of two classes, in which a CpG island occurred near the 5'-end of the gene or covered the whole gene region. The position of a plant CpG island within its associated gene appeared to be related to the extent of tissue-specific expression of the gene; the CpG islands of most of the widely expressed rice genes occurred near the 5'-end of the genes.

Surveying CpG methylation at 5'-CCGG in the genomes of rice cultivars.

We investigated the CpG methylation status of the sequence CCGG in the rice genome by using methylation-sensitive AFLP and subsequent Southern analyses with the isolated AFLP fragments as probes. CpGs located in single- or low-copy-sequence regions could be grouped into two classes on the basis of their methylation status: methylation status at the class 1 CpG sites was conserved among genetically diverse rice cultivars, whereas cultivar-specific differential methylation was frequently detected among the cultivars at the class 2 CpG sites. The frequency of occurrence of methylation polymorphism between a pair of cultivars was not related to the genetic distance between the two. Through mapping, five class 2 CpG sites were localized on different chromosomes and were not clustered together in the genome. Segregation analysis of the cultivar-specific methylations with their target sites indicated that the differential methylation was stably inherited in a Mendelian fashion over 6 generations, although alterations in the methylation status at the class 2 CpG sites were observed with a low frequency.

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.

Yeast artificial chromosome clones of rice chromosome 2 ordered using DNA markers.

Yeast artificial chromosome (YAC) clones were ordered for the physical mapping of rice chromosome 2, the last of the 12 rice chromosomes to be assigned YACs by the Rice Genome Research Program. A total of 128 restriction fragment length polymorphism markers and 4 sequence-tagged site (STS) markers located on our high-density genetic map were used for YAC clone landing. By colony/Southern hybridization and polymerase chain reaction screening, a total of 239 individual YACs were selected from our YAC library of 6934 clones covering six genome equivalents. The YACs located on the corresponding marker positions in the linkage map formed 43 contigs and islands and were estimated to encompass about 50% of the length of rice chromosome 2.

Ordered YAC clone contigs assigned to rice chromosomes 3 and 11.

Yeast artificial chromosome (YAC) clones were arranged on the positions of restriction fragment length polymorphism (RFLP) and sequence-tagged site (STS) markers already mapped on the high-resolution genetic maps of rice chromosomes 3 and 11. From a total of 416 and 242 YAC clones selected by colony/Southern hybridization and polymerase chain reaction (PCR) analysis, 238 and 135 YAC clones were located on chromosomes 3 and 11, respectively. For chromosomes 3 and 11, 24 YAC contigs and islands with total coverage of about 46% and 12 contigs and islands with coverage of about 40%, respectively, were assigned. Although many DNA fragments of multiple copy marker sequences could not be mapped to their original locations on the genetic map by Southern hybridization because of a lack of RFLP, the physical mapping of YAC clones could often assign specific locations of such multiple copy sequences on the genome. The information provided here on contig formation and similar sequence distribution revealed by ordering YAC clones will help to unravel the genome organization of rice as well as being useful in isolation of genes by map-based cloning.

Physical mapping of rice chromosomes 4 and 7 using YAC clones.

Physical maps of rice chromosomes 4 and 7 were constructed by landing yeast artificial chromosomes (YACs) along our high-density molecular linkage map. Using 114 DNA markers, 258 individual YACs were located on chromosome 4. Sixty-two out of 258 YACs carried two or more DNA marker positions and formed 16 contigs which covered a total length of 17.1 cM. The other YACs were arranged to 23 positions. On chromosome 7, 203 individual YACs were landed on 109 DNA markers. Sixty-four out of 203 YACs formed 15 contigs which covered a total length of 21.8 cM and 139 YACs were localized to 26 positions. Chromosomes 4 and 7 were covered with minimum tiling paths of 45 and 48 YACs, respectively. Taking the average size of YAC insert DNA to be 350 kb and the entire genome size to be 430 Mb, about 16-18 Mb of each chromosome or an estimated 50% of their total lengths have been covered with YACs. Physical maps of these 2 chromosomes should be of great help in identifying useful trait genes and unraveling genetic and biological characteristics in rice.

Physical mapping of rice chromosomes 8 and 9 with YAC clones.

First efforts for physical mapping of rice chromosomes 8 and 9 were carried out by ordering YAC clones of a rice genomic DNA library covering six genome equivalents with mapped DNA markers. A total of 79 and 74 markers from chromosomes 8 and 9, respectively, were analyzed by YAC colony and Southern hybridization using RFLP markers of cDNA and genomic clones, and by polymerase chain reaction (PCR) screening using PCR-derived and sequence-tagged site (STS) markers. As a result, 252 YAC clones were confirmed to contain the mapped DNA fragments on both chromosomes. A contig map was constructed by ordering these YAC clones and about 53% and 43% genome coverage was obtained for chromosomes 8 and 9, respectively, assuming a YAC clone size of 350 kb and overlap between neighboring YACs of 50%. A continuous array of YAC clones with minimum overlap gave a total size of 18.9 Mb for chromosome 8 and 15.6 Mb for chromosome 9, which are close to previous estimates. These contig maps may provide valuable information that can be useful in understanding chromosome structure and isolating specific genes by map-based cloning.

Assignment of YAC clones spanning rice chromosomes 10 and 12.

Yeast artificial chromosome (YAC) clones were assigned on rice (Oryza saliva L. cv. Nipponbare) chromosomes 10 and 12 using DNA markers from our high-density linkage map. Out of 1,383 markers localized in this genetic map, 68 and 74 markers were located on chromosomes 10 and 12, respectively. Screening of the YAC genomic library was conducted by colony hybridization and Southern hybridization using restriction fragment length polymorphism (RFLP) markers or by polymerase chain reaction (PCR) using sequence-tagged site (STS) markers. We have completed the screening of 68 markers on chromosomes 10 and 74 markers on chromosome 12. A total of 134 and 103 YACs were assigned to chromosomes 10 and 12, respectively, with an estimated coverage of more than 60% for chromosome 10 and about 47% for chromosome 12. As rice is considered a model plant for genome analysis, the ordered YAC clones on chromosomes 10 and 12 as well as other chromosomes will certainly be helpful for isolation of agronomically and biologically important genes and for understanding the genome structure of these chromosomes.

An ordered yeast artificial chromosome library covering over half of rice chromosome 6.

Yeast artificial chromosome (YAC) clones carrying DNA marker sequences located on the rice genetic map of chromosome 6 were ordered for physical mapping. A total of 122 restriction fragment length polymorphism markers, 16 sequence-tagged site markers, and five random amplified polymorphic DNA markers located, on average, at 0.9-cM intervals, were used for YAC clone screening by colony/Southern hybridization and PCR screening, respectively. A total of 216 individual YACs were selected from our YAC library of 7000 clones covering six genome equivalents. Each DNA marker could select, on average, 4.8 YAC clones, with 11 clones being the maximum. The YACs localized to the corresponding linkage map positions form 43 contigs and encompass about 60% of rice chromosome 6. This is the first step in constructing a physical map covering the whole rice genome by chromosome landing with YAC clones. These YACs and data will be used soon to isolate phenotypical trait genes by map-based cloning.

Physical mapping of rice chromosome 1 with yeast artificial chromosomes (YACs).

We have constructed a physical map of rice chromosome 1 using yeast artificial chromosomes (YACs). A YAC library of 350 kb average insert size, covering about 6 rice haploid genome equivalents, was screened using 182 DNA markers which we had previously located on chromosome 1, by colony hybridization and polymerase chain reaction (PCR) amplification. One hundred and sixty-two DNA markers identified at least one YAC each carrying one, two or more marker sequences, for a total of 476 clones. Of these identified YACs, 284 were located in their original positions on chromosome 1. These 284 YACs defined 69 YAC contigs or islands which are estimated to cover more than 60% of the total chromosome length. The use of mapped DNA markers in constructing a physical map facilitates the integration of genetic and physical maps, as well as fine ordering of the DNA markers, especially at sites where the markers are clustered tightly on the genetic map. Our high density molecular map has been proven, by chromosome landing with YACs using mapped DNA markers, to cover more than half of the entire length of chromosome 1. The remaining 192 YACs were selected by other copies of DNA markers that mapped on chromosome 1. This description of the YAC contigs formed on chromosome 1 constitutes the second report of rice physical mapping, following that for chromosome 6.

Construction of YAC contigs on rice chromosome 5.

A physical map of rice chromosome 5 was constructed with yeast artificial chromosome (YAC) clones along a high-resolution molecular linkage map carrying 118 DNA markers distributed over 123.7 cM of genomic DNA. YAC clones have been identified by colony and Southern hybridization for 105 restriction fragment length polymorphism (RFLP) markers and by polymerase chain reaction (PCR) screening for 8 sequence-tagged site (STS) markers and 5 randomly amplified polymorphic DNA (RAPD) markers. Of 458 YACs, 235 individual YACs with an average insert length of 350 kb were selected and ordered on chromosome 5 from the YAC library. Forty-eight contigs covering nearly 21 Mb were formed on the chromosome 5; the longest one was 6 cM and covered 1.5 Mb. The length covered with YAC clones corresponded to 62% of the total length, of chromosome 5. There were many multicopy sequences of expressed genes on chromosome 5. The distribution of many copies of these expressed gene sequences was determined by YAC Southern hybridization and is discussed. A physical map with these characteristics provides a powerful tool for elucidation of genome structure and extraction of useful genetic information in rice.

Molecular organization and dynamics in bacteriorhodopsin-rich reconstituted membranes: discrimination of lipid environments by the oxygen transport parameter using a pulse ESR spin-labeling technique.

Molecular organization and dynamics in protein-rich membranes have been studied by investigating transport (diffusion-concentration product) of molecular oxygen at various locations in reconstituted membranes of bacteriorhodopsin (BR) and L-alpha-dimyristoylphosphatidylcholine. Oxygen transport was evaluated by monitoring the bimolecular collision of molecular oxygen with four types of nitroxide lipid spin labels placed at various locations in the membrane. The collision rate was estimated from the spin-lattice relaxation times (T1's) measured at various oxygen partial pressures by analyzing the short-pulse saturation recovery ESR signals. CD spectra and decay of polarized flash-induced photodichroism of bacteriorhodopsin indicated that BR molecules are monomers in reconstituted membranes with a lipid/BR molar ratio of 80 (80-rec) and are 25% monomers and 75% trimers plus oligomers of trimers when the lipid/BR ratio is 40 (40-rec). In the 80-rec, the lipid environment is homogeneous on a microsecond scale (T1), probably because the exchange rate of lipids between the bulk and the boundary regions is greater than the T1 relaxation rate (approximately 10(6) s-1). The oxygen collision rate in the hydrophobic region of the 80-rec membrane is smaller by a factor of 1.6 than in that of the lipid membrane without BR, and the effect of BR in decreasing the collision rate is independent of the "depth" in the hydrophobic region. In the 40-rec, two collision rates were observed, one of which is close to those for purple membrane (or the gel-phase membrane), while the other is about the same as was measured in the 80-rec.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and mapping of telomere-associated sequences from rice.

We have isolated three telomere-associated sequences from rice using cassette-ligation-mediated polymerase chain reaction (PCR). Each of the obtained clones hybridized to the terminal of one or several rice chromosome arms. The telomeres recognized by the clones displayed a high level of polymorphism between two rice varieties, Nipponbare (a japonica variety) and Kasalath (an indica variety). Variability in the chromosome termini was also detected among individual F2 progeny plants, which were derived from a cross between the two rice varieties. One clone containing telomere-associated sequences was located to one end of chromosome 5, and another clone to one end of chromosome 11. For another clone, non-allelic segregation of polymorphic hybridization bands was observed between japonica and indica rice; this clone was mapped to one end of chromosome 12 in japonica and to one end of chromosome 11 in indica rice. This indicates an exchange of termini between nonhomologous chromosomes.

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.

Drug-induction decreases the mobility of cytochrome P-450 in rat liver microsomes: protein rotation study.

Effect of drug-induction on the rotation of cytochrome P-450 and on lipid fluidity in rat liver microsomes was examined. Rotational diffusion of cytochrome P-450 was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. Microsomal lipid fluidity was measured by observing fluorescence anisotropy of DPH incorporated in the lipid bilayer. The absorption anisotropy decayed within 2 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the drug-induction with PB, MC, and PCB when compared with non-induced CON-microsomes. The observed values for the normalized time-independent anisotropy r(infinity)/r(0) are r(infinity)/r(0) = 0.41 (CON-microsomes), 0.54 (PB-microsomes), 0.52 (MC-microsomes), and 0.57 (PCB-microsomes). The average rotational relaxation time phi = 580-690 microseconds was almost unchanged over all microsomes presently examined. A significantly high value of r(infinity)/r(0) = 0.41-0.57 implies the co-existence of mobile and immobile populations of cytochrome P-450. Based on the assumption that the heme tilts about 55 degrees from the membrane plane for all species of P-450s besides P-450PB, 59% (CON-microsomes), 46% (PB-microsomes), 48% (MC-microsomes), and 43% (PCB-microsomes), respectively, of the cytochrome P-450 in microsomes is calculated to be mobile. Upon drug-induction the microsomal membrane was fluidized to some extent as judged by the steady-state fluorescence anisotropy of 0.156 for CON-microsomes and 0.139-0.148 for drug-induced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope.

Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.